Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media {LLC}  

Bifidobacteria are among the first colonizers of the infant gut, and human milk oligosaccharides (HMOs) in breastmilk are instrumental for the formation of a bifidobacteria-rich microbiota. However, little is known about the assembly of bifidobacterial communities. Here, by applying assembly theory to a community of four representative infant-gut associated Bifidobacterium species that employ varied strategies for HMO consumption, we show that arrival order and sugar consumption phenotypes significantly affected community formation. Bifidobacterium bifidum and Bifidobacterium longum subsp. infantis, two avid HMO consumers, dominate through inhibitory priority effects. On the other hand, Bifidobacterium breve, a species with limited HMO-utilization ability, can benefit from facilitative priority effects and dominates by utilizing fucose, an HMO degradant not utilized by the other bifidobacterial species. Analysis of publicly available breastfed infant faecal metagenome data showed that the observed trends for B. breve were consistent with our in vitro data, suggesting that priority effects may have contributed to its dominance. Our study highlights the importance and history dependency of initial community assembly and its implications for the maturation trajectory of the infant gut microbiota.

DOI： 10.1038/s41396-022-01270-3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.carres.2022.108626

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Japanese Society of Applied Glycoscience  

DOI： 10.5458/jag.jag.jag-2021_0012

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Human milk oligosaccharides (HMOs), which are natural bifidogenic prebiotics, were recently commercialized to fortify formula milk. However, HMO assimilation phenotypes of bifidobacteria vary by species and strain, which has not been fully linked to strain genotype. We have recently shown that specialized uptake systems, particularly for the internalization of major HMOs (fucosyllactose [FL]), are associated with the formation of a Bifidobacterium-rich gut microbial community. Phylogenetic analysis revealed that FL transporters have diversified into two clades harboring four clusters within the Bifidobacterium genus, but the underpinning functional diversity associated with this divergence remains underexplored. In this study, we examined the HMO consumption phenotypes of two bifidobacterial species, Bifidobacterium catenulatum subsp. kashiwanohense and Bifidobacterium pseudocatenulatum, both of which possess FL-binding proteins that belong to phylogenetic clusters with unknown specificities. Growth assays, heterologous gene expression experiments, and HMO consumption analyses showed that the FL transporter type from B. catenulatum subsp. kashiwanohense JCM 15439T conferred a novel HMO uptake pattern that includes complex fucosylated HMOs (lacto-N-fucopentaose II and lacto-N-difucohexaose I/II). Further genomic landscape analyses of FL transporter-positive bifidobacterial strains revealed that the H-antigen- or Lewis antigen-specific fucosidase gene(s) and FL transporter specificities were largely aligned. These results suggest that bifidobacteria have acquired FL transporters along with the corresponding gene sets necessary to utilize the imported HMOs. Our results provide insight into the species- and strain-dependent adaptation strategies of bifidobacteria in HMO-rich environments. IMPORTANCE The gut of breastfed infants is generally dominated by health-promoting bifidobacteria. Human milk oligosaccharides (HMOs) from breast milk selectively promote the growth of specific taxa such as bifidobacteria, thus forming an HMO-mediated host-microbe symbiosis. While the coevolution of humans and bifidobacteria has been proposed, the underpinning adaptive strategies employed by bifidobacteria require further research. Here, we analyzed the divergence of the critical fucosyllactose (FL) HMO transporter within Bifidobacterium. We have shown that the diversification of the solute-binding proteins of the FL transporter led to uptake specificities of fucosylated sugars ranging from simple trisaccharides to complex hexasaccharides. This transporter and the congruent acquisition of the necessary intracellular enzymes allow bifidobacteria to consume different types of HMOs in a predictable and strain-dependent manner. These findings explain the adaptation and proliferation of bifidobacteria in the competitive and HMO-rich infant gut environment and enable accurate specificity annotation of transporters from metagenomic data.

DOI： 10.1128/AEM.01437-21

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DOI： 10.1016/j.jbc.2021.101324

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DOI： 10.1016/j.carres.2021.108402

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1007/s10570-021-03954-z

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Other Link： https://link.springer.com/article/10.1007/s10570-021-03954-z/fulltext.html

Publishing type：Research paper (scientific journal)   Publisher：Informa UK Limited  

DOI： 10.1080/19490976.2021.1973835

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Levoglucosan (LG) is an anhydrosugar produced through glucan pyrolysis and is widely found in nature. We previously isolated an LG-utilizing thermophile, Bacillus smithii S-2701M, and suggested that this bacterium may have a metabolic pathway from LG to glucose, initiated by LG dehydrogenase (LGDH). Here, we completely elucidated the metabolic pathway of LG involving three novel enzymes in addition to LGDH. In the S-2701M genome, three genes expected to be involved in the LG metabolism were found in the vicinity of the LGDH gene locus. These four genes including LGDH gene (lgdA, lgdB1, lgdB2, and lgdC) were expressed in Escherichia coli and purified to obtain functional recombinant proteins. Thin layer chromatography analyses of the reactions with the combination of the four enzymes elucidated the following metabolic pathway: LgdA (LGDH) catalyzes 3-dehydrogenation of LG to produce 3-keto-LG, which undergoes β-elimination of 3-keto-LG by LgdB1, followed by hydration to produce 3-keto-D-glucose by LgdB2; next, LgdC reduces 3-keto-D-glucose to glucose. This sequential reaction mechanism resembles that proposed for an enzyme belonging to glycoside hydrolase family 4, and results in the observational hydrolysis of LG into glucose with coordination of the four enzymes.

DOI： 10.1038/s41598-020-77133-8

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.carres.2020.108129

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Publishing type：Research paper (scientific journal)   Publisher：The Japanese Society of Applied Glycoscience  

DOI： 10.5458/jag.jag.jag-2020_0003

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© 2019, Springer Nature B.V. The local structure on the surface of plate-like cellulose II oligomer crystals (CIIOCs) was investigated by NMR spectroscopy. CIIOCs with reducing ends that were selectively labeled with 13C were enzymatically synthesized using cellodextrin phosphorylase. The solid-state 13C cross polarization/magic angle spinning spectra of the 13C-labeled CIIOCs clearly exhibited two resonance peaks (labeled as C1Rα and C1Rβ) derived from the C1 atoms of the reducing ends. The 2D 13C double quantum/13C single quantum homonuclear correlation spectrum indicated that two magnetically nonequivalent glucopyranose rings (Rα and Rβ units) coexisted at the reducing end units. The 1H/13C heteronuclear correlation spectrum suggested that there was a large difference between the local environments around the anomeric carbons of Rα and Rβ units. The abundance ratio of C1Rβ to C1Rα in the solid state was 4:1, whereas that of C1α and C1β in solution was 1:1. When the oligomer chains are packed in the cellulose II crystal, the reducing end units located on the surface of the plate-like crystal may tend to have β-anomeric structure, which would be more sterically stable than the α-anomeric structure.

DOI： 10.1007/s10570-019-02896-x

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Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

The infant’s gut microbiome is generally rich in the Bifidobacterium genus. The mother’s milk contains natural prebiotics, called human milk oligosaccharides (HMOs), as the third most abundant solid component after lactose and lipids, and of the different gut microbes, infant gut-associated bifidobacteria are the most efficient in assimilating HMOs. Indeed, the fecal concentration of HMOs was found to be negatively correlated with the fecal abundance of Bifidobacterium in infants. Given these results, two HMO molecules, 2′-fucosyllactose and lacto-N-neotetraose, have recently been industrialized to fortify formula milk. As of now, however, our knowledge about the HMO consumption pathways in infant gut-associated bifidobacteria is still incomplete. The recent studies indicate that HMO assimilation abilities significantly vary among different Bifidobacterium species and strains. Therefore, to truly maximize the effects of prebiotic and probiotic supplementation in commercialized formula, we need to understand HMO consumption behaviors of bifidobacteria in more detail. In this review, we summarized how different Bifidobacterium species/strains are equipped with varied gene sets required for HMO assimilation. We then examined the correlation between the abundance of the HMO-related genes and bifidobacteria-rich microbiota formation in the infant gut through data mining analysis of a deposited fecal microbiome shotgun sequencing dataset. Finally, we shortly described future perspectives on HMO-related studies.

DOI： 10.3390/nu12010071

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The human gut microbiota established during infancy has persistent effects on health. In vitro studies have suggested that human milk oligosaccharides (HMOs) in breast milk promote the formation of a bifidobacteria-rich microbiota in infant guts; however, the underlying molecular mechanism remains elusive. Here, we characterized two functionally distinct but overlapping fucosyllactose transporters (FL transporter-1 and -2) from Bifidobacterium longum subspecies infantis. Fecal DNA and HMO consumption analyses, combined with deposited metagenome data mining, revealed that FL transporter-2 is primarily associated with the bifidobacteria-rich microbiota formation in breast-fed infant guts. Structural analyses of the solute-binding protein (SBP) of FL transporter-2 complexed with 2'-fucosyllactose and 3-fucosyllactose, together with phylogenetic analysis of SBP homologs of both FL transporters, highlight a unique adaptation strategy of Bifidobacterium to HMOs, in which the gain-of-function mutations enable FL transporter-2 to efficiently capture major fucosylated HMOs. Our results provide a molecular insight into HMO-mediated symbiosis and coevolution between bifidobacteria and humans.

DOI： 10.1126/sciadv.aaw7696

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Infant gut-associated bifidobacteria has a metabolic pathway that specifically utilizes lacto-N-biose I (Gal-β1,3-GlcNAc) and galacto-N-biose (Gal-β1,3-GalNAc) from human milk and mucin glycans. UDP-glucose 4-epimerase (GalE) from Bifidobacterium longum (bGalE) catalyzes epimerization reactions of UDP-Gal into UDP-Glc and UDP-GalNAc into UDP-GlcNAc with the same level of activity that is required to send galacto-hexoses into glycolysis. Here, we determined the crystal structures of bGalE in three ternary complex forms: NAD+/UDP, NAD+/UDP-GlcNAc, and NAD+/UDP-Glc. The broad specificity of bGalE was explained by structural features of the binding pocket for the N-acetyl or C2 hydroxy group of the substrate. Asn200 is located in a pocket of the C2 group, and its side chain adopts different conformations in the complex structures with UDP-Glc and UDP-GlcNAc. On the other side, Cys299 forms a large pocket for the C5 sugar ring atom. The flexible C2 pocket and the large C5 pocket of bGalE are suitable for accommodating both the hydroxy and N-acetyl groups of the substrate during sugar ring rotation in the catalytic cycle. The substrate specificity and active site structure of bGalE were distinct from those of Esherichia coli GalE but similar to those of human GalE.

DOI： 10.1038/s41598-019-47591-w

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DOI： 10.5458/jag.jag.JAG-2018_0002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Levoglucosan is the 1,6-anhydrosugar of d-glucose formed by pyrolysis of glucans and is found in the environment and industrial waste. Two types of microbial levoglucosan metabolic pathways are known. Although the eukaryotic pathway involving levoglucosan kinase has been well-studied, the bacterial pathway involving levoglucosan dehydrogenase (LGDH) has not been well-investigated. Here, we identified and cloned the lgdh gene from the bacterium Pseudarthrobacter phenanthrenivorans and characterized the recombinant protein. The enzyme exhibited high substrate specificity toward levoglucosan and NAD(+) for the oxidative reaction and was confirmed to be LGDH. LGDH also showed weak activities (approximate to 4%) toward l-sorbose and 1,5-anhydro-d-glucitol. The reverse (reductive) reaction using 3-keto-levoglucosan and NADH exhibited significantly lower K-m and higher k(cat) values than those of the forward reaction. The crystal structures of LGDH in the apo and complex forms with NADH, NADH + levoglucosan, and NADH + l-sorbose revealed that LGDH has a typical fold of Gfo/Idh/MocA family proteins, similar to those of scyllo-inositol dehydrogenase, aldose-aldose oxidoreductase, 1,5-anhydro-d-fructose reductase, and glucose-fructose oxidoreductase. The crystal structures also disclosed that the active site of LGDH is distinct from those of these enzymes. The LGDH active site extensively recognized the levoglucosan molecule with six hydrogen bonds, and the C3 atom of levoglucosan was closely located to the C4 atom of NADH nicotinamide. Our study is the first molecular characterization of LGDH, providing evidence for C3-specific oxidation and representing a starting point for future biotechnological use of LGDH and levoglucosan-metabolizing bacteria.

DOI： 10.1074/jbc.RA118.004963

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Gut microbiota of breast-fed infants are generally rich in bifidobacteria. Recent studies show that infant gut-associated bifidobacteria can assimilate human milk oligosaccharides (HMOs) specifically among the gut microbes. Nonetheless, little is known about how bifidobacterial-rich communities are shaped in the gut. Interestingly, HMOs assimilation ability is not related to the dominance of each species. Bifidobacterium longum susbp. longum and Bifidobacterium breve are commonly found as the dominant species in infant stools; however, they show limited HMOs assimilation ability in vitro. In contrast, avid in vitro HMOs consumers, Bifidobacterium bifidum and Bifidobacterium longum subsp. infantis, are less abundant in infant stools. In this study, we observed altruistic behaviour by B. bifidum when incubated in HMOs-containing faecal cultures. Four B. bifidum strains, all of which contained complete sets of HMO-degrading genes, commonly left HMOs degradants unconsumed during in vitro growth. These strains stimulated the growth of other Bifidobacterium species when added to faecal cultures supplemented with HMOs, thereby increasing the prevalence of bifidobacteria in faecal communities. Enhanced HMOs consumption by B. bifidum-supplemented cultures was also observed. We also determined the complete genome sequences of B. bifidum strains JCM7004 and TMC3115. Our results suggest B. bifidum-mediated cross-feeding of HMOs degradants within bifidobacterial communities.

DOI： 10.1038/s41598-018-32080-3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer New York LLC  

SPase is widely used in the food, cosmetics, and pharmaceutical industries. Previously, a SPase gene was cloned from Bifidobacterium longum JCM1217 and constructed into Escherichia coli BL21. In this paper, its expression conditions were optimized. The results showed that several induction factors determined the expression efficiency of SPase. The initial cell density, IPTG concentration, and induction time and temperature significantly (p OpenSPiltSPi 0.01) affected the total protein content and activity of expressed SPase. The highest expression efficiency was obtained at an initial cell density of OD600 = 0.5, with 0.05 mM IPTG, followed by shaking at 180 rpm and incubation at 30 °C for 15 h. The purified SPase had a specific activity of 122.1 U/mg, which was raised by 1.85 -fold more than that before optimization, and its recovery yield was 86%. Furthermore, SPase also showed higher thermostability. The results of this study provide essential information for the industrial production of SPase.

DOI： 10.1007/s10930-017-9754-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Enzymes of the glycoside hydrolase family 42 (GH42) are widespread in bacteria of the human gut microbiome and play fundamental roles in the decomposition of both milk and plant oligosaccharides. All GH42 enzymes characterized so far have beta-galactosidase activity. Here, we report the existence of a GH42 subfamily that is exclusively specific for beta-L-arabinopyranoside and describe the first representative of this subfamily. We found that this enzyme (BlArap42B) from a probiotic Bifidobacterium species cannot hydrolyze beta-galactosides. However, BlArap42B effectively hydrolyzed paeonolide and ginsenoside Rb2, plant glycosides containing an aromatic aglycone conjugated to beta-L-arabinopyranosyl-(1,6)-beta-D-glucopyranoside. Paeonolide, a natural glycoside from the roots of the plant genus Paeonia, is not hydrolyzed by classicalGH42 beta-galactosidases. X-ray crystallography revealed a unique Trp345-X12-Trp358 sequence motif at the BlArap42B active site, as compared with a Phe-X12-His motif in classicalGH42 beta-galactosidases. This analysis also indicated that the C6 position of galactose is blocked by the aromatic side chains, hence allowing accommodation only of Arap lacking this carbon. Automated docking of paeonolide revealed that it can fit into the BlArap42B active site. The Glcp moiety of paeonolide stacks onto the aromatic ring of the Trp252 at subsite beta 1 and C4-OH is hydrogen bonded with Asp249. Moreover, the aglycone stacks against Phe421 from the neighboring monomer in the BlArap42B trimer, forming a proposed subsite beta 2. These results further support the notion that evolution of metabolic specialization can be tracked at the structural level in key enzymes facilitating degradation of specific glycans in an ecological niche.

DOI： 10.1074/jbc.M117.792598

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Breast-fed infants generally have a bifidobacteriarich microbiota with recent studies indicating that human milk oligosaccharides (HMOs) selectively promote bifidobacterial growth. Bifidobacterium bifidum possesses a glycoside hydrolase family 20 lacto-N-biosidase for liberating lacto-N-biose I from lacto-N-tetraose, an abundant HMO unique to human milk, while Bifidobacterium longum subsp. longum has a non-classified enzyme (LnbX). Here, we determined the crystal structure of the catalytic domain of LnbX and provide evidence for creation of a novel glycoside hydrolase family, GH136. The structure, in combination with inhibition and mutation studies, provides insight into the molecular mechanism and broader substrate specificity of this enzyme. Moreover, through genetic studies, we show that lnbX is indispensable for B. longum growth on lacto-N-tetraose and is a key genetic factor for persistence in the gut of breast-fed infants. Overall, this study reveals possible evolutionary routes for the emergence of symbiosis between humans and bifidobacterial species in the infant gut.

DOI： 10.1016/j.chembiol.2017.03.012

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Other Link： http://orcid.org/0000-0002-0083-1838

Language：English   Publisher：The Japanese Society of Applied Glycoscience  

<p>3-Keto-levoglucosan (3ketoLG) has been postulated to be the product of a reaction catalyzed by levoglucosan dehydrogenase (LGDH), a bacterial enzyme involved in the metabolism of levoglucosan (LG). To investigate the LG metabolic pathway catalyzed by LGDH, 3ketoLG is needed. However, 3ketoLG has not been successfully isolated from the LGDH reaction. This study investigated the ability of pyranose oxidase to convert LG into 3ketoLG by oxidizing the C3 hydroxyl group. During the oxidation of LG, 3ketoLG was spontaneously crystallized in the reaction mixture. Starting with 500 mM LG, the isolation yield of 3ketoLG was 80 %. Nuclear magnetic resonance analyses revealed that a part of 3ketoLG dimerized in aqueous solution, explaining its poor solubility. Even under normal conditions, 3ketoLG was unstable in aqueous solution, with a half-life of 16 h at pH 7.0 and 30 °C. The decomposition proceeded through β-elimination of the C–O bonds at both C1 and C5, as evidenced by decomposition products. This instability explains the difficulty in obtaining 3ketoLG via the LGDH reaction.</p>

DOI： 10.5458/jag.jag.JAG-2017_013

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE SOC APPLIED GLYCOSCIENCE  

Cellobiose phosphorylase from Cellvibrio gilvus was used to prepare 1,5-anhydro-4-O-beta-D-glucopyranosyl-D-fructose [beta Glc(1 -> 4)AF] from 1,5-anhydro-D-fructose and alpha-D-glucose 1-phosphate. beta Glc(1 -> 4)AF decomposed into D-glucose and ascopyrone T via beta-elimination. Higher pH and temperature caused faster decomposition. However, decomposition proceeded significantly even under mild conditions. For instance, the half-life of beta Glc(1 -> 4)AF was 17 h at 30 degrees C and pH 7.0. Because beta Glc(1 -> 4)AF is a mimic of cellulose, in which the C2 hydroxyl group is oxidized, such decomposition may occur in oxidized cellulose in nature. Here we propose a possible oxidizing pathway by which this occurs.

DOI： 10.5458/jag.jag.JAG-2017_010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Galacto-N-biose (GNB: Gal beta 1-3GalNAc) is an O-glycan disaccharide core moiety that is a core component of mucin in the gastrointestinal tract; however, the physiological properties of GNB are not well understood. Glutamate excitotoxicity causes neuronal death in acute neurological disorders including stroke, trauma, and neurodegenerative disease. Therefore the discovery of drugs to treat glutamate excitotoxicity is an important goal. Here, we report that GNB is neuroprotective against glutamate-induced excitotoxicity. We treated 14-15 days in vitro cultured rat cortical neurons with 0.1-1000 nM GNB together with 30 mu m glutamate for various durations. Short-term (3 h) GNB treatments showed a modest neuroprotective effect against glutamate neurotoxicity, however, long-term (24 h) GNB treatment conferred significant neuroprotective effects, as shown by both MTT and immunocytochemical assays. Prolonged GNB treatment did not alter glutamate-induced calcium influx, but did induce antioxidant-related gene expression. Furthermore, GNB treatment did not induce cell death or alter synaptic connections. These data suggest that GNB is a potential candidate drug that protects against glutamate excitotoxicity without affecting cell viability and synaptic connections.

DOI： 10.1016/j.ejphar.2016.10.010

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Other Link： http://orcid.org/0000-0002-0083-1838

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS INC  

Fuc alpha 1-2 Gal linkages, or H-antigens, constitute histo-blood group antigens and are involved in various physiological processes. In addition, recent studies have shown that the H-antigen-containing glycans play an important role, not only in establishing harmonious relationship between gut microbes and the host, but also in preventing gut dysbiosis-related diseases. Therefore, development of an efficient method for introducing Fuc residue at Gal residue at the nonreducing end of glycans via alpha-(1 -&gt; 2) linkage is desired for research as well as medicinal purposes. In this study, we succeeded in derivatizing inverting 1,2-alpha-L-fucosidase (AfcA) into a highly efficient 1,2-alpha-L-fucosynthase. The synthase specifically synthesized H type 1-, type 2-, type 3- and type 4-chaincontaining oligosaccharides with yields of 57-75% based on acceptor depletion. The synthase was also able to specifically introduce Fuc residues into Lewis a/x antigens to produce Lewis b/y antigens, with yields of 43% and 62%, respectively. In addition, the enzyme efficiently introduced Hantigens into sugar chains of porcine gastric mucins, as revealed by lectin blotting and mass spectroscopy analysis of the sugars. Detailed acceptor specificity analysis using various monosaccharides and oligosaccharides unraveled unique substrate recognition feature of this synthase at the subsite (+1), which can be explained by our previous X-ray crystallographic study of AfcA. These results show that the synthase developed in this study could serve as an alternative to other H-antigen synthesis methods involving alpha-1,2-fucosyltransferases and retaining alpha-fucosidase.

DOI： 10.1093/glycob/cww085

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Other Link： http://orcid.org/0000-0002-0083-1838

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

We created a glycosynthase from a GH19 chitinase from rye seeds (RSC-c), that has a long-extended binding cleft consisting of eight subsites; -4, -3, -2, -1, + 1, + 2, + 3 and + 4. When wild-type RSC-c was incubated with alpha-(G1cNAc)(3)-F [alpha-(GleNAc)(3) fluoride], (G1cNAc)(3) and hydrogen fluoride were produced through the Hehre resynthesis-hydrolysis mechanism. Glu89, which acts as a catalytic base, and Ser120, which fixes a nucleophilic water molecule, were mutated to produce two single mutants, E89G and S120A, and a double mutant, E89G/S120A. E89G only produced a small amount of (G1cNAc)(7) from alpha-(GlcNAc)(3)-F in the presence of (G1cNAc)(4). S120A, with the highest F- -releasing activity, produced a larger amount of (G1cNAc)(7), a fraction of which was decomposed by its own residual hydrolytic activity. However, the double mutant E89G/S120A, of which the hydrolytic activity was completely abolished while its F--releasing activity was only moderately affected, produced the largest amount of (G1eNAc)(7) from alpha-(GleNAc)(3)-F and (G1cNAc)(4) without decomposition. We concluded that E89G/S120A was an efficient glycosynthase, that enabled the addition of a three-sugar unit.

DOI： 10.1093/jb/mvw014

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Language：English   Publishing type：Part of collection (book)   Publisher：Pan Stanford Publishing Pte. Ltd.  

DOI： 10.4032/9789814669795

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

In Ruminococcus albus, 4-Omicron-beta-D-mannosyl-D-glucose phosphorylase (RaMP1) and beta-(1,4)-mannooligosaccharide phosphorylase (RaMP2) belong to two subfamilies of glycoside hydrolase family 130. The two enzymes phosphorolyze beta-mannosidic linkages at the nonreducing ends of their substrates, and have substantially diverse substrate specificity. The differences in their mechanism of substrate binding have not yet been fully clarified. In the present study, we report the crystal structures of RaMP1 with/without 4-Omicron-beta-D-mannosyl-D-glucose and RaMP2 with/without beta-(1 -&gt; 4)-mannobiose. The structures of the two enzymes differ at the +1 subsite of the substrate-binding pocket. Three loops are proposed to determine the different substrate specificities. One of these loops is contributed from the adjacent molecule of the oligomer structure. In RaMP1, His245 of loop 3 forms a hydrogen-bond network with the substrate through a water molecule, and is indispensible for substrate binding.

DOI： 10.1002/1873-3468.12105

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Despite the presence of beta-1,2-glucan in nature, few beta-1,2-glucan degrading enzymes have been reported to date. Recently, the Lin1839 protein from Listeria innocua was identified as a 1,2-beta-oligoglucan phosphorylase. Since the adjacent lin1840 gene in the gene cluster encodes a putative glycoside hydrolase family 3 beta-glucosidase, we hypothesized that Lin1840 is also involved in beta-1,2-glucan dissimilation. Here we report the functional and structural analysis of Lin1840. A recombinant Lin1840 protein (Lin1840r) showed the highest hydrolytic activity toward sophorose (Glc-beta-1,2-Glc) among beta-1,2-glucooligosaccharides, suggesting that Lin1840 is a beta-glucosidase involved in sophorose degradation. The enzyme also rapidly hydrolyzed laminaribiose (beta-1,3), but not cellobiose (beta-1,4) or gentiobiose (beta-1,6) among beta-linked gluco-disaccharides. We determined the crystal structures of Lin1840r in complexes with sophorose and laminaribiose as productive binding forms. In these structures, Arg572 forms many hydrogen bonds with sophorose and laminaribiose at subsite + 1, which seems to be a key factor for substrate selectivity. The opposite side of subsite + 1 from Arg572 is connected to a large empty space appearing to be subsite + 2 for the binding of sophorotriose (Glc-beta-1,2-Glc-beta-1,2-Glc) in spite of the higher Km value for sophorotriose than that for sophorose. The conformations of sophorose and laminaribiose are almost the same on the Arg572 side but differ on the subsite + 2 side that provides no interaction with a substrate. Therefore, Lin1840r is unable to distinguish between sophorose and laminaribiose as substrates. These results provide the first mechanistic insights into beta-1,2-glucooligosaccharide recognition by beta-glucosidase.

DOI： 10.1371/journal.pone.0148870

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PORTLAND PRESS LTD  

Exo-beta-D-glucosaminidase (EC 3.2.1.165) from Photobacterium profundum (PpGlcNase) is an inverting GH (glycoside hydrolase) belonging to family 9. We have determined the three-dimensional structure of PpGlcNase to describe the first structure-function relationship of an exo-type GH9 glycosidase. PpGlcNase has a narrow and straight active-site pocket, in contrast with the long glycan-binding cleft of a GH9 endoglucanase. This is because PpGlcNase has a long loop, which blocks the position corresponding to subsites -4 to -2 of the endoglucanase. The pocket shape of PpGlcNase explains its substrate preference for a beta 1,4-linkage at the non-reducing terminus. Asp(139), Asp(143) and Glu(555) in the active site were located near the beta-O1 hydroxy group of GlcN (D-glucosamine), with Asp(139) and Asp(143) holding a nucleophilic water molecule for hydrolysis. The D139A, D143A and E555A mutants significantly decreased hydrolytic activity, indicating their essential role. Of these mutants, D139A exclusively exhibited glycosynthase activity using alpha-GlcN-F (alpha-D-glucosaminyl fluoride) and GlcN as substrates, to produce (GlcN)(2). Using saturation mutagenesis at Asp(139), we obtained D139E as the best glycosynthase. Compared with the wild-type, the hydrolytic activity of D139E was significantly suppressed (&lt;0.1%), and the F--release activity also decreased (&lt;3%). Therefore the glycosynthase activity of D139E was lower than that of glycosynthases created previously from other inverting GHs. Mutation at the nucleophilic water holder is a general strategy for creating an effective glycosynthase from inverting GHs. However, for GH9, where two acidic residues seem to share the catalytic base role, mutation of Asp(139) might inevitably reduce F--release activity.

DOI： 10.1042/BJ20150966

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Glycoside hydrolase family 130 consists of phosphorylases and hydrolases for beta-mannosides. Here, we characterized beta-1,2-mannobiose phosphorylase from Listeria innocua (Lin0857) and determined its crystal structures complexed with beta-1,2-linked mannooligosaccharides. beta-1,2-Mannotriose was bound in a U-shape, interacting with a phosphate analog at both ends. Lin0857 has a unique dimer structure connected by a loop, and a significant open-close loop displacement was observed for substrate entry. A long loop, which is exclusively present in Lin0857, covers the active site to limit the pocket size. A structural basis for substrate recognition and phosphorolysis was provided. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2015.11.034

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The glycoside hydrolase family (GH) 130 is composed of inverting phosphorylases that catalyze reversible phosphorolysis of beta-D-mannosides. Here we report a glycoside hydrolase as a new member of GH130. Dfer_3176 from Dyadobacter fermentans showed no synthetic activity using alpha-D-mannose 1-phosphate but it released alpha-D-mannose from beta-1,2-mannooligosaccharides with an inversion of the anomeric configuration, indicating that Dfer_3176 is a beta-1,2-mannosidase. Mutational analysis indicated that two glutamic acid residues are critical for the hydrolysis of beta-1,2-mannotriose. The two residues are not conserved among GH130 phosphorylases and are predicted to assist the nucleophilic attack of a water molecule in the hydrolysis of the beta-D-mannosidic bond. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2015.10.008

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Other Link： http://orcid.org/0000-0002-0083-1838

Language：English   Publisher：SPRINGER  

Phosphorylases are useful catalysts for the practical preparation of various sugars. The number of known specificities was 13 in 2002 and is now 30. The drastic increase in available genome sequences has facilitated the discovery of novel activities. Most of these novel phosphorylase activities have been identified through the investigations of glycoside hydrolase families containing known phosphorylases. Here, the diversity of phosphorylases in each family is described in detail.

DOI： 10.1007/s00253-015-6927-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The microbial oxidative cellulose degradation system is attracting significant research attention after the recent discovery of lytic polysaccharide mono-oxygenases. A primary product of the oxidative and hydrolytic cellulose degradation system is cellobionic acid (CbA), the aldonic acid form of cellobiose. We previously demonstrated that the intracellular enzyme belonging to glycoside hydrolase family 94 from cellulolytic fungus and bacterium is cellobionic acid phosphorylase (CBAP), which catalyzes reversible phosphorolysis of CbA into glucose 1-phosphate and gluconic acid (GlcA). In this report, we describe the biochemical characterization and the three-dimensional structure of CBAP from the marine cellulolytic bacterium Saccharophagus degradans. Structures of ligand-free and complex forms with CbA, GlcA, and a synthetic disaccharide product from glucuronic acid were determined at resolutions of up to 1.6 angstrom. The active site is located near the dimer interface. At subsite + 1, the carboxylate group of GlcA and CbA is recognized by Arg-609 and Lys-613. Additionally, one residue from the neighboring protomer (Gln-190) is involved in the carboxylate recognition of GlcA. A mutational analysis indicated that these residues are critical for the binding and catalysis of the aldonic and uronic acid acceptors GlcA and glucuronic acid. Structural and sequence comparisons with other glycoside hydrolase family 94 phosphorylases revealed that CBAPs have a unique subsite + 1 with a distinct amino acid residue conservation pattern at this site. This study provides molecular insight into the energetically efficient metabolic pathway of oxidized sugars that links the oxidative cellulolytic pathway to the glycolytic and pentose phosphate pathways in cellulolytic microbes.

DOI： 10.1074/jbc.M115.664664

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

The aerobic soil bacterium Cellvibrio vulgaris has a beta-mannan-degradation gene cluster, including unkA, epiA, man5A, and aga27A. Among these genes, epiA has been assigned to encode an epimerase for converting d-mannose to d-glucose, even though the amino acid sequence of EpiA is similar to that of cellobiose 2-epimerases (CEs). UnkA, whose function currently remains unknown, shows a high sequence identity to 4-O-beta-d-mannosyl-d-glucose phosphorylase. In this study, we have investigated CE activity of EpiA and the general characteristics of UnkA using recombinant proteins from Escherichia coli. Recombinant EpiA catalyzed the epimerization of the 2-OH group of sugar residue at the reducing end of cellobiose, lactose, and beta-(1 -&gt; 4)-mannobiose in a similar manner to other CEs. Furthermore, the reaction efficiency of EpiA for beta-(1 -&gt; 4)-mannobiose was 5.5x10(4)-fold higher than it was for d-mannose. Recombinant UnkA phosphorolyzed beta-d-mannosyl-(1 -&gt; 4)-d-glucose and specifically utilized d-glucose as an acceptor in the reverse reaction, which indicated that UnkA is a typical 4-O-beta-d-mannosyl-d-glucose phosphorylase.

DOI： 10.1080/09168451.2015.1012146

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We describe the novel substrate specificities of two independently evolved lacto-N-biosidases (LnbX and LnbB) towards the sugar chains of globo-and ganglio-series glycosphingolipids. LnbX, a non-classified member of the glycoside hydrolase family, isolated from Bifidobacterium longum subsp. longum, was shown to liberate galacto-N-biose (GNB: Gal beta 1-3GalNAc) and 2'-fucosyl GNB (a type-4 trisaccharide) from Gb5 pentasaccharide and globo H hexasaccharide, respectively. LnbB, a member of the glycoside hydrolase family 20 isolated from Bifidobacterium bifidum, was shown to release GNB from Gb5 and GA1 oligosaccharides. This is the first report describing enzymatic release of beta-linked GNB from natural substrates. These unique activities may play a role in modulating the microbial composition in the gut ecosystem, and may serve as new tools for elucidating the functions of sugar chains of glycosphingolipids. (C) 2015 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.carres.2015.03.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Japanese Society of Applied Glycoscience  

1,2-β-Glucan was produced enzymatically from 1.0 M sucrose and 0.5 M glucose by the combination of sucrose phosphorylase and 1,2-β-oligoglucan phosphorylase in the presence of 100 mM inorganic phosphate. Accumulation of 1,2-β-glucan in 2 L of the reaction mixture reached over 800 mM (glucose equivalent). Sucrose, glucose and fructose were removed after the reaction by yeast treatment. 1,2-β-Glucan was precipitated with ethanol to obtain 167 g of 1,2-β-glucan from 1 L of the reaction mixture.

DOI： 10.5458/jag.jag.JAG-2014_011

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Other Link： https://jlc.jst.go.jp/DN/JLC/20010850716?from=CiNii

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Infant gut-associated bifidobacteria possess a metabolic pathway to utilize lacto-N-biose (Gal-beta 1,3-G1cNAc) and galacto-N-biose (Gal-beta 1,3-GalNAc) from human milk and glycoconjugates specifically. In this pathway, N-acetylhexosamine 1-kinase (NahK) catalyzes the phosphorylation of GlcNAc or GalNAc at the anomeric Cl position with ATP. Crystal structures of NahK have only been determined in the closed state. In this study, we determined open state structures of NahK in three different forms (apo, ADP complex, and ATP complex). A comparison of the open and closed state structures revealed an induced fit structural change defined by two rigid domains. ATP binds to the small N-terminal domain, and binding of the N-acetylhexosamine substrate to the large C-terminal domain induces a closing conformational change with a rotation angle of 16 degrees. In the nucleotide binding site, two magnesium ions bridging the alpha-gamma and beta-gamma phosphates were identified. A mutational analysis indicated that a residue coordinating both of the two magnesium ions (Asp228) is essential for catalysis. The involvement of two magnesium ions in the catalytic machinery is structurally similar to the catalytic structures of protein kinases and aminoglycoside phosphotransferases, but distinct from the structures of other anomeric kinases or sugar 6-kinases. These findings help to elucidate the possible evolutionary adaptation of substrate specificities and induced fit mechanism. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbapap.2015.01.011

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Three sugar 1-phosphates that are donor substrates for phosphorylases were produced at the gram scale from phosphoenolpyruvic acid and the corresponding sugars by the combined action of pyruvate kinase and the corresponding anomeric kinases in good yields. These sugar 1-phosphates were purified through two electrodialysis steps. alpha-D-Galactose 1-phosphate was finally isolated as crystals of dipotassium salts. alpha-D-Mannose 1-phosphate and 2-acetamido-2-deoxy-alpha-D-glucose 1-phosphate were isolated as crystals of bis(cyclohexylammonium) salts. (C) 2014 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.carres.2014.10.014

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

The Bifidobacterium genus harbours several health promoting members of the gut microbiota. Bifidobacteria display metabolic specialization by preferentially utilizing dietary or host-derived -galactosides. This study investigates the biochemistry and structure of a glycoside hydrolase family 42 (GH42) -galactosidase from the probiotic Bifidobacterium animalis subsp. lactisBl-04 (BlGal42A). BlGal42A displays a preference for undecorated 1-6 and 1-3 linked galactosides and populates a phylogenetic cluster with close bifidobacterial homologues implicated in the utilization of N-acetyl substituted 1-3 galactosides from human milk and mucin. A long loop containing an invariant tryptophan in GH42, proposed to bind substrate at subsite +1, is identified here as specificity signature within this clade of bifidobacterial enzymes. Galactose binding at the subsite -1 of the active site induced conformational changes resulting in an extra polar interaction and the ordering of a flexible loop that narrows the active site. The amino acid sequence of this loop provides an additional specificity signature within this GH42 clade. The phylogenetic relatedness of enzymes targeting 1-6 and 1-3 galactosides likely reflects structural differences between these substrates and 1-4 galactosides, containing an axial galactosidic bond. These data advance our molecular understanding of the evolution of sub-specificities that support metabolic specialization in the gut niche.

DOI： 10.1111/mmi.12815

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We characterized Teth514_1788 and Teth514_1789, belonging to glycoside hydrolase family 130, from Thermoanaerobacter sp. X-514. These two enzymes catalyzed the synthesis of 1,2-beta-oligomannan using beta-1,2-mannobiose and Dmannose as the optimal acceptors, respectively, in the presence of the donor alpha-Dmannose 1-phosphate. Kinetic analysis of the phosphorolytic reaction toward 1,2-beta-oligomannan revealed that these enzymes followed a typical sequential Bi Bi mechanism. The kinetic parameters of the phosphorolysis of 1,2-beta-oligomannan indicate that Teth514_1788 and Teth514_1789 prefer 1,2-beta-oligomannans containing a DP &gt;= 3 and beta-1,2-Man(2), respectively. These results indicate that the two enzymes are novel inverting phosphorylases that exhibit distinct chain-length specificities toward 1,2-beta-oligomannan. Here, we propose 1,2-beta-oligomannan: phosphate alpha-D-mannosyltransferase as the systematic name and 1,2 beta- oligomannan phosphorylase as the short name for Teth514_1788 and beta-1,2-mannobiose: phosphate alpha-D-mannosyltransferase as the systematic name and beta-1-,2-mannobiosephosphorylase as the short name for Teth514_1789.

DOI： 10.1371/journal.pone.0114882

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DOI： 10.5458/jag.jag.JAG-2013_012

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

2-O-alpha-Glucosylglycerol phosphorylase (GGP) from Bacillus selenitireducens catalyzes both the reversible phosphorolysis of 2-O-alpha-glucosylglycerol (GG) and the hydrolysis of beta-D-glucose 1-phosphate (beta Glc1P). GGP belongs to the glycoside hydrolase (GH) family 65 and can efficiently and specifically produce GG. However, its structural basis has remained unclear. In this study, the crystal structures of GGP complexed with glucose and the glucose analog isofagomine and glycerol were determined. Subsite -1 of GGP is similar to those of other GH65 enzymes, maltose phosphorylase and kojibiose phosphorylase, whereas subsite +1 is largely different and is well designed for GG recognition. An automated docking analysis was performed to complement these crystal structures, beta Glc1P being docked at an appropriate position. To investigate the importance of residues at subsite +1 in the bifunctionality of GGP, we constructed mutants at these residues. Y327F and K587A did not show detectable activities for either reverse phosphorolysis or beta Glc1P hydrolysis. Y572F also showed significantly reduced activities for both of these reactions. In contrast, W381F showed significantly reduced reverse phosphorolytic activity but retained beta Glc1P hydrolysis. The mode of substrate recognition and the reaction mechanisms of GGP were proposed based on these analyses. Specifically, an extensive hydrogen bond network formed by Tyr-327, Tyr-572, Lys-587, and water molecules contributes to fixing the acceptor molecule in both reverse phosphorolysis( glycerol) and beta Glc1P hydrolysis(water) for a glycosyl transfer reaction. This study will contribute to the development of a large scale production system of GG by facilitating the rational engineering of GGP.

DOI： 10.1074/jbc.M114.573212

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Applied Glycoscience  

DOI： 10.5458/jag.jag.JAG-2013_013

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

In vitro synthesis of (1 -&gt; 3)-beta-D-glucan was performed using laminaribiose phosphorylase obtained by an extraction of Euglena gracilis with sucrose phosphorylase. The synthetic product was a linear (1 -&gt; 3)-beta-D-glucan with a narrow distribution of degree of polymerization (DP) centered on DP =30. X-ray diffraction and electron microscopy revealed that the glucan molecules obtained were self-organized as highly crystalline hexagonal lamellae. This synthetic product has quite high structural homogeneity at every level from primary to higher-order structure, which is a great advantage for the detailed analyses of physiological functions of (1 -&gt; 3)-(beta-D-glucan. (C) 2013 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.ijbiomac.2013.12.027

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We characterized recombinant Lin1839 protein (Lin1839r) belonging to glycoside hydrolase family 94 from Listeria innocua. Lin1839r catalyzed the synthesis of a series of 1,2-beta-oligoglucans (Sop(n): n denotes degree of polymerization) using sophorose (Sop(2)) as the acceptor and alpha-D-glucose 1-phosphate (Glc1P) as the donor. Lin1839r recognized glucose as a very weak acceptor substrate to form polymeric 1,2-beta-glucan. The degree of polymerization of the 1,2-beta-glucan gradually decreased with long-term incubation to generate a series of Sop(n)s. Kinetic analysis of the phosphorolytic reaction towards sophorotriose revealed that Lin1839r followed a sequential Bi Bi mechanism. The kinetic parameters of the phosphorolysis of sophorotetraose and sophoropentaose were similar to those of sophorotriose, although the enzyme did not exhibit significant phosphorolytic activity on Sop(2). These results indicate that the Lin1839 protein is a novel inverting phosphorylase that catalyzes reversible phosphorolysis of 1,2-beta-glucan with a degree of polymerization of &gt;= 3. We propose 1,2-beta-oligoglucan: phosphate a-glucosyltransferase as the systematic name and 1,2-beta-oligoglucan phosphorylase as the short name for this Lin1839 protein.

DOI： 10.1371/journal.pone.0092353

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS INC  

Glycoside hydrolase family 42 (GH42) includes beta-galactosidases catalyzing the release of galactose (Gal) from the non-reducing end of different beta-d-galactosides. Health-promoting probiotic bifidobacteria, which are important members of the human gastrointestinal tract microbiota, produce GH42 enzymes enabling utilization of beta-galactosides exerting prebiotic effects. However, insight into the specificity of individual GH42 enzymes with respect to substrate monosaccharide composition, glycosidic linkage and degree of polymerization is lagging. Kinetic analysis of natural and synthetic substrates resembling various milk and plant galactooligosaccharides distinguishes the three GH42 members, Bga42A, Bga42B and Bga42C, encoded by the probiotic B. longum subsp. infantis ATCC 15697 and revealed the glycosyl residue at subsite +1 and its linkage to the terminal Gal at subsite -1 to be key specificity determinants. Bga42A thus prefers the beta 1-3-galactosidic linkage from human milk and other beta 1-3- and beta 1-6-galactosides with glucose or Gal situated at subsite +1. In contrast, Bga42B very efficiently hydrolyses 4-galactosyllactose (Gal beta 1-4Gal beta 1-4Glc) as well as 4-galactobiose (Gal beta 1-4Gal) and 4-galactotriose (Gal beta 1-4Gal beta 1-4Gal). The specificity of Bga42C resembles that of Bga42B, but the activity was one order of magnitude lower. Based on enzyme kinetics, gene organization and phylogenetic analyses, Bga42C is proposed to act in the metabolism of arabinogalactan-derived oligosaccharides. The distinct kinetic signatures of the three GH42 enzymes correlate to unique sequence motifs denoting specific clades in a GH42 phylogenetic tree providing novel insight into GH42 subspecificities. Overall, the data illustrate the metabolic adaptation of bifidobacteria to the beta-galactoside-rich gut niche and emphasize the importance and diversity of beta-galactoside metabolism in probiotic bifidobacteria.

DOI： 10.1093/glycob/cwt104

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

4-O-beta-D-Mannosyl-D-glucose phosphorylase (MGP), found in anaerobes, converts 4-O-beta-D-mannosylD- glucose (Man-Glc) to alpha-D-mannosyl phosphate and D-glucose. It participates in mannan metabolism with cellobiose 2-epimerase (CE), which converts beta-1,4-mannobiose to Man-Glc. A putative MGP gene is present in the genome of the thermophilic aerobe Rhodothermus marinus (Rm) upstream of the gene encoding CE. Konjac glucomannan enhanced production by R. marinus of MGP, CE, and extracellular mannan endo-1,4-beta-mannosidase. Recombinant RmMGP catalyzed the phosphorolysis of Man-Glc through a sequential bi-bi mechanism involving ternary complex formation. Its molecular masses were 45 and 222 kDa under denaturing and nondenaturing conditions, respectively. Its pH and temperature optima were 6.5 and 75 degrees C, and it was stable between pH 5.5-8.3 and below 80 degrees C. In the reverse reaction, RmMGP had higher acceptor preferences for 6-deoxy-D-glucose and D-xylose than R. albus NE1 MGP. In contrast to R. albus NE1 MGP, RmMGP utilized methyl beta-D-glucoside and 1,5-anhydro- D-glucitol as acceptor substrates.

DOI： 10.1080/09168451.2014.882760

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The glycoside hydrolase family (GH) 65 is a family of inverting phosphorylases that act on alpha-glucosides. A GH65 protein (Bsel_2816) from Bacillus selenitireducens MLS10 exhibited inorganic phosphate (Pi)-dependent hydrolysis of kojibiose at the rate of 0.43 s(-1). No carbohydrate acted as acceptor for the reverse phosphorolysis using beta-D-glucose 1-phosphate (beta Glc1P) as donor. During the search for a suitable acceptor, we found that Bsel_2816 possessed hydrolytic activity on bGlc1P with a k(cat) of 2.8 s(-1); moreover, such significant hydrolytic activity on sugar 1-phosphate had not been reported for any inverting phosphorylase. The (H2O)-O-18 incorporation experiment and the anomeric analysis during the hydrolysis of bGlc1P revealed that the hydrolysis was due to the glucosyl-transferring reaction to a water molecule and not a phosphatase-type reaction. Glycerol was found to be the best acceptor to generate 2-O-alpha-D-glucosylglycerol (GG) at the rate of 180 s(-1). Bsel_2816 phosphorolyzed GG through sequential Bi-Bi mechanism with a kcat of 95 s(-1). We propose 2-O-alpha-D-glucopyranosylglycerol: phosphate beta-D-glucosyltransferase as the systematic name and 2-O-alpha-D-glucosylglycerol phosphorylase as the short name for Bsel_2816. This is the first report describing a phosphorylase that utilizes polyols, and not carbohydrates, as suitable acceptor substrates.

DOI： 10.1371/journal.pone.0086548

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Humana Press Inc.  

We describe a simple and easy protocol to introduce random mutations into plasmid DNA: error-prone rolling circle amplification. A template plasmid is amplified via rolling circle amplification with decreased fidelity in the presence of MnCl&lt
inf&gt
2&lt
/inf&gt
and is used to transform a host strain resulting in a mutant library with several random point mutations per kilobase through the entire plasmid. The primary advantage of this method is its simplicity. This protocol does not require the design of specific primers or thermal cycling. The reaction mixture can be used for direct transformation of a host strain. This method allows rapid preparation of randomly mutated plasmid libraries, enabling wider application of random mutagenesis. © 2014 Springer Science+Business Media New York.

DOI： 10.1007/978-1-4939-1053-3_2

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Although proteins can be artificially improved by random insertion and deletion mutagenesis methods, these procedures are technically difficult. Here we describe a simple method called random insertional-deletional strand exchange mutagenesis (RAISE). This method is based on gene shuffling and can be used to introduce a wide variety of insertions, deletions, and substitutions. RAISE involves three steps: DNA fragmentation, attachment of a random short sequence, and reconstruction. This yields unique mutants and can be a powerful technique for protein engineering. © 2014 Springer Science+Business Media New York.

DOI： 10.1007/978-1-4939-1053-3_10

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A novel phosphorylase was characterized as new member of glycoside hydrolase family 94 from the cellulolytic bacterium Xanthomonas campestris and the fungus Neurospora crassa. The enzyme catalyzed reversible phosphorolysis of cellobionic acid. We propose 4-O-β-d-glucopyranosyl-d-gluconic acid: phosphate α-d-glucosyltransferase as the systematic name and cellobionic acid phosphorylase as the short names for the novel enzyme. Several cellulolytic fungi of the phylum Ascomycota also possess homologous proteins. We, therefore, suggest that the enzyme plays a crucial role in cellulose degradation where cellobionic acid as oxidized cellulolytic product is converted into α-d-glucose 1-phosphate and d-gluconic acid to enter glycolysis and the pentose phosphate pathway, respectively. © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2013.09.014

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

A novel phosphorylase was characterized as new member of glycoside hydrolase family 94 from the cellulolytic bacterium Xanthomonas campestris and the fungus Neurospora crassa. The enzyme catalyzed reversible phosphorolysis of cellobionic acid. We propose 4-O-beta-D-glucopyranosyl-D-gluconic acid: phosphate alpha-D-glucosyltransferase as the systematic name and cellobionic acid phosphorylase as the short names for the novel enzyme. Several cellulolytic fungi of the phylum Ascomycota also possess homologous proteins. We, therefore, suggest that the enzyme plays a crucial role in cellulose degradation where cellobionic acid as oxidized cellulolytic product is converted into alpha-D-glucose 1-phosphate and D-gluconic acid to enter glycolysis and the pentose phosphate pathway, respectively. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2013.09.014

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We discovered a potassium ion-dependent trehalose phosphorylase (Bsel_1207) belonging to glycoside hydrolase family 65 from halophilic Bacillus selenitireducens MLS10. Under high potassium ion concentrations, the recombinant Bsel_1207 produced in Escherichia coli existed as an active dimeric form that catalyzed the reversible phosphorolysis of trehalose in a typical sequential bi bi mechanism releasing beta-D-glucose 1-phosphate and D-glucose. Decreasing potassium ion concentrations significantly reduced thermal and pH stabilities, leading to formation of inactive monomeric Bsel_1207.
Structured summary of protein interactions:
Bsel_1207 and Bsel_1207 bind by molecular sieving (View interaction)
(C) 2013 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

DOI： 10.1016/j.febslet.2013.08.038

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) is the key enzyme in the enzymatic production of lacto-N-biose I. For the purpose of industrial use, we improved the thermostability of GLNBP by evolutionary engineering in which five substitutions in the amino acid sequence were selected from a random mutagenesis GLNBP library constructed using error-prone polymerase chain reaction. Among them, C236Y and D576V mutants showed considerably improved thermostability. Structural analysis of C236Y revealed that the hydroxyl group of Tyr236 forms a hydrogen bond with the carboxyl group of E319. The C236Y and D576V mutations together contributed to the thermostability. The C236Y/D576V mutant exhibited 20C higher thermostability than the wild type.

DOI： 10.1093/protein/gzt049

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A gene cluster involved in N-glycan metabolism was identified in the genome of Bacteroides thetaiotaomicron VPI-5482. This gene cluster encodes a major facilitator superfamily transporter, a starch utilization system-like transporter consisting of a TonB-dependent oligosaccharide transporter and an outer membrane lipoprotein, four glycoside hydrolases (α-mannosidase, β-N-acetylhexosaminidase, exo-α-sialidase, and endo-β-N-acetylglucosaminidase), and a phosphorylase (BT1033) with unknown function. It was demonstrated that BT1033 catalyzed the reversible phosphorolysis of β-1,4-D-mannosyl-N-acetyl-Dglucosamine in a typical sequential Bi Bi mechanism. These results indicate that BT1033 plays a crucial role as a key enzyme in the N-glycan catabolism where β-1,4-D-mannosyl-N-acetyl-D-glucosamine is liberated from N-glycans by sequential glycoside hydrolase-catalyzed reactions, transported into the cell, and intracellularly converted into α-D-mannose 1-phosphate and N-acetyl-D-glucosamine. In addition, intestinal anaerobic bacteria such as Bacteroides fragilis, Bacteroides helcogenes, Bacteroides salanitronis, Bacteroides vulgatus, Prevotella denticola, Prevotella dentalis, Prevotella melaninogenica, Parabacteroides distasonis, and Alistipes finegoldii were also suggested to possess the similar metabolic pathway for N-glycans. A notable feature of the new metabolic pathway for N-glycans is the more efficient use of ATP-stored energy, in comparison with the conventional pathway where β-mannosidase and ATP-dependent hexokinase participate, because it is possible to directly phosphorylate the D-mannose residue of β-1,4-D-mannosyl-Nacetyl-D-glucosamine to enter glycolysis. This is the first report of a metabolic pathway for N-glycans that includes a phosphorylase. We propose 4-O-β-D-mannopyranosyl-N-acetyl-Dglucosamine: phosphate α-D-mannosyltransferase as the systematic name and β-1,4-D-mannosyl-N-acetyl-D-glucosamine phosphorylase as the short name for BT1033. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1074/jbc.M113.469080

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Proteins belonging to glycoside hydrolase family63 (GH63) are found in bacteria, archaea and eukaryotes. Although the eukaryotic GH63 proteins have been identified as processing -glucosidaseI, the substrate specificities of the bacterial and archaeal GH63 proteins are not clear. Here, we converted a bacterial GH63 enzyme, Escherichiacoli YgjK, to a glycosynthase to probe its substrate specificity. Two mutants of YgjK (E727A and D324N) were constructed, and both mutants showed glycosynthase activity. The reactions of E727A with -d-glucosyl fluoride and monosaccharides showed that the largest amount of glycosynthase product accumulated when galactose was employed as an acceptor molecule. The crystal structure of E727A complexed with the reaction product indicated that the disaccharide bound at the active site was 2-O--d-glucopyranosyl--d-galactopyranose (Glc12Gal). A comparison of the structures of E727A-Glc12Gal and D324N-melibiose showed that there were two main types of conformation: the open and closed forms. The structure of YgjK adopted the closed form when subsite -1 was occupied by glucose. These results suggest that sugars containing the Glc12Gal structure are the most likely candidates for natural substrates of YgjK.
DatabaseThe coordinates and structure factors for E727A-Glc12Gal and D324N-melibiose have been deposited in the Protein Data Bank under accession numbers 3W7W and 3W7X, respectively

DOI： 10.1111/febs.12424

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Infant gut-associated bifidobacteria possess species-specific enzymatic sets to assimilate human milk oligosaccharides, and lacto-N-biosidase (LNBase) is a key enzyme that degrades lacto-N- tetraose (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc), the main component of human milk oligosaccharides, to lacto-N-biose I (Gal beta 1-3GlcNAc) and lactose. We have previously identified LNBase activity in Bifidobacterium bifidum and some strains of Bifidobacterium longum subsp. longum (B. longum). Subsequently, we isolated a glycoside hydrolase family 20 (GH20) LNBase from B. bifidum; however, the genome of the LNBase(+) strain of B. longum contains no GH20 LNBase homolog. Here, we reveal that locus tags BLLJ_1505 and BLLJ_1506 constitute LNBase from B. longum JCM1217. The gene products, designated LnbX and LnbY, respectively, showed no sequence similarity to previously characterized proteins. The purified enzyme, which consisted of LnbX only, hydrolyzed via a retaining mechanism the GlcNAc beta 1-3Gal linkage in lacto-N-tetraose, lacto-N-fucopentaose I (Fuc alpha 1-2Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc), and sialyllacto-N-tetraose a (Neu5Ac alpha 2-3Gal beta 1-3GlcNAc beta 1-3-Gal beta 1-4Gal); the latter two are not hydrolyzed by GH20 LNBase. Among the chromogenic substrates examined, the enzyme acted on p-nitrophenyl (pNP)-beta-lacto-N-bioside I (Gal beta 1-3-GlcNAc beta-pNP) and GalNAc beta 1-3GlcNAc beta-pNP.GalNAc beta 1-3-GlcNAc beta linkage has been found in O-mannosyl glycans of alpha-dystroglycan. Therefore, the enzyme may serve as a new tool for examining glycan structures. In vitro refolding experiments revealed that LnbY and metal ions (Ca2+ and Mg2+) are required for proper folding of LnbX. The LnbX and LnbY homologs have been found only in B. bifidum, B. longum, and a few gut microbes, suggesting that the proteins have evolved in specialized niches.

DOI： 10.1074/jbc.M113.484733

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Japanese Society of Applied Glycoscience  

The BLLJ_1391 protein from <i>Bifidobacterium longum</i> subsp. <i>longum</i> JCM1217, a cytosolic β-<i>N</i>-acetylglucosaminidase belonging to glycoside hydrolase family (GH) 20, hydrolyzed lacto-<i>N</i>-triose II (LNTri) as well as chitin oligosaccharides. Its reaction was found to follow a substrate-assisted mechanism with anomeric retention, which is common for GH 20 enzymes. Homologous enzymes are found in genomic sequences of <i>B. longum</i> subsp. <i>infantis</i>, <i>Bifidobacterium bifidum</i>, and <i>Bifidobacteium breve</i>, all of which are infant gut-associated species of <i>Bifidobacterium</i>. The distribution resembles that of 1,3-β-galactosyl-<i>N</i>-acetylhexosamine phosphorylase, suggesting that the enzyme plays a role in metabolism of human milk oligosaccharides by hydrolyzing LNTri generated via the cytosolic hydrolysis of lacto-<i>N</i>-tetraose (LNT) by LNT 1,3-β-galactosidase.

DOI： 10.5458/jag.jag.JAG-2013_001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Japanese Society of Applied Glycoscience  

We developed an enzymatic colorimetric method for the quantification of <i>α</i>-D-mannose 1-phosphate by adding phosphomannomutase, mannose 6-phosphate isomerase and glucose 6-phosphate isomerase to a conventional glucose 6-phosphate assay using glucose 6-phosphate dehydrogenase. In this method, <i>α</i>-D-mannose 1-phosphate is converted into D-glucose 6-phosphate <i>via </i>D-mannose 6-phosphate and D-fructose 6-phosphate and the resultant D-glucose 6-phosphate is ultimately converted into 6-phosphogluconolactone under concomitant reduction of thio-NAD⁺ to thio-NADH, which can be quantified by its wavelength of 400 nm. This method is not altered by the presence of D-mannose, D-mannosamine, <i>N</i>-acetyl-D-mannosamine, L-mannose, <i>β</i>-1,4-mannobiose, <i>α</i>-1,2-mannobiose, methyl <i>α</i>-D-mannoside or dimethyl sulfoxide and it would be useful in studies involving enzymes such as phosphorylases belonging to glycoside hydrolase family 130, which release <i>α</i>-D-mannose 1-phosphate as the reaction product.

DOI： 10.5458/jag.jag.JAG-2012_019

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Language：English   Publisher：ELSEVIER SCI LTD  

Phosphorylases are one group of carbohydrate active enzymes involved in the cleavage and formation of glycosidic linkages together with glycoside hydrolases and sugar nucleotide-dependent glycosyltransferases. Noticeably, the catalyzed phosphorolysis is reversible, making phosphorylases suitable catalysts for efficient synthesis of particular oligosaccharides from a donor sugar 1-phosphate and suitable carbohydrate acceptors with strict regioselectivity. Although utilization of phosphorylases for oligosaccharide synthesis has been limited because only few different enzymes are known, recently the number of reported phosphorylases has gradually increased, providing the variation making these enzymes useful tools for efficient synthesis of diverse oligosaccharides.

DOI： 10.1016/j.cbpa.2013.01.006

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Lacto-N-biose I (LNB) is a potential factor for the selective growth of bifidobacteria. We previously reported that the species of bifidobacteria predominant in infant intestines might use LNB. We aimed to assess the prebiotic properties of LNB in comparison to other oligosaccharides using an in vitro fermentation system. Stool samples from formula-fed infants were inoculated with media containing a sole carbon source of 1% LNB, lactulose, raffinose, galactooligosaccharide, or mannanoligosaccharides. LNB significantly increased the total bifidobacterial population similarly to other oligosaccharides, but induced a significantly higher level of Bifidobacterium bifidum in comparison to other oligosaccharides. Furthermore, significantly lower concentrations of lactic acid and significantly higher concentrations of acetic acid were produced in cultures containing LNB in comparison to cultures that contained other oligosaccharides. In conclusion, LNB might have a beneficial effect on the fecal microbiota of infants and is a potential prebiotic for application in infant foods or supplements. (C) 2013 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.anaerobe.2012.12.007

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We identified a glycoside hydrolase family 94 homolog (ACL0729) from Acholeplasma laidlawii PG-8A as a laminaribiose (1,3-beta-D-glucobiose) phosphorylase (EC 2.4.1.31). The recombinant ACL0729 produced in Escherichia coli catalyzed phosphorolysis of laminaribiose with inversion of the anomeric configuration in a typical sequential bi bi mechanism releasing alpha-D-glucose 1-phosphate and D-glucose. Laminaritriose (1,3-beta-D-glucotriose) was not an efficient substrate for ACL0729. The phosphorolysis is reversible, enabling synthesis of 1,3-beta-D-glucosyl disaccharides by reverse phosphorolysis with strict regioselectivity from alpha-D-glucose 1-phosphate as the donor and suitable monosaccharide acceptors (D-glucose, 2-deoxy-D-arabino-hexopyranose, D-xylose, D-glucuronic acid, 1,5-anhydro-D-glucitol, and D-mannose) with C-3 and C-4 equatorial hydroxyl groups. The D-glucose and 2-deoxy-D-arabino-hexopyranose caused significantly strong competitive substrate inhibition compared with other glucobiose phosphorylases reported, in which the acceptor competitively inhibited the binding of the donor substrate. By contrast, none of the examined disaccharides served as acceptor in the synthetic reaction. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.carres.2012.08.006

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We discovered an inverting maltose phosphorylase (Bsel2056) belonging to glycoside hydrolase family 65 from Bacillus selenitireducens MLS10, which possesses synthetic ability for alpha-D-glucosyl disaccharides and trisaccharides through the reverse phosphorolysis with beta-D-glucose 1-phosphate as the donor. Bsel2056 showed the flexibility for monosaccharide acceptors with alternative C2 substituent (2-amino-2-deoxy-D-glucose, 2-deoxy-D-arabino-hexose, 2-acetamido-2-deoxy-D-glucose, D-mannose), resulting in production of 1,4-alpha-D-glucosyl disaccharides with strict regioselectivity. In addition, Bsel2056 synthesized two maltose derivatives possessing additional D-glucosyl residue bound to C2 position of the D-glucose residue at the reducing end, 1,4-alpha-D-glucopyranosyl-[1,2-alpha-D-glucopyranosyl]-D-glucose and 1,4-alpha-D-glucopyranosyl-[1,2-beta-D-glucopyranosyl]-D-glucose, from 1,2-alpha-D-glucopyranosyl-D-glucose (kojibiose) and 1,2-beta-D-glucopyranosyl-D-glucose (sophorose), respectively, as the acceptors. These results suggested that Bsel2056 possessed a binding space to accommodate the bulky C2 substituent of D-glucose. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.carres.2012.07.014

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PORTLAND PRESS LTD  

BcChi-A, a GH19 chitinase from the moss Bryum coronatum, is an endo-acting enzyme that hydrolyses the glycosidic bonds of chitin, (G1cNAc)(n) [a beta-1,4-linked polysaccharide of G1cNAc (N-acetylglucosamine) with a polymerization degree of n], through an inverting mechanism. When the wild-type enzyme was incubated with alpha-(G1cNAc)(2)-F [alpha-(G1cNAc)(2) fluoride] in the absence or presence of (G1cNAc)(2), (G1cNAc)(2) and hydrogen fluoride were found to be produced through the Hehre resynthesis-hydrolysis mechanism. To convert BcChi-A into a glycosynthase, we employed the strategy reported by Honda et al. [(2006) J. Biol. Chem. 281, 1426-1431; (2008) Glycobiology 18, 325-330] of mutating Seri(102), which holds a nucleophilic water molecule, and Glu(70), which acts as a catalytic base, producing S102A, S102C, 5102D, S102G, S102H, S102T, E7OG and E70Q. all of the mutated enzymes, except S102T, hydrolytic activity towards (G1cNAc)(6) was not detected under the conditions we used. Among the inactive BcChi-A mutants, S102A, S102C, S1020G and E70G were found to successfully synthesize (G1cNAc)4 as a major product from alpha-(G1eNAc)(2)-F in the presence of (G1cNAc)(2). The SIO2A mutant showed the greatest glycosynthase activity owing to its enhanced F-releasing activity and its suppressed hydrolytic activity. This is the first report on a glycosynthase that employs amino sugar fluoride as a donor substrate.

DOI： 10.1042/BJ20120036

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

alpha-L-Fucosyl residues attached at the non-reducing ends of glycoconjugates constitute histo-blood group antigens Lewis (Le) and ABO and play fundamental roles in various biological processes. Therefore, establishing a method for synthesizing the antigens is important for functional glycomics studies. However, regiospecific synthesis of glycosyl linkages, especially alpha-L-fucosyl linkages, is quite difficult to control both by chemists and enzymologists. Here, we generated an alpha-L-fucosynthase that specifically introduces Le(a) and Le(x) antigens into the type-1 and type-2 chains, respectively; i.e. the enzyme specifically accepts the disaccharide structures (Gal beta 1-3/4GlcNAc) at the non-reducing ends and attaches a Fuc residue via an alpha-(1,4/3)-linkage to the GlcNAc. X-ray crystallographic studies revealed the structural basis of this strict regio- and acceptor specificity, which includes the induced fit movement of the catalytically important residues, and the difference between the active site structures of 1,3-1,4-alpha-L-fucosidase (EC 3.2.1.111) and alpha-L-fucosidase (EC 3.2.1.51) in glycoside hydrolase family 29. The glycosynthase developed in this study should serve as a potentially powerful tool to specifically introduce the Le(a/x) epitopes onto labile glycoconjugates including glycoproteins. Mining glycosidases with strict specificity may represent the most efficient route to the specific synthesis of glycosidic bonds.

DOI： 10.1074/jbc.M111.333781

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Intestinal colonization of bifidobacteria is important for the health of infants. Human milk oligosaccharides (HMO) have been identified as growth factors for bifidobacteria. Recently, a bifidobacterial enzymatic system to metabolize HMO was identified. 1,3-beta-Galactosyl-N-acetylhexosamine phosphorylase (GLNBP, EC 2.4.1.211), which catalyzes the reversible phosphorolysis of galacto-N-biose (GNB) (Gal beta 1,-&gt; 3GalNAc)] and lacto-N-biose I (LNB) (Gal beta 1 -&gt; 3GlcNAc), is a key enzyme to explain the metabolism of HMO. Infant-type bifidobacteria possess the intracellular pathway to specifically metabolize GNB and LNB (GNB/LNB pathway). Bifidobacterium bifidum possesses extracellular enzymes to liberate [NB from HMO. However, Bindobactenum longum subsp. infantis imports intact HMO to be hydrolyzed by intracellular enzymes. Bifidobacterial enzymes related to the metabolism of HMO are useful tools for preparing compounds related to HMO. For instance, LNB and GNB were produced from sucrose and GlcNAc/GalNAc in 1 pot using 4 bifidobacterial enzymes, including GLNBP. [NB is expected to be a selective bifidus factor for infant-type strains. Adv. Nutr. 3: 422S-429S, 2012.

DOI： 10.3945/an.111.001420

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DOI： 10.12938/bmfh.31.47

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We found an unreported activity of phosphorylase catalyzed by a protein (Cphy1019) belonging to glycoside hydrolase family 65 (GH65) from Clostridium phytofermentans. The recombinant Cphy1019 produced in Escherichia coli did not phosphorolyze alpha-linked glucobioses, such as trehalose (alpha 1-alpha 1), kojibiose (alpha 1-2), nigerose (alpha 1-3), and maltose (alpha 1-4), which are typical substrates for GH65 enzymes. In reverse phosphorolysis, Cphy1019 utilized only L-rhamnose as the acceptor among various sugars examined with beta-D-glucose 1-phosphate as the donor. The reaction product was determined to be 3-O-alpha-D-glucopyranosyl-L-rhamnose, indicating strict alpha 1-3 regioselectivity. We propose 3-O-alpha-D-glucopyranosyl-L-rhamnose: phosphate beta-D-glucosyltransferase as the systematic name and 3-O-alpha-D-glucopyranosyl-L-rhamnose phosphorylase as the short name for this novel GH65 phosphorylase. (C) 2011 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.carres.2011.12.019

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The breast-fed infant intestine is often colonized by particular bifidobacteria, and human milk oligosaccharides (HMOs) are considered to be bifidogenic. Recent studies showed that Bifidobacterium longum subsp. infantis can grow on HMOs as the sole carbon source. This ability has been ascribed to the presence of a gene cluster (HMO cluster-1) contained in its genome. However, the metabolism of HMOs by the organism remains unresolved because no enzymatic studies have been completed. In the present study, we characterized beta-galactosidases of this subspecies to understand how the organism degrades type-1 (Gal beta 1-3GlcNAc) and type-2 (Gal beta 1-4GlcNAc) isomers of HMOs. The results revealed that the locus tag Blon_2016 gene, which is distantly located from the HMO cluster-1, encodes a novel beta-galactosidase (Bga42A) with a significantly higher specificity for lacto-N-tetraose (LNT; Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc) than for lacto-N-biose I (Gal beta 1-3GlcNAc), lactose (Lac) and type-2 HMOs. The proposed name of Bga42A is LNT beta-1,3-galactosidase. The Blon_2334 gene (Bga2A) located within the HMO cluster-1 encodes a beta-galactosidase specific for Lac and type-2 HMOs. Real-time quantitative reverse transcription-polymerase chain reaction analysis revealed the physiological significance of Bga42A and Bga2A in HMO metabolism. The organism therefore uses two different beta-galactosidases to selectively degrade type-1 and type-2 HMOs. Despite the quite rare occurrence in nature of beta-galactosidases acting on type-1 chains, the close homologs of Bga42A were present in the genomes of infant-gut associated bifidobacteria that are known to consume LNT. The predominance of type-1 chains in HMOs and the conservation of Bga42A homologs suggest the coevolution of these bifidobacteria with humans.

DOI： 10.1093/glycob/cwr116

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Bacterial laminaribiose phosphorylase (LBPbac) was first identified and purified from cell-free extract of Paenibacillus sp. YM-1. It phosphorolyzed laminaribiose into a-glucose 1-phosphate and glucose, but did not phosphorolyze other glucobioses. It slightly phosphorolyzed laminaritriose and higher laminarioligosaccharides. The specificity of the degree of polymerization of the substrate was clearly different from that of the enzyme of Euglena gracilis (LBPEug): LBPbac was more specific to laminaribiose than LBPEug. It showed acceptor specificity in reverse phosphorolysis similar to LBPEug. Cloning of the gene encoding LBPbac (IbpA) has revealed that LBPbac is a member of the glucoside hydrolase family 94, which includes cellobiose phosphorylase, cellodextrin phosphorylase, and N,N'-diacetylchitobiose phosphorylase. The genes that encode the components of an ATP-binding cassette sugar transporter specific to laminarioligosaccharides were identified upstream of IbpA, suggesting that the role of LBPbac, is to utilize laminaribiose generated outside the cell. This role is different from that of LBPEug, which participates in the utilization of paramylon, the intracellular storage 1,3-beta-glucan.

DOI： 10.1271/bbb.110772

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A novel phosphorylase from Clostridium phytofermentans belonging to the glycoside hydrolase family (GH) 65 (Cphy1874) was characterized. The recombinant Cphy1874 protein produced in Escherichia coli showed phosphorolytic activity on nigerose in the presence of inorganic phosphate, resulting in the release of d-glucose and beta-d-glucose 1-phosphate (beta-G1P) with the inversion of the anomeric configuration. Kinetic parameters of the phosphorolytic activity on nigerose were k (cat) = 67 s(-1) and K (m) = 1.7 mM. This enzyme did not phosphorolyze substrates for the typical GH65 enzymes such as trehalose, maltose, and trehalose 6-phosphate except for a weak phosphorolytic activity on kojibiose. It showed the highest reverse phosphorolytic activity in the reverse reaction using d-glucose as the acceptor and beta-G1P as the donor, and the product was mostly nigerose at the early stage of the reaction. The enzyme also showed reverse phosphorolytic activity, in a decreasing order, on d-xylose, 1,5-anhydro-d-glucitol, d-galactose, and methyl-alpha-d-glucoside. All major products were alpha-1,3-glucosyl disaccharides, although the reaction with d-xylose and methyl-alpha-d-glucoside produced significant amounts of alpha-1,2-glucosides as by-products. We propose 3-alpha-d-glucosyl-d-glucose:phosphate beta-d-glucosyltransferase as the systematic name and nigerose phosphorylase as the short name for this Cphy1874 protein.

DOI： 10.1007/s00253-011-3515-9

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Three amino acid residues of 1,3-beta-galactosyl-N-acetylhexosamine phosphorylase (GalHexNAcP) were assigned as the determinants of substrate preference for galacto-N-biose (GNB) and lacto-N-biose I (LNB) based on the three-dimensional structure of the protein. Mutants of GalHexNAcP from Bifidobacterium longum, which acts similarly on both GNB and LNB, were constructed and characterized. V162T mutation led to an increase in the selectivity on GNB. P161S and S336A mutations independently enhanced the selectivity on LNB. The alignment of amino acid sequences suggests that the activities of most homologous sequences are predictable by comparing the corresponding three residues. (C) 2011 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.molcatb.2011.09.004

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Bifidobacteria inhabit the lower intestine of mammals including humans where the mucin gel layer forms a space for commensal bacteria. We previously identified that infant-associated bifidobacteria possess an extracellular membrane-bound endo-alpha-N-acetylgalactosaminidase (EngBF) that may be involved in degradation and assimilation of mucin-type oligosaccharides. However, EngBF is highly specific for core-1-type O-glycan (Gal beta 1-3GalNAc alpha 1-Ser/Thr), also called T antigen, which is mainly attached onto gastroduodenal mucins. By contrast, core-3-type O-glycans (GlcNAc beta 1-3GalNAc alpha 1-Ser/Thr) are predominantly found on the mucins in the intestines. Here, we identified a novel alpha-N-acetylgalactosaminidase (NagBb) from Bifidobacterium bifidum JCM 1254 that hydrolyzes the Tn antigen (GalNAc alpha 1-Ser/Thr). Sialyl and galactosyl core-3 (Gal beta 1-3/4GlcNAc beta 1-3(Neu5Ac alpha 2-6)GalNAc alpha 1-Ser/Thr), a major tetrasaccharide structure on MUC2 mucin primarily secreted from goblet cells in human sigmoid colon, can be serially hydrolyzed into Tn antigen by previously identified bifidobacterial extracellular glycosidases such as alpha-sialidase (SiaBb2), lacto-N-biosidase (LnbB), beta-galactosidase (BbgIII), and beta-N-acetylhexosaminidases (BbhI and BbhII). Because NagBb is an intracellular enzyme without an N-terminal secretion signal sequence, it is likely involved in intracellular degradation and assimilation of Tn antigen-containing polypeptides, which might be incorporated through unknown transporters. Thus, bifidobacteria possess two distinct pathways for assimilation of O-glycans on gastroduodenal and intestinal mucins. NagBb homologs are conserved in infant-associated bifidobacteria, suggesting a significant role for their adaptation within the infant gut, and they were found to form a new glycoside hydrolase family 129.

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DOI： 10.4032/9789814303484

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2-Acetamido-2-deoxy-D-galactose (GalNAc) is a common monosaccharide found in biologically functional sugar chains, but its availability is often limited due to the lack of abundant natural sources. In order to produce GalNAc from abundantly available sugars, 2-acetamido-2-deoxy-D-glucose (GlcNAc) was converted to GalNAc by a one-pot reaction using three enzymes involved in the galacto-N-biose/lacto-N-biose I pathway of bifidobacteria. Starting the reaction with 600 mM GlcNAc, 170 mM GalNAc was produced at equilibrium in the presence of catalytic amounts of ATP and UDP-Glc under optimized conditions. GalNAc was separated from GlcNAc using water-eluting cation-exchange chromatography with a commonly available cation-exchange resin. (C) 2011 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.carres.2011.08.032

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Japanese Society of Applied Glycoscience  

The time profile of an enzymatic reaction at an early stage is generally considered to be linear. We observed that the time profile of the hydrolysis of 1 mM <i>p</i>-nitrophenyl-β-D-glucoside (pNP-Glc) by a β-glucosidase obtained from <i>Agrobacterium tumefaciens</i> (Cbg1) was not linear before 5% of the substrate was consumed, even though the initial concentration of the substrate was much higher than its <i>K</i><small>m</small> value. The time profiles obtained with higher concentrations of pNP-Glc suggested that the time profile was the function of the absolute concentration of <i>p</i>-nitrophenol (pNP). The addition of various alcohols made the time profile linear. A self-transferring product inhibition model was constructed in which the pNP generated during the hydrolysis acts as an acceptor substrate to inhibit the hydrolysis. The theoretical curve agreed well with the experimental data.

DOI： 10.5458/jag.jag.JAG-2011_003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The bifidogenic effect of human milk oligosaccharides (HMOs) has long been known, yet the precise mechanism underlying it remains unresolved. Recent studies show that some species/subspecies of Bifidobacterium are equipped with genetic and enzymatic sets dedicated to the utilization of HMOs, and consequently they can grow on HMOs; however, the ability to metabolize HMOs has not been directly linked to the actual metabolic behavior of the bacteria. In this report, we clarify the fate of each HMO during cultivation of infant gut-associated bifidobacteria. Bifidobacterium bifidum JCM1254, Bifidobacterium longum subsp. infantis JCM1222, Bifidobacterium longum subsp. longum JCM1217, and Bifidobacterium breve JCM1192 were selected for this purpose and were grown on HMO media containing a main neutral oligosaccharide fraction. The mono-and oligosaccharides in the spent media were labeled with 2-anthranilic acid, and their concentrations were determined at various incubation times using normal phase high performance liquid chromatography. The results reflect the metabolic abilities of the respective bifidobacteria. B. bifidum used secretory glycosidases to degrade HMOs, whereas B. longum subsp. infantis assimilated all HMOs by incorporating them in their intact forms. B. longum subsp. longum and B. breve consumed lacto-N-tetraose only. Interestingly, B. bifidum left degraded HMO metabolites outside of the cell even when the cells initiate vegetative growth, which indicates that the different species/subspecies can share the produced sugars. The predominance of type 1 chains in HMOs and the preferential use of type 1 HMO by infant gut-associated bifidobacteria suggest the coevolution of the bacteria with humans.

DOI： 10.1074/jbc.M111.248138

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DOI： 10.5458/jag.jag.JAG-2011_001

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DOI： 10.5458/jag.jag.JAG-2010_024

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Japanese Society of Applied Glycoscience  

Quantification of orthophosphate (Pi) in the presence of labile phosphate esters is required for biochemical assays. We developed a method for the enzymatic colorimetric quantification of Pi using pyruvate oxidase and peroxidase. The calibration curve was not affected by the presence of labile phosphate esters. Furthermore, this method allows continuous monitoring of the reaction of Pi-releasing enzymes.

DOI： 10.5458/jag.jag.JAG-2011_002

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Other Link： https://jlc.jst.go.jp/DN/JALC/00376567404?from=CiNii

Language：English   Publishing type：Part of collection (book)   Publisher：Elsevier Inc.  

Mammalian milk/colostrum contains from trace to ∼13% of carbohydrate, of which lactose (Gal(β1-4)Glc) usually constitutes more than 80%. Milk/colostrum of most mammals also contains a variety of oligosaccharides, many of which have N-acetylglucosamine, galactose, fucose, and/or sialic acid residues attached to lactose, which is always located at the reducing end. The ratio of milk oligosaccharides to free lactose in milk/colostrum varies, depending on the mammalian species. Most human milk oligosaccharides (HMOs) cannot be digested within the small intestine, and reach the colon where they stimulate the growth of bifidobacteria and act as possible antiadhesion factors against pathogenic microorganisms in the human infant. The biological functions of milk oligosaccharides have recently been an active field of research. In this article, we describe the chemical structures and methods for the structural analysis of milk oligosaccharides, quantitative aspects and methods for their quantification, their gastrointestinal digestion and biosynthesis, and their biological significance as prebiotics, anti-infection factors, receptor analogues, and immunomodulators, with special reference to the oligosaccharides of human milk. We also describe the milk oligosaccharides of domestic farm animals and discuss comparative aspects of milk oligosaccharides among mammalian species, as well as future trends and the industrial utilization of milk oligosaccharides.

DOI： 10.1016/B978-0-12-374407-4.00279-X

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS INC  

Bifidobacteria are predominant in the intestines of breast-fed infants and offer health benefits to the host. Human milk oligosaccharides (HMOs) are considered to be one of the most important growth factors for intestinal bifidobacteria. HMOs contain two major structures of core tetrasaccharide: lacto-N-tetraose (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc; type 1 chain) and lacto-N-neotetraose (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc; type 2 chain). We previously identified the unique metabolic pathway for lacto-N-tetraose in Bifidobacterium bifidum. Here, we clarified the degradation pathway for lacto-N-neotetraose in the same bifidobacteria. We cloned one beta-galactosidase (BbgIII) and two beta-N-acetylhexosaminidases (BbhI and BbhII), all of which are extracellular membrane-bound enzymes. The recombinant BbgIII hydrolyzed lacto-N-neotetraose into Gal and lacto-N-triose II, and furthermore the recombinant BbhI, but not BbhII, catalyzed the hydrolysis of lacto-N-triose II to GlcNAc and lactose. Since BbgIII and BbhI were highly specific for lacto-N-neotetraose and lacto- N-triose II, respectively, they may play essential roles in degrading the type 2 oligosaccharides in HMOs.

DOI： 10.1093/glycob/cwq101

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Exo-1,5-alpha-L-arabinofuranosidases belonging to glycoside hydrolase family 43 have strict substrate specificity. These enzymes hydrolyze only the alpha-1,5-linkages of linear arabinan and arabino-oligosaccharides in an exo-acting manner. The enzyme from Streptomyces avermitilis contains a core catalytic domain belonging to glycoside hydrolase family 43 and a C-terminal arabinan binding module belonging to carbohydrate binding module family 42. We determined the crystal structure of intact exo-1,5-alpha-L-arabinofuranosidase. The catalytic module is composed of a 5-bladed beta-propeller topologically identical to the other family 43 enzymes. The arabinan binding module had three similar subdomains assembled against one another around a pseudo-3-fold axis, forming a beta-trefoil-fold. A sugar complex structure with alpha-1,5-L-arabinofuranotriose revealed three subsites in the catalytic domain, and a sugar complex structure with alpha-L-arabinofuranosyl azide revealed three arabinose-binding sites in the carbohydrate binding module. A mutagenesis study revealed that substrate specificity was regulated by residues Asn-159, Tyr-192, and Leu-289 located at the aglycon side of the substrate-binding pocket. The exo-acting manner of the enzyme was attributed to the strict pocket structure of subsite -1, formed by the flexible loop region Tyr-281-Arg-294 and the side chain of Tyr-40, which occupied the positions corresponding to the catalytic glycon cleft of GH43 endo-acting enzymes.

DOI： 10.1074/jbc.M110.164251

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PORTLAND PRESS LTD  

beta-Glucosidase from Kluyveromyces marxianus (KmBgII) belongs to the GH3 (glycoside hydrolase family 3). The enzyme is particularly unusual in that a PA14 domain (pf07691), for which a carbohydrate-binding role has been claimed, is inserted into the catalytic core sequence. In the present study, we determined the enzymatic properties and crystal structure of KmBgII in complex with glucose at a 2.55 angstrom (1 angstrom = 0.1 nm) resolution. A striking characteristic of KmBgII was that the enzyme activity is essentially limited to disaccharides, and when trisaccharides were used as the substrates the activity was drastically decreased. This chain-length specificity is in sharp contrast with the preferred action on oligosaccharides of barley beta-D-glucan glucohydrolase (Exol), which does not have a PA14 domain insertion. The structure of subsite (-1) of KmBgII is almost identical with that of Thermotoga neapolitana beta-glucosidase and is also similar to that of Exol, however, the structures of subsite (+1) significantly differ among them. In KmBgII, the loops extending from the PA14 domain cover the catalytic pocket to form subsite (+1), and hence simultaneously become a steric hindrance that could limit the chain length of the substrates to be accommodated. Mutational studies demonstrated the critical role of the loop regions in determining the substrate specificity. The active-site formation mediated by the PA14 domain of KmBgII invokes alpha-complementation of beta-galactosidase exerted by its N-terminal domain, to which the PA14 domain shows structural resemblance. The present study is the first which reveals the structural basis of the interaction between the PA14 domain and a carbohydrate.

DOI： 10.1042/BJ20100351

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

beta-D-Galactopyranosyl-(1 -&gt; 3)-2-acetamido-2-deoxy-D-glucose (LNB) and beta-D-galactopyranosyl-(1 -&gt; 3)-2-acetamido-2-deoxy-D-galactose (GNB) decompose rapidly upon heating into D-galactose and mono-dehydrated derivatives of the corresponding 2-acetamido-2-deoxy-D-hexoses, including 2-acetamido-2,3-dideoxy-hex-2-enofuranoses and bicyclic 2-acetamido-3,6-anhydro-2-deoxy-hexofuranoses. The decomposition is conducted under neutral conditions where glycosyl linkages are generally believed to be stable. The half-lives of LNB and GNB were 8.1 min and 20 min, respectively, at 90 degrees C and pH 7.5. The pH dependency of decomposition rates suggests that the instabilities are an extension of the conditions for the peeling reaction, often observed with glycans of O-linked glycoproteins under alkaline conditions. Such decomposition under the neutral conditions is commonly observed with 3-O-linked reducing aldoses. (C) 2010 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.carres.2010.06.003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

D-Galactosyl-beta 1 -&gt; 4-L-rhamnose (GalRha) was produced enzymatically from 1.1 M sucrose and 1.0 M L-rhamnose by the concomitant actions of four enzymes (sucrose phosphorylase, UDP-glucose-hexose 1-phosphate uridylyltransferase, UDP-glucose 4-epimerase, and D-galactosyl-beta 1 -&gt; 4-L-rhamnose phosphorylase) in the presence of 1.0 mm UDP-glucose and 30 mm inorganic phosphate. The accumulation of GalRha in 1 liter of the reaction mixture reached 230 g (the reaction yield was 71% from L-rhamnose). Sucrose and fructose in the reaction mixture were removed by yeast treatment, but isolation of GalRha by crystallization after yeast treatment was unsuccessful. Finally, 49 g of GalRha was isolated from part of the reaction mixture with yeast treatment by gel-filtration chromatography.

DOI： 10.1271/bbb.100263

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Language：English   Publishing type：Part of collection (book)   Publisher：John Wiley and Sons  

DOI： 10.1002/9780470608524.ch23

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Language：English   Publishing type：Part of collection (book)   Publisher：John Wiley and Sons  

DOI： 10.1002/9780470608524.ch2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

We characterized a glycoside hydrolase family 112 protein from Opitutus terrae (Oter_1377 protein). The enzyme phosphorolyzed D-galactosyl-beta 1 -&gt; 4-L-rhamnose (GalRha) and also showed phosphorolytic activity on D-galactosyl-beta 1 -&gt; 3-D-glucose as a minor substrate. In the reverse reaction, the enzyme showed higher activity on L-rhamnose derivatives than Oil D-glucose derivatives. The enzyme was stable up to 45 degrees C and at pH 6.0-7.0. The values of k(cat) and K-m of the phosphorolytic activity of the enzyme on GalRha were 60 s(-1) and 2.1 mM, respectively. Thus, Oter_1377 protein was identified as D-galactosyl-beta 1 -&gt;-4-L-rhamnose phosphorylase (GalRhaP). The presence of GalRhaP in O. terrae suggests that genes encoding GalRhaP are widely distributed in different organisms. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.enzmictec.2009.12.007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KOREAN SOC BIOTECHNOLOGY & BIOENGINEERING  

In vivo folding of many proteins can be facilitated by growth temperature, extent of induction, and molecular chaperones, which prevent over-expressed protein from being trapped into insoluble inclusion bodies. In the present report, we describe the role of molecular chaperones and growth temperature on the solubilization of over-expressed Cellobiose Phosphorylase (CBP) in Escherichia coli. The growth of host at low temperature enhanced enzyme in soluble fraction. Similarly, induction of target gene at low level of IPTG also yielded higher enzyme in soluble fraction. The synergistic effect of low temperature and induction on the prevention of inclusion bodies was also evident from our results. In addition, co-expression of the target gene with two types of molecular chaperones (GroESL and KODHsp) was also attempted. However, none of these chaperones enhanced the solubilization under in vivo conditions. Nevertheless, effective role of low growth temperature coupled with low level of induction appeared to be an attractive feature for producing recombinant protein.

DOI： 10.1007/s12257-009-0023-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Glycosynthases are engineered glycoside hydrolases (GHs) that catalyse the synthesis of glycoside from glycosyl-fluoride donors and suitable acceptors. We have determined five crystal structures of the glycosynthase mutants reducing-end xylose-releasing exo-oligoxylanase, an inverting GH, that exhibit various levels of glycosynthetic activities. At the active site of the Y198F mutant, the most efficient glycosynthase, a water molecule is observed at the same position as nucleophilic water (NW) in the parent enzyme, and the loss of the fixation of the direction of the lone pair of water molecules in the mutant drastically decreases hydrolytic activity. Water molecules were also observed at each active site of the general base mutant, but they were shifted 1.0-3.0 A from the NW in the wild type. Their positions exhibited a strong correlation with the strength of glycosynthase activity. Here, we propose that a structural prerequisite for the sufficient glycosynthase reaction is the presence of a water molecule at the NW position, and mutation at the NW holder provides a general strategy for inverting GHs. The idea on the position of a water molecule may also be applicable to the design of efficient glycosynthases from retaining GHs.

DOI： 10.1093/jb/mvp159

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Language：English   Publisher：JAPAN BIFIDUS FOUNDATION  

Many bifidobacteria produce an endo-α- <i>N</i>-acetylgalactosaminidase that liberates the <i>O</i>-linked galactosyl β-1,3<i>N-</i>acetylgalactosamine (GNB) from intestinal mucin glycoproteins. The molecular cloning of the <i>Bifidobacterium</i> <i>longum</i> enzyme was completed using information in public databases. The enzyme constitutes a novel glycoside hydrolase (GH) family 101 member. The gene encoding a specific 1,2-α -L-fucosidase was cloned from <i>B.bifidum</i>. The recombinant enzyme specifically hydrolyzes the terminal α-1,2-fucosidic linkages of various oligosaccharides, including human milk oligosaccharides and blood group substances. Analysis of its primary structure revealed that this enzyme constitutes a novel GH family 95 member. We also solved the crystal structure of its catalytic domain. We assumed that these bifidobacterial enzymes are involved in the metabolism of oligosaccharides in mucin glycoproteins that are abundant in the intestine. Some bifidobacteria strains produce a <b>lacto-N-</b>biosidase that releases galactosyl β-1,3<i>N</i>-acetylglucosamine (LNB) from human milk oligosaccharides, but the other enteric bacteria do not. This disaccharide is one of the building blocks in human milk oligosaccharides and is rarely found in other mammalian milks. The <b>lacto-N-</b>biosidase gene was cloned from <i>B.bifidum</i> and we hypothesized that this enzyme is crucially involved in the degradation of human milk oligosaccharides. The genes encoding sialidase and α-1,3/4-L-fucosidase were also cloned from <i>B.bifidum.</i> These enzymes release modified sialic acid and L-fucose from human milk oligosaccharides, respectively. A solute-binding protein of a putative ABC transporter specific for GNB and LNB was also discovered, and its gene was cloned from <i>B.longum</i>. We named it GNB/LNB-binding protein and crystallized it. Isothermal titration calorimetry measurements revealed that this protein specifically binds GNB and LNB. We speculate that bifidobacteria have a novel GNB/LNB metabolic pathway.<br>

DOI： 10.12938/bifidus.29.23

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Other Link： http://search.jamas.or.jp/link/ui/2010222393

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

This study investigated the potential utilization of lacto-N-biose I (LNB) by individual strains of bifidobacteria. LNB is a building block for the human milk oligosaccharides, which have been suggested to be a factor for selective growth of bifidobacteria. A total of 208 strains comprising 10 species and 4 subspecies were analyzed for the presence of the galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) gene (lnpA) and examined for growth when LNB was used as the sole carbohydrate source. While all strains of Bifidobacterium longum subsp. longum, B. longum subsp. infantis, B. breve, and B. bifidum were able to grow on LNB, none of the strains of B. adolescentis, B. catenulatum, B. dentium, B. angulatum, B. animalis subsp. lactis, and B. thermophilum showed any growth. In addition, some strains of B. pseudo-catenulatum, B. animalis subsp. animalis, and B. pseudolongum exhibited the ability to utilize LNB. With the exception for B. pseudocatenulatum, the presence of lnpA coincided with LNB utilization in almost all strains. These results indicate that bifidobacterial species, which are the predominant species found in infant intestines, are potential utilizers of LNB. These findings support the hypothesis that GLNBP plays a key role in the colonization of bifidobacteria in the infant intestine.

DOI： 10.1128/AEM.01683-09

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Language：Japanese   Publisher：日本応用糖質科学会  

DOI： 10.11541/jsag.2010.0.81.0

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Language：Japanese   Publisher：The Japanese Society of Applied Glycoscience  

DOI： 10.11541/jsag.2010.0.120.0

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Language：Japanese   Publisher：The Japanese Society of Applied Glycoscience  

DOI： 10.11541/jsag.2010.0.131.0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

beta-D-Galactopyranosyl-(1--&gt;3)-2-acetamido-2-deoxy-D-galactose (galacto-N-biose, GNB) is an important core structure in functional sugar chains such as T-antigen disaccharide and the core I sugar chain in mucin glycoproteins. We successfully developed a one-pot enzymatic production of GNB from sucrose and GalNAc by the concomitant action of four enzymes: sucrose phosphorylase, UDP-glucose-hexose I-phosphate uridylyltransferase, UDP-glucose 4-epimerase, and galacto-N-biose/lacto-N-biose I phosphorylase in the presence of UDP-glucose and phosphate, by modifying the method of lacto-N-biose I production [Nishimoto, M.; Kitaoka, M., Biosci. Biotechnol. Biochem., 2007, 71, 2101-2104]. The reaction yield of GNB was 88% from GalNAc. GNB was isolated from the reaction mixture by crystallization after yeast treatment to obtain approximately 45 g of GNB in 95% purity from a 280-mL reaction mixture. (C) 2009 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.carres.2009.09.031

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

Synthesis of cellulose in vitro is expected to afford tailor-made cellulosic materials with highly homogeneous structure compared to natural cellulosic materials. Here we report the enzymatic synthesis of cellulose II with high crystallinity from glucose and alpha-glucose 1-phosphate (alpha G1P) by cellodextrin phosphorylase (CDP). Although glucose had been believed not to act as a glucosyl acceptor of CDP, a significant amount of insoluble cellulose was precipitated without accumulation of soluble cello oligosaccharides when glucose was mixed with alpha G1P and CDP. This phenomenon can be explained in terms of the large difference in acceptor reactivity between glucose and cello oligosaccharides. (1)H NMR spectrometric analysis revealed that this insoluble cellulose had an average degree of polymerization (DP) of nine. TEM observation, together with electron and X-ray diffraction studies, indicated that the insoluble cellulose formed platelet-shaped single lamellar crystals of cellulose II, several mu m in length and several hundred nm in width; this is large compared to reported cellulose crystals. The thickness of the lamellar crystal is 4.5 nm, which is equivalent to a chain length of a cello oligosaccharide with DP nine and is consistent with the (1)H NMR spectroscopic results. These results suggest that cello oligosaccharides having an average DP of nine are synthesized in vitro by CDP when glucose is used as an acceptor, and the product forms highly crystalline cellulose II when it precipitates. (C) 2009 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.carres.2009.10.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

The intracellular beta-glucosidase from Kluyveromyces marxianus NBRC1777 (KmBglI) belongs to glycoside hydrolase family 3 and has a unique domain architecture. Selenomethionine-labelled KmBglI was purified and crystallized by the hanging-drop vapour-diffusion method using the purified enzyme at 30 mg ml(-1), 0.04 M potassium dihydrogen phosphate pH 5.1, 16%(w/v) PEG 8000 and 20%(v/v) glycerol. The crystal belonged to space group C2, with unit-cell parameters a = 245.8, b = 148.7, c = 119.9 angstrom, beta = 112.9 degrees. Multiple-wavelength anomalous dispersion data were collected at 2.4 and 2.5 angstrom resolution. A tetramer was assumed to be present in the asymmetric unit, which gave a Matthews coefficient of 2.6 angstrom(3) Da(-1).

DOI： 10.1107/S1744309109042948

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Language：English   Publisher：AMER SOC MICROBIOLOGY  

DOI： 10.1128/AEM.01847-09

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Cellobiose was enzymatically synthesized from starch using two phosphorylases. Under the presence of 1 m Pi (inorganic phosphate), glucan phosphorylase converted 40% of glucose residues in the starch molecule into G1P (glucose-1-phosphate). By electrodialysis fitted with an ion exchange membrane having molecular weight cutoff of 100, Pi was effectively dialyzed out and GIP was recovered with 80% yield. G1P and glucose were incubated with cellobiose phosphorylase in the presence of magnesium acetate at an alkaline condition. Inorganic phosphate coformed with cellobiose was immediately removed as insoluble magnesium ammonium phosphate and 85% of added GIP was converted into cellobiose. On the whole, cellobiose was produced with 60% yield from G1P and, at least, 23.7% yield from starch.

DOI： 10.1016/j.nbt.2009.07.004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

We report herein a bacterial N-acetylhexosamine kinase, NahK, with broad substrate specificity towards structurally modified GalNAc analogues, and the production of a GalNAc-1-phosphate library using this kinase. (C) 2009 Published by Elsevier Ltd.

DOI： 10.1016/j.bmcl.2009.07.104

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Endo-alpha-N-acetylgalactosaminidase (endo-alpha-GalNAc-ase), a member of the glycoside hydrolase (GH) family 101, hydrolyses the O-glycosidic bonds in mucin-type O-glycan between alpha-GalNAc and Ser/Thr. Endo-alpha-GalNAc-ase from Bifidobacterium longum JCM1217 (EngBF) is highly specific for the core 1-type O-glycan to release the disaccharide Gal beta 1-3GalNAc (GNB), whereas endo-alpha-GalNAe-ase from Clostridium perfringens (EngCP) exhibits. broader substrate specificity. We determined the crystal structure of EngBF at 2.0 angstrom resolution and performed automated docking analysis to investigate possible binding modes of GNB. Mutational analysis revealed important residues for substrate binding, and two Trp residues (Trp748 and Trp750) appeared to form stacking interactions with the beta-faces of sugar rings of GNB by substrate-induced fit. The difference in substrate specificities between EngBF and EngCP is attributed to the variations in amino acid sequences in the regions forming the substrate-binding pocket. Our results provide a structural basis for substrate recognition by GH101 endo-alpha-GalNAe-ases and will help structure-based engineering of these enzymes to produce various kinds of neo-glycoconjugates.

DOI： 10.1093/jb/mvp086

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

In the title compound, C14H25NO11 center dot 2H(2)O, the primary hydroxyl group connected to the anomeric C atom of the N-acetyl-beta-D-glucopyranose residue exhibits positional disorder, with occupancy factors for the alpha and beta anomers of 0.77 and 0.23, respectively. The two torsion angles (Phi and Psi) and the bridge angle (tau) that describe conformation of the glycosidic linkage between the galactopyranose and glucopyranose rings are Phi = -81.6 (3)degrees, Psi = 118.1 (2)degrees and tau = 115.2 (2)degrees. Two water molecules stabilize the molecular packing by forming hydrogen bonds with the saccharide residues.

DOI： 10.1107/S1600536809024775

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

We characterized three D-galactosyl-beta 1 -&gt; 3-N-acetyl-D-hexosamine phosphorylase (EC 2.4.1.211) homologs from Clostridium phytofermentans (Cphy0577, Cphy1920, and Cphy3030 proteins). Cphy0577 and Cphy3030 proteins exhibited similar activity on galacto-N-biose (GNB; D-Gal-beta 1 -&gt; 3-D-GalNAc) and lacto-N-biose I (LNB; D-Gal-beta 1 -&gt; 3-D-GlcNAc), thus indicating that they are D-galactosyl-beta 1 -&gt; 3-N-acetyl-D-hexosamine phosphorylases, subclassified as GNB/LNB phosphorylase. In contrast, Cphy1920 protein phosphorolyzed neither GNB nor LNB. It showed the highest activity with L-rhamnose as the acceptor in the reverse reaction using alpha-D-galactose 1-phosphate as the donor. The reaction product was D-galactosyl-beta 1 -&gt; 4-L-rhamnose. The enzyme also showed activity on L-mannose, L-lyxose, D-glucose, 2-deoxy-D-glucose, and D-galactose in this order. When D-glucose derivatives were used as acceptors, reaction products were beta-1,3-galactosides. Kinetic parameters of phosphorolytic activity on D-galactosyl-beta 1 -&gt; 4-L-rhamnose were k(cat) = 45 s(-1) and K-m = 7.9 mM, thus indicating that these values are common among other phosphorylases. We propose D-galactosyl-beta 1 -&gt; 4-L-rhamnose phosphorylase as the name for Cphy1920 protein.

DOI： 10.1074/jbc.M109.007666

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Homologs of the beta-1,3-galactosyl-N-acetylhexosamine phosphorylase (GalHexNAcP) gene (gnpA) were cloned from the genomic DNA of Propionibacterium acnes JCM6425 and P. acnes JCM6473, showing 99.9% and 97.9% nucleotide sequence identity, respectively, with the ppa0083 gene from the genome-sequenced P. acnes KPA171202. No gnpA gene was detected in the genomic DNA of type strain P. acnes ATCC25746. The recombinant enzyme from P. acnes JCM6425 (GnpA) showed approximately 70 times higher specific activity of phosphorolysis on galacto-N-biose (Gal beta 1 -&gt; 3GalNAc, GNB) than that on lacto-N-biose I (Gal beta 1 -&gt; 3GlcNAc). K (m) value for GnpA on GNB was high, but GnpA did not exhibit activity on any derivatives of GNB examined. These results indicate that GnpA is GalHexNAcP which should be classified as galacto-N-biose phosphorylase. The large k (cat) value of GnpA on GalNAc suggests that GnpA would be a useful catalyst for the synthesis of GNB.

DOI： 10.1007/s00253-008-1838-y

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

We demonstrated the prebiotic effect of lacto-N-biose I (Gal beta 1-3GlcNAc) on bifidobacteria in vitro. Lacto-N-biose I, a building unit of the type-I milk oligosaccharides, enhanced the growth of many bifidobacteria, especially Bifidobacterium bifidum, B. breve, and B. longum, which are predominant in the intestines of breast-fed infants. It might be a substantial, natural prebiotic in human colostrums.

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Publisher：日本応用糖質科学会  

還元末端オリゴキシラナーゼ［EC 3.2.1.156］（REX）はキシロオリゴ糖を還元末端側から加水分解するアノマー反転型酵素である。野生型REXにα－X2-FとX1を作用させると、いわゆる&quot;Hehre resynthesis-hydrolysis&quot;と呼ばれる反応を触媒した。REXをグライコシンターゼに変換するために、REXの触媒塩基であるD263にsaturation mutagenesisによって種々のアミノ酸に置換した変異型酵素を作製し、α－X2-FとX1を作用させてグライコシンターゼ反応を調べた。9種のD263変異型酵素がグライコシンターゼ反応産物であるX3を生成したが、これらの酵素の中でD263Cが良いグライコシンターゼであった。しかし、これらの変異型酵素は、X3が生成するとともに加水分解産物であるX2を生成した。REXの活性部位構造を詳細にみると、求核試薬として作用する水分子がD263およびY198と水素結合を形成していることが判明した。次にY198に変異を導入したY198FとD263C／Y198FおよびD263N／Y198Fを作製した。これらの変異型酵素によるX3加水分解活性は、野生型酵素と比較して大幅に減少した。またY198Fのα－X2-F消費能は野生型の1.5倍であったが、D263N／Y198Fは1／10以下であり、D263C／Y198Fは微弱な活性しか示さなかった。次にY198FとD263N／Y198Fによるグライコシンターゼ反応の経時変化を調べると、X3が効率良く蓄積され、分解産物であるX2がほとんど検出されなかった。さらに、両酵素によるα－X2-F消費能の比活性を比較すると、Y198FがD263N／Y198Fより約20倍程度高かったので、Y198Fが最も良いグライコシンターゼであると総合的に判断した。

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Glycoside hydrolase family 55 consists of beta-1,3-glucanases mainly from filamentous fungi. A beta-1,3-glucanase (Lam55A) from the Basidiomycete Phanerochaete chrysosporium hydrolyzes beta-1,3-glucans in the exo-mode with inversion of anomeric configuration and produces gentiobiose in addition to glucose from beta-1,3/1,6-glucans. Here we report the crystal structure of Lam55A, establishing the three-dimensional structure of a member of glycoside hydrolase 55 for the first time. Lam55A has two beta-helical domains in a single polypeptide chain. These two domains are separated by a long linker region but are positioned side by side, and the overall structure resembles a rib cage. In the complex, a gluconolactone molecule is bound at the bottom of a pocket between the two beta-helical domains. Based on the position of the gluconolactone molecule, Glu-633 appears to be the catalytic acid, whereas the catalytic base residue could not be identified. The substrate binding pocket appears to be able to accept a gentiobiose unit near the cleavage site, and a long cleft runs from the pocket, in accordance with the activity of this enzyme toward various beta-1,3-glucan oligosaccharides. In conclusion, we provide important features of the substrate-binding site at the interface of the two beta-helical domains, demonstrating an unexpected variety of carbohydrate binding modes.

DOI： 10.1074/jbc.M808122200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) from Bifidobacterium longum, a key enzyme for intestinal growth, phosphorolyses galacto-N-biose and lacto-N-biose I with anomeric inversion. GLNBP homologues are often found in human pathogenic and commensal bacteria, and their substrate specificities potentially define the nutritional acquisition ability of these microbes in their habitat. We report the crystal structures of GLNBP in five different ligand-binding forms. This is the first three-dimensional structure of glycoside hydrolase (GH) family 112. The GlcNAc-and GalNAc-bound forms provide structural insights into distinct substrate preferences of GLNBP and its homologues from pathogens. The catalytic domain consists of a partially broken TIM barrel fold that is structurally similar to a thermophilic beta-galactosidase, strongly supporting the current classification of GLNBP homologues as one of the GH families. Anion binding induces a large conformational change by rotating a half-unit of the barrel. This is an unusual example of molecular adaptation of a TIM barrel scaffold to substrates.

DOI： 10.1074/jbc.M808525200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：GAKUSHIN PUBL CO  

Reducing-end xylose-releasing exo-oligoxylanase (Rex, EC. 3.2.1.156) is an inverting xylanolytic enzyme, belonging to the glycoside hydrolase (GH) family 8, which hydrolyzes xylooligosaccharides to release xylose (X-1) from its reducing end. Rex hydrolyzes alpha-xylobiosyl fluoride (alpha-X2F) to yield xylobiose (X-2) only in the presence of X-1, confirming the Hehre resynthesis-hydrolysis mechanism. A library of mutant Rex at the catalytic base (D263) was constructed by saturation mutagenesis, in which D263C accumulated the highest level of xylotriose (X-3) from alpha-X2F and X-1. However, F-releasing activities of the mutants were much less than that of the wild type. Next, Y198 residue of Rex that forms a hydrogen bond with nucleophilic water was substituted with phenylalanine, causing a marked decrease in hydrolytic activity and a small increase in the F- releasing activity from alpha-X2F in the presence of X-1. Y198F of Rex accumulated more product during the glycosynthase reaction than D263C. Recently, an inverting alpha-1,2-fucosidase belonging to GH95 was converted into glycosynthase by mutating a catalytic base residue. In both cases, the catalytic base should be intact.

DOI： 10.4052/tigg.21.23

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Honda, Y, Fushinobu, S, Hidaka, M, Wakagi, T, Shoun, H, Taniguchi, H &amp; Kitaoka, M, 2009, &#039;Conversion of an Inverting Glycoside Hydrolase into Glycosynthase&#039;, &lt;i&gt;Journal of Applied Glycoscience&lt;/i&gt;, vol. 291, no. 3, pp. 883-125.

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Language：English   Publishing type：Part of collection (book)   Publisher：Springer New York  

Mammalian milk contains up to 10% carbohydrate, of which the disaccharide, lactose (Gal(β1-4)Glc), is usually a prominent component. Milk and colostrum also contain lesser amounts of other saccharides, referred to as milk oligosaccharides, nearly all of which have a lactose unit at their reducing end to which GlcNAc, Gal, Fuc and/or Neu5Ac or Neu5Gc residues can be attached (Jenness et al., 1964
Newburg and Neubauer, 1995
Boehm and Stahl, 2003
Urashima et al., 2001
Messer and Urashima, 2002). Pronounced heterogeneity as well as homology of milk oligosaccharide structures among different mammalian species has been documented (Urashima et al., 2001
Messer and Urashima, 2002). © 2009 Springer Science+Business Media, LLC.

DOI： 10.1007/978-0-387-84865-5_8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROYAL SOC CHEMISTRY  

Reports an efficient chemoenzymatic production of an N-acetylhexosamine 1-phophate analogues library by N-acetylhexosamine 1-kinase (NahK) and describes the respective substrate specificity on this enzyme.

DOI： 10.1039/b904853g

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Language：Japanese   Publisher：The Japanese Society of Applied Glycoscience  

DOI： 10.11541/jsag.2009.0.84.0

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Language：Japanese   Publisher：The Japanese Society of Applied Glycoscience  

DOI： 10.11541/jsag.2009.0.131.0

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Language：Japanese   Publisher：The Japanese Society of Applied Glycoscience  

DOI： 10.11541/jsag.2009.0.71.0

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Language：Japanese   Publisher：The Japanese Society of Applied Glycoscience  

DOI： 10.11541/jsag.2009.0.72.0

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Language：Japanese   Publisher：The Japanese Society of Applied Glycoscience  

DOI： 10.11541/jsag.2009.0.53.0

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Language：Japanese   Publisher：The Japanese Society of Applied Glycoscience  

DOI： 10.11541/jsag.2009.0.52.0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Fucosyloligosaccharides have great therapeutic potential. Here we present a new route for synthesizing a Fuc alpha 1,2-Gal linkage by introducing glycosynthase technology into 1,2-alpha-L-fucosidase. The enzyme adopts a unique reaction mechanism, in which asparagine-423 activated by aspartic acid-766 acts as a base while asparagine-421 fixes both a catalytic water and glutamic acid-566 ( an acid) in the proper orientations. Glycosynthase activity of N421G, N423G, and D766G mutants was examined using beta-fucosyl fluoride and lactose, and among them, the D766G mutant most effectively synthesized 2&apos;-fucosyllactose. 1,2-alpha-L-Fucosynthase is the first glycosynthase derived from an inverting alpha-glycosidase and from a glycosidase with an unusual reaction mechanism. (C) 2008 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

DOI： 10.1016/j.febslet.2008.09.054

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

To understand microbial communities in petroleum crude oils, we precipitated DNA using high concentrations of 2,2,4-trimethylpentane (isooctane) and purified. Samples of DNA from five crude oils, (Middle East, 3; China, 1; and Japan, 1) were characterized based upon their 16S rRNA gene sequences after PCR amplification and the construction of clone libraries. We detected 48 eubacterial species, one cyanobacterium, and one archaeon in total. The microbial constituents were diverse in the DNA samples. Most of the bacteria affiliated with the sequences of the three oils from the Middle East comprised similar mesophilic species. Acinetobacter, Propionibacterium, Sphingobium and a Bacillales were common. In contrast, the bacterial communities in Japanese and Chinese samples were unique. Thermophilic Petrotoga-like bacteria (11%) and several anaerobic-thermophilic Clostridia- and Synergistetes-like bacteria (20%) were detected in the Chinese sample. Different thermophiles (12%) and Clostridia (2%) were detected in the Japanese sample.

DOI： 10.1271/bbb.80227

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

The specificity of 1,3-1,4-beta-glucanase from Synechocystis PCC6803 (SsGlc) was investigated using novel substrates 1,3-1,4-beta-glucosyl oligosaccharides, in which 1,3- and 1,4-linkages are located in various arrangements. After the enzymatic reaction, the reaction products were separated and determined by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). As a result, SsGlc was found to hydrolyze the pentasaccharides, which possess three contiguous 1,4-beta-glycosidic linkages (cellotetraose sequence) adjacent to 1,3-beta-linkage, but none of the other oligosaccharides were hydrolyzed. To further analyze the specificity, kinetic measurements were performed using polymeric substrates and 4-methylumbelliferyl derivatives of laminaribiose and cellobios e (1,3-beta-(Glc)(2)-MU and 1,4-beta-(Glc)(2)-MU). The k(cat)/K-m value obtained for barley beta-glucan was considerably larger than that for lichenan, indicating that SsGlc prefers 1,3-1,4-beta-glucan possessing a larger amount of cellotetraose sequence. This is consistent with the data obtained for 1,3-1,4-beta-glucosyl oligosaccharides. However, the k(cat)/K-m value obtained for 1,4-beta-(Glu)(2)-MU was considerably lower than that for 1,3-beta-(Glc)(2)-MU, suggesting inconsistency with the data obtained from the other natural substrates. It is likely that the kinetic data obtained from such chromophoric substrates do not always reflect the true enzymatic properties. (c) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.abb.2008.07.019

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

A beta-1,3-galactosyl-N-acetylhexosamine phosphorylase (GalGlyNAcP) homolog gene was cloned from Vibrio vulnificus CMCP6. In synthetic reactions, the recombinant enzyme acted only with GlcNAc and GalNAc as acceptors in the presence of alpha-D-galactose-1-phosphate as a donor to form lacto-N-biose I (LNB) (Gal beta 1 -&gt; 3GlcNAc) and galacto-N-biose (GNB) (Gal beta 1 -&gt; 3GalNAc), respectively. GlcNAc was a much better acceptor than GalNAc. The enzyme also phosphorolysed LNB faster than it phosphorolysed GNB, and the k(cat)/K(m) for LNB was approximately 60 times higher than the k(cat)/K(m) for GNB. This result indicated that the enzyme was remarkably different from GalGlyNAcP from Bifidobacterium longum, which has similar activities with LNB and GNB, and GalGlyNAcP from Clostridium perfringens, which is a GNB-specific enzyme. The enzyme is the first LNB-specific enzyme that has been found and was designated lacto-N-biose I phosphorylase. The discovery of an LNB-specific GalGlyNAcP resulted in recategorization of bifidobacterial GalGlyNAcPs as galacto-N-biose/lacto-N-biose I phosphorylases.

DOI： 10.1128/AEM.02846-07

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

A family-4 alpha-galactosidase Mel4A of Bacillus halodurans was expressed in Escherichia coli and characterized. Recombinant enzyme rMel4A depended on NAD(+), some divalent cations such as Mn2(+), and reducing reagents such as dithiothreitol. rMel4A was active on small saccharides such as raffinose but not on highly polymerized galactomannan. Immunological analysis indicated that raffinose induced the production of Mel4A in B. halodurans.

DOI： 10.1271/bbb.80242

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Breast-fed infants often have intestinal microbiota dominated by bifidobacteria in contrast to formula-fed infants. We found that several bifidobacterial strains produce a lacto-N-biosidase that liberates lacto-N-biose I (Gal beta 1,3GlcNAc; type 1 chain) from lacto-N-tetraose (Gal beta 1,3GlcNAc beta 1,3Gal beta 1,4G1c), which is a major component of human milk oligosaccharides, and subsequently isolated the gene from Bifidobacterium bifidum JCM1254. The gene, designated lnbB, was predicted to encode a protein of 1,112 amino acid residues containing a signal peptide and a membrane anchor at the N and C termini, respectively, and to possess the domain of glycoside hydrolase family 20, carbohydrate binding module 32, and bacterial immunoglobulin-like domain 2, in that order, from the N terminus. The recombinant enzyme showed substrate preference for the unmodified P-linked lacto-N-biose I structure. Lacto-N-biosidase activity was found in several bifidobacterial strains, but not in the other enteric bacteria, such as clostridia, bacteroides, and lactobacilli, under the tested conditions. These results, together with our recent finding of a novel metabolic pathway specific for lacto-N-biose I in bifidobacterial cells, suggest that some of the bifidobacterial strains are highly adapted for utilizing human milk oligosaccharides with a type I chain.

DOI： 10.1128/AEM.00149-08

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

GH94 cellobiose phosphorylase (CBP) catalyzes the phosphorolysis of cellobiose into alpha-D-glucose 1-phosphate (G1P) and D-glucose with inversion of anomeric configuration. The complex crystal structure of CBP from Cellvibrio gilvus had previously been determined; glycerol, glucose, and phosphate are bound to subsites -1, +1, and the anion binding site, respectively. We performed computational analyses to elucidate the conformational itinerary along the reaction pathway of this enzyme. AUTODOCK was used to dock cellobiose with its glycon glucosyl residue in various conformations and with its aglycon glucosyl residue in the low-energy C-4(1) conformer. An oxocarbenium ion-like glucose molecule mimicking the transition state was also docked. Based on the clustering analysis, docked energies, and comparison with the crystallographic ligands, we conclude that the reaction proceeds from S-1(3) as the pre-transition state conformer (Michaelis complex) via E-3 as the transition state candidate to C-4(1) as the GIP product conformer. The predicted reaction pathway of the inverting phosphorylase is similar to that proposed for the first-half glycosylation reaction of retaining cellulases, but is different from those for inverting cellulases. NAMD was used to simulate molecular dynamics of the enzyme. The S-1(3) pre-transition state conformer is highly stable compared with other conformers, and a conformational change from C-4(1) to B-1,B-4 was observed. (c) 2008 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.carres.2008.02.026

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Recently, a gene cluster involving a phosphorylase specific for lacto-N-biose I (LNB; Gal beta 1-3GlcNAc) and galacto-N-biose (GNB; Gal beta 1-3GalNAc) has been found in Bifidobacterium longum. We showed that the solute-binding protein of a putative ATP-binding cassette-type transporter encoded in the cluster crystallizes only in the presence of LNB or GNB, and therefore we named it GNB/LNB-binding protein (GL-BP). Isothermal titration calorimetry measurements revealed that GL-BP specifically binds LNB and GNB with K-d values of 0.087 and 0.010 mu M, respectively, and the binding process is enthalpy-driven. The crystal structures of GL-BP complexed with LNB, GNB, and lacto-N-tetraose ( Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc) were determined. The interactions between GL-BP and the disaccharide ligands mainly occurred through water-mediated hydrogen bonds. In comparison with the LNB complex, one additional hydrogen bond was found in the GNB complex. These structural characteristics of ligand binding are in agreement with the thermodynamic properties. The overall structure of GL-BP was similar to that of maltose-binding protein; however, the mode of ligand binding and the thermodynamic properties of these proteins were significantly different.

DOI： 10.1074/jbc.M709777200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS INC  

The tyrosine residue Y198 is known to support a nucleophilic water molecule with the general base residue, D263, in the reducing-end xylose-releasing exo-oligoxylanase (Rex). A mutation in the tyrosine residue changing it into phenylalanine caused a drastic decrease in the hydrolytic activity and a small increase in the F-releasing activity from alpha-xylobiosyl fluoride in the presence of xylose. In contrast, mutations at D263 resulted in the decreased F-releasing activity. As a result of the high F-releasing activity and low hydrolytic activity, Y198F of Rex accumulates a large amount of product during the glycosynthase reaction. We propose a novel method for producing a glycosynthase from an inverting glycoside hydrolase by mutating a residue that holds the nucleophilic water molecule with the general base residue while keeping the general base residue intact.

DOI： 10.1093/glycob/cwn011

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

A chitinase gene belonging to the glycoside hydrolase family 19 from Vibrio proteolyticus (chi19) was cloned. The recombinant enzyme (Chi19) showed weak activities against polymeric substrates and considerable activities against fully N-acetylated chitooligosaccharides, (GlcNAc)(n) , whose degree of polymerization was greater than or equal to five. It hydrolyzed (GlcNAc) (n) at the second linkage position from the reducing ends of the chitooligosaccharides. The hydrolytic products of colloidal chitin were mainly (GlcNAc)(2) from the initial stage of the reaction. The hydrolytic pattern of reduced colloidal chitin clearly suggested that the enzyme hydrolyzed the polymeric substrate from the reducing end.

DOI： 10.1007/s00253-008-1352-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Lacto-N-biose phosphorylase (LNBP) from bifidobacteria is involved in the metabolism of lacto-N-biose I (Gal beta 1 -&gt; 3GlcNAc, LNB) and galacto-N-biose (Gal beta 1 -&gt; 3GalNAc, GNB). A homologous gene of LNBP (CPF0553 protein) was identified in the genome of Clostridium perfringens ATCC13124, which is a gram-positive anaerobic intestinal bacterium. In the present study, we cloned the gene and compared the substrate specificity of the CPF0553 protein with LNBP from Bifidobacterium longum JCM1217 (LNBPBl). In the presence of alpha-galactose 1-phosphate (Gal 1-P) as a donor, the CPF0553 protein acted only on GlcNAc and GalNAc, and GalNAc was a more effective acceptor than GlcNAc. The reaction product from GlcNAc/GalNAc and Gal 1-P was identified as LNB or GNB. The CPF0553 protein also phosphorolyzed GNB much faster than LNB, which suggests that the protein should be named galacto-N-biose phosphorylase (GNBP). GNBP showed a k (cat)/K-m value for GNB that was approximately 50 times higher than that for LNB, whereas LNBPBl showed similar k(cat)/K-m values for both GNB and LNB. Because C. perfringens possesses a gene coding endo-alpha-N-acetylgalactosaminidase, GNBP may play a role in the intestinal residence by metabolizing GNB that is available as a mucin core sugar.

DOI： 10.1007/s00253-007-1319-8

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Language：Japanese   Publisher：日本応用糖質科学会  

DOI： 10.11541/jsag.2008.0.149.0

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Language：Japanese   Publisher：日本応用糖質科学会  

DOI： 10.11541/jsag.2008.0.105.0

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：日本応用糖質科学会  

DOI： 10.5458/jag.jag.JAG-2010_022

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Language：English   Publishing type：Part of collection (book)   Publisher：Elsevier Ltd.  

We found a novel inverting xylanolytic enzyme belonging to GH8, reducing end xylose-releasing exo-oligoxylanase (Rex, EC. 3.2.1.156), that hydrolyzed xylooligosaccharides (X3 or larger) to release X1 at their reducing end. Rex hydrolyzed α-X2F into X2 only in the presence of X1, clearly proving the Hehre-resynthesis hydrolysis mechanism. A library of mutant Rex at the catalytic base (Asp263) was constructed by saturation mutagenesis. Among them, D263C showed the highest level of X3 production, and D263N exhibited the fastest consumption of α-X2F. However, F- releasing activities of the mutants were much less than that of wild type. Next, Y198 of Rex that forms a hydrogen bond with the nucleophilic water was substituted with phenylalanine, causing a drastic decrease in the hydrolytic activity and a small increase in F- releasing activity from α-xylobiosyl fluoride in the presence of xylose. Y198F of Rex accumulates much more product during the glycosynthase reaction than D263C. We here conclude that an inverting glycosidase is effectively converted into glycosynthase by mutating a residue holding the nucleophilic water molecule with the general base residue while keeping the general base residue intact. © 2008 Woodhead Publishing Limited. All rights reserved.

DOI： 10.1533/9781845695750.2.193

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Language：Japanese   Publisher：The Japanese Society of Applied Glycoscience  

DOI： 10.11541/jsag.2008.0.107.0

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Language：Japanese   Publisher：The Japanese Society of Applied Glycoscience  

DOI： 10.11541/jsag.2008.0.137.0

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Language：Japanese   Publisher：The Japanese Society of Applied Glycoscience  

DOI： 10.11541/jsag.2008.0.106.0

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Language：Japanese   Publisher：The Japanese Society of Applied Glycoscience  

DOI： 10.11541/jsag.2008.0.147.0

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DOI： 10.11541/jsag.2008.0.19.0

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Language：English   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

DOI： 10.1016/j.ab.2007.07.012

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

We have determined the functions of the enzymes encoded by the lnpB, InpC, and InpD genes, located downstream of the lacto-N-biose phosphorylase gene (lnpA), in Bifidobacterium longum JCM1217. The lnpB gene encodes a novel kinase, N-acetylhexosamine 1-kinase, which produces N-acetylhexosamine I-phosphate; the InpC gene encodes UDP-glucose hexose 1-phosphate uridylyltransferase, which is also active on N-acetylhexosamine I-phosphate; and the lnpD gene encodes a UDP-glucose 4-epimerase, which is active on both UDPgalactose and UDP-N-acetylgalactosamine. These results suggest that the gene operon lnpABCD encodes a previously undescribed lacto-N-biose I/galacto-N-biose metabolic pathway that is involved in the intestinal colonization of bifidobacteria and that utilizes lacto-N-biose I from human milk oligosaccharides or galacto-N-biose from mucin sugars.

DOI： 10.1128/AEM.01425-07

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A recombinant galacto-N-biose-/lacto-N-biose I-binding protein (GL-BP) from Bifidobacterium longum JCM1217 has been prepared and crystallized by the hanging-drop vapour-diffusion method using 10 mg ml-1 purified enzyme, 0.01 M zinc sulfate, 0.1 M MES buffer pH 5.9-6.4 and 20-22%(v/v) PEG MME 550 in the presence of 5 mM disaccharide ligands. Suitable crystals grew after 10 d incubation at 293 K. The crystals belong to space group C2221, with unit-cell parameters a = 106.3, b = 143.6, c = 114.6 Å for the lacto-N-biose I complex and a = 106.4, b = 143.4, c = 115.5 Å for the galacto-N-biose complex, and diffracted to 1.85 and 1.99 Å resolution, respectively. © International Union of Crystallography 2007.

DOI： 10.1107/S1744309107036263

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

A one-pot enzymatic reaction to produce lacto-N-biose I (LNB), which is supposed to represent the bifidus factor in human milk oligosaccharides, was demonstrated. Approximately 500 mM of LNB was generated in 10-liter of reaction mixture initially containing 660 m,mM of sucrose and 600 mm of GlcNAc by the concurrent actions of four enzymes, sucrose phosphorylase, UDP-glucose-hexose-1-phospate uridylyltransferase, UDPglucose 4-epimerase, and lacto-N-biose phosphorylase, in the presence of UDP-Glc and phosphate, indicating a reaction yield of 83%. LNB was isolated from the mixture by crystallization after yeast treatment. Finally, 1.4 kg of LNB of 99.6% purity was recovered after recrystallization.

DOI： 10.1271/bbb.70320

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A putative endoglucanase gene corresponding to locus TM 1752 (AAD36817.1, Q9X274) of Thermotoga maritima MSB8 was cloned and expressed in Escherichia coli. The enzyme contains a NEP (Asn-Glu-Pro) motif but lacks a PCD (proposed catalytic domain) block. This endoglucanase is grouped under family 5 of the glycosyl hydrolases members of which predominantly hydrolyze β-1,4-linkages of carbohydrate polymers. The enzyme efficiently hydrolyzes the glycosidic bonds of mixed β-1,3-1,4 linkages in lichenan and barley β-glucan, but did not display any activity on crystalline cellulose or laminarin. However, negligible amount of hydrolysis was observed with CM-cellulose. This enzyme produced glucosyl β-1,3 glucosyl β-1,4 glucose as the major end product and glucosyl β-1,4 glucosyl β-1,3 glucose was not detected. The putative endoglucanase of T. maritima appears to be a unique endoglucanase being the first lichenase producing glucosyl β-1,3 glucosyl β-1,4 glucose to be placed into family 5 of the glucosyl hydrolases.

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Two regions in xylanase A from Bacillus halodurans C-125 (XynA), an alkaliphilic xylanase, were identified to be responsible for its activity at basic pH by comparing the dissociation constants of the XynA proton donor Glu residue (pKe2 and pKes2) with those of xylanase B from Clostridium stercorarium F9 (XynB) and their mutants constructed by substituting either Ser137/Asn127 of XynA/XynB or the 4th loop, designed based on the structural difference close to the proton donor. The substitution of XynB at Asn127 into Ser increased pKe2 by 0.37. The effect is explained that the positive charge of His126 likely affects the proton donor via Asnl27 and a water molecule in XynB, resulting in a decrease in pKe2, whereas such interactions were not observed with Ser. The substitution of XynB at the 4th loop into XynA (XynB Loop4A) increased the pKe2 and pKes2 values by 0.29 and 0.62, respectively. The effect of the 4th loop in XynA is likely due to a hydrogen bond between Asp 199 in the loop and Tyr239, which interacts with both the proton donors Glu195 and Arg204, with flexibility of the loop. Both the mutations independently affected the increases in pK e2. © 2007 The Japanese Biochemical Society.

DOI： 10.1093/jb/mvm072

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

The putative raffinose synthase gene from rice was cloned and expressed in Escherichia coli. The enzyme displayed an optimum activity at 45 degrees C and pH 7.0, and a sulfhydryl group was required for its activity. The enzyme was specific for galactinol and p-nitrophenyl-alpha-D-galactoside as galactosyl donors, and sucrose, lactose, 4-beta-galactobiose, N-acetyl-D-lactosamine, trehalose and lacto-N-biose were recognized as galactosyl acceptors.

DOI： 10.1007/s10529-006-9268-3

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：AMER CHEMICAL SOC  

Phosphorylases are one of the three main types of enzymes involved in the formation and cleavage of glycosyl linkages. They are expected to function as unique catalysts in the production of oligosaccharides, owing to their reactions being reversible and highly regio-specific. However, they have not been studied as extensively as the other two types, the hydrolases and synthases. Pairs of phosphorylases have been used to prepare oligosaccharides, including trehalose, cellobiose, and laminaribiose. New processes using phosphorylases to produce oligosaccharides with alpha-1,2 glucosyl linkage and amylose are reported.

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Two lacto-N-biose phosphorylase (LNBP) isozyme genes were cloned from Bifidobacterium bifidum JCM1254. Alignment of the amino acid sequences of LNBP and its homologs identified 24 completely conserved acidic amino acid residues. All single mutants of Bifidobacterium longum LNBP at residues other than D313N retained considerable activity, suggesting that Asp313 is the putative proton donor residue in LNBP.

DOI： 10.1271/bbb.70064

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DOI： 10.1038/nprot.2006.403

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Microbial Ecology  

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Cellobiose phosphorylase, a member of the glycoside hydrolase family 94, catalyses the reversible phosphorolysis of cellobiose into α-D-glucose 1-phosphate and D-glucose with inversion of the anomeric configuration. The substrate specificity and reaction mechanism of cellobiose phosphorylase from Cellvibrio gilvus have been investigated in detail. We have determined the crystal structure of the glucose-sulphate and glucose-phosphate complexes of this enzyme at a maximal resolution of 2.0 Å (1 Å = 0.1 nm). The phosphate ion is strongly held through several hydrogen bonds, and the configuration appears to be suitable for direct nucleophilic attack to an anomeric centre. Structural features around the sugar-donor and sugar-acceptor sites were consistent with the results of extensive kinetic studies. When we compared this structure with that of homologous chitobiose phosphorylase, we identified key residues for substrate discrimination between glucose and N-acetylglucosamine in both the sugar-donor and sugar-acceptor sites. We found that the active site pocket of cellobiose phosphorylase was covered by an additional loop, indicating that some conformational change is required upon substrate binding. Information on the three-dimensional structure of cellobiose phosphorylase will facilitate engineering of this enzyme, the application of which to practical oligosaccharide synthesis has already been established. © 2006 Biochemical Society.

DOI： 10.1042/BJ20060274

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When Phanerochaete chrysosporium was grown with laminarin (a beta-1,3/1,6-glucan) as the sole carbon source, a beta-1,3-glucanase with a molecular mass of 36 kDa was produced as a major extracellular protein. The cDNA encoding this enzyme was cloned, and the deduced amino acid sequence revealed that this enzyme belongs to glycoside hydrolase family 16; it was named Lam16A. Recombinant Lam16A, expressed in the methylotrophic yeast Pichia pastoris, randomly hydrolyzes linear beta-1,3-glucan, branched beta-1,3/1,6-glucan, and beta-1,3-1,4-glucan, suggesting that the enzyme is a typical endo-1,3(4)-beta-glucanase (EC 3.2.1.6) with broad substrate specificity for (3-1,beta-glucans. When laminarin and lichenan were used as substrates, Lam16A produced 6-O-glucosyl-laminaritriose (beta-D-Glcp-(1-&gt; 6)-(beta-D-Glcp-(1-&gt; 3)-(3-D-Glcp-(1-&gt; 3)-D-Glc) and 4-O-glucosyl-laminaribiose (beta-D-Glcp-(1-&gt; 4)-(3-D-Glcp(1-&gt; 3)-D-Glc), respectively, as one of the major products. These results suggested that the enzyme strictly recognizes (3-D-Glcp-(1-&gt; 3)-D-Glcp at subsites -2 and -1, whereas it permits 6-O-glucosyl substitution at subsite +1 and a beta-1,4-glucosidic linkage at the catalytic site. Consequently, Lam16A generates non-branched oligosaccharide from branched beta-1,3/1,6-glucan and, thus, may contribute to the effective degradation of such molecules in combination with other extracellular beta-1,3-glucanases.

DOI： 10.1007/s00253-005-0214-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Four xylanases belonging to glycoside hydrolase family 10-Thermotoga maritima XylB (TM), Clostridium stercorarium XynB (CS), Bacillus halodurans XynA (BH), and Cellulonumas fimi Cex (CF)-were converted to glycosynthases by substituting the nucleophilic glutamic acid residues with glycine, alanine, and serine. The glycine mutants exhibited the highest levels of glycosynthase activity with all four enzymes. All the glycine mutants formed polymeric beta-1,4-linked xylopyranose as a precipitate during reaction with alpha-xylobiosyl fluoride. Two glycine mutants (TM and CF) recognized X-2 as an effective acceptor molecule to prohibit the formation of the polymer, while the other two (CS and BH) did not. The difference in acceptor specificity is considered to reflect the difference in substrate affinity at their +2 subsites. The results agreed with the structural predictions of the subsite, where TM and CF exhibit high affinity at subsite 2, suggesting that the glycosynthase technique is useful for investigating the affinity of +subsites.

DOI： 10.1271/bbb.70.1210

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Although proteins may be artificially improved by random insertion and deletion mutagenesis methods, these procedures are technically difficult, and the mutations introduced are no more variable than those introduced by the introduction of random point mutations. We describe here a three-step method called RAISE, which is based on gene shuffling and can introduce a wide variety of insertions, deletions and substitutions. To test the efficacy of this method, we used it to mutate TEM β-lactamase to generate improved antibiotic resistance. Some unique insertion or deletion mutations were observed in the improved mutants, some of which caused higher activities than point mutations. Our findings indicate that the RAISE method can yield unique mutants and may be a powerful technique of protein engineering. © 2006 Oxford University Press.

DOI： 10.1093/nar/gnj032

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

Four derivatives of 2(11)-deoxycellobiose were synthesized from D-glucal and acceptor sugars (D-glucose, D-xylose, D-mannose, and 2-deoxy-D-arabino-hexose) using a cellobiose phosphorylase from Cellvibrio gilvus. The enzyme was found to be an effective catalyst to synthesize the beta-(1 -&gt; 4) linkage of 2-dcoxy-D-arabhio-hexopyranoside. The acceptor specificity for the D-glucal reaction was identical to that for the alpha-D-glucose 1-phosphate reaction, but the activity Of (D)-glucal was approximately 500 times less than that Of alpha-D-glucose 1-phosphate, using 10 mM substrates. (c) 2006 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.carres.2006.01.003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Reducing end xylose-releasing exooligoxylanase (Rex, EC 3.2.1.156) is an inverting GH that hydrolyzes xylooligosaccharides (&gt;= X-3) to release X-1 at their reducing end. The wild-type enzyme exhibited the Hehre resynthesis hydrolysis mechanism, in which alpha-X2F was hydrolyzed to X-2 and HF in the presence of X-1 as an acceptor molecule. However, the transglycosidation product (X-3) was not detectable in the reaction. To convert reducing end xylose-releasing exooligoxylanase to glycosynthase, derivatives with mutations in the catalytic base (Asp-263) were constructed by saturation random mutagenesis. Nine amino acid residue mutants (Asp-263 to Gly, Ala, Val, Thr, Leu, Asn, Cys, Pro, or Ser) were found to possess glycosynthase activity forming X-3 from alpha-X2F and X-1. Among them, D263C showed the highest level of X-3 production, and D263N exhibited the fastest consumption of alpha-X2F. The D263C mutant showed 10-fold lower hydrolytic activity than D263N, resulting in the highest yield of X-3. X-2 was formed from the early stage of the reaction of the D263C mutant, indicating that a portion of the X-3 formed by condensation was hydrolyzed before its release from the enzyme. To acquire glycosynthase activity from inverting enzymes, it is important to minimize the decrease in F--releasing activity while maximizing the decrease in the hydrolytic activity. The present study expands the possibility of conversion of glycosynthases from inverting enzymes.

DOI： 10.1074/jbc.M511202200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

A simple protocol to introduce random mutations, named error-prone rolling circle amplification (RCA), is described. A template plasmid is amplified by RCA in the presence of MnCl(2) and used for transformation of a host strain to give a mutant library with three to four random point mutations per kilobase throughout the entire plasmid. The prime advantage of this method is its simplicity. This protocol requires neither the design of specific primers nor the exploration of thermal cycling conditions. It takes just 10 min to prepare the reaction mixture, followed by overnight incubation and transformation of a host strain. This method permits rapid preparation of randomly mutated plasmid libraries, and will enable the wider adoption of random mutagenesis.

DOI： 10.1038/nprot.2006.403

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

A new carbohydrate synthesis method using a special hydroxy protecting group (uni-chemo protection = UCP) in both solution and solid phases was developed. The UCP group was comprised of polymerized amino acid derivatives. Each hydroxyl group was protected by a UCP group with a different degree of polymerization, which allowed them to be uniquely identified. To deprotect the UCP group, one cycle of Edman degradation was performed as follows: 1) removal of the amino protecting group; 2) phenyl isothiocyanate coupling; 3) removal of the N-terminal phenyl thiocarbamoyl mono-amino acid derivative by treatment with trifluoroacetic acid; and 4) re-protection of the newly exposed amino group with a Boc group. The hydroxyl groups were deprotected successively from the UCP group with the lowest to the highest degree of polymerization by repeating this Edman degradation cycle. First, commercially available N-alpha-t-Boc-sarcosine, N-alpha-t-Boc-N-alpha-methyl-L-alanine, and N-alpha-t-Boc-L-phenylglycine were examined as UCP groups. Despite successfully protecting and selectively deprotecting the hydroxyl groups, there were problems with stability or reactivity. To address these problems, N-alpha-1-ethylpropylglycine was chosen as the UCP group, and we successfully synthesized two sialyl-T antigen analogues. Tri-UCP and mono-UCP were attached to the 6- and 3-positions of the D-galactosamine (GalN) derivative, respectively, using cyanuric chloride. To selectively deprotect the mono-UCP group on the 3-position of the GalN derivative, one cycle of Edman degradation was performed. As a result of this cycle, the tri-UCP on the 6-position of the GalN derivative was degraded to di-UCP, and the mono-UCP on the 3-position was selectively deprotected to yield a GalN derivative with a free 3-position. Glycosylation of this selectively deprotected free 3-position GalN derivative with a suitably protected D-galactose (Gal) derivative in which the 3-position was protected by a mono-UCP group using N-iodosuccinimide (NIS) and trifluoromethanesulfonic acid (TfOH) in dichloromethane yielded the desired disaccharide in high yield in both a position- and stereoselective manner. By repeating one cycle of Edman degradation, the X-position-free disaccharide in which the 6-position was protected by a mono-UCP group was prepared selectively. Subsequent acetylation and a final cycle of Edman degradation (except the third and fourth steps) yielded the 6-position-free disaccharide selectively. Two disaccharides, one with a free X-position and another with a free 6-position, were coupled with a sialic acid donor using NIS and TfOH in acetonitrile to obtain sialyl (2 -&gt; 3) T and sialyl (2 -&gt; 6) T antigen derivatives, respectively. Solid-phase synthesis was demonstrated using polystyrene-type beads and a new linker, 2-{4 - (hydroxymethyl)benzamido) acetic acid (HMBA-Gly), to synthesize Galp (1 -&gt; 3) Gal from a suitably protected Gal derivative in which the 3-position was protected by mono-UCR Solid-phase glycosylation was successfully monitored by measuring the removal of the Fmoc group, which protected the amino group on UCP, similar to the method for monitoring solid-phase Fmoc peptide synthesis. This UCP hydroxy protecting group was suitable for solid-phase synthesis, and will be the key technique in both automated oligosaccharide synthesis and oligosaccharide library synthesis. ((c) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005).

DOI： 10.1002/ejoc.200500382

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

A lacto-N-biose phosphorylase (LNBP) was purified from the cell extract of Bifidobacterium bifidum. Its N-terminal and internal amino acid sequences were homologous with those of the hypothetical protein of Bifidobacterium longum NCC2705 encoded by the BL1641 gene. The homologous gene of the type strain B. longum JCM1217, lnpA, was expressed in Escherichia coli to confirm that it encoded LNBP. No significant identity was found with any proteins with known function, indicating that LNBP should be classified in a new family. The lnpA gene is located in a novel putative operon for galactose metabolism that does not contain a galactokinase gene. The operon seems to be involved in intestinal colonization by bifidobacteria mediated by metabolism of mucin sugars. In addition, it may also resolve the question of the nature of the bifidus factor in human milk as the lacto-N-biose structure found in milk oligosaccharides.

DOI： 10.1128/AEM.71.6.3158-3162.2005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

An arginine residue in loop 4 connecting beta strand 4 and et-helix 4 is conserved in glycoside hydrolase family 10 (GH10) xylanases. The arginine residues, Arg(204) in xylanase A from Bacillus halodurans C-125 (XynA) and Arg(196) in xylanase B from Clostridium stercorarium F9 (XynB), were replaced by glutamic acid, lysine, or glutamine residues (XynA R204E, K and Q, and XynB R196E, K and Q). The pH-k(cat)/K-m and the pH-k(cat) relationships of these mutant enzymes were measured. The pK(e2) and pK(es2) values calculated from these curves were 8.59 and 8.29 (R204E), 8.59 and 8.10 (R204K), 8.61 and 8.19 (R204Q), 7.42 and 7.19 (R196E), 7.49 and 7.18 (R196K), and 7.86 and 7.38 (R196Q) respectively. Only the pK(es2) value of arginine derivatives was less than those of the wild types (8.49 and 9.39 [XynA] and 7.62 and 7.82 [XynB]). These results suggest that the conserved arginine residue in GH10 xylanases increases the pK(a) value of the proton donor Glu during substrate binding. The arginine residue is considered to clamp the proton donor and subsite +1 to prevent structural change during substrate binding.

DOI： 10.1271/bbb.69.904

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Reducing end xylose-releasing exo-oligoxylanase from Bacillus halodurans C-125 (Rex) hydrolyzes xylooligosaccharides whose degree of polymerization is greater than or equal to 3, releasing the xylose unit at the reducing end. It is a unique exo-type glycoside hydrolase that recognizes the xylose unit at the reducing end in a very strict manner, even discriminating the beta-anomeric hydroxyl configuration from the alpha-anomer or 1-deoxyxylose. We have determined the crystal structures of Rex in unliganded and complex forms at 1.35 - 2.20-angstrom resolution and revealed the structural aspects of its three subsites ranging from - 2 to + 1. The structure of Rex was compared with those of endo-type enzymes in glycoside hydrolase subfamily 8a (GH-8a). The catalytic machinery of Rex is basically conserved with other GH-8a enzymes. However, subsite + 2 is blocked by a barrier formed by a kink in the loop before helix alpha(10). His-319 in this loop forms a direct hydrogen bond with the beta-hydroxyl of xylose at subsite + 1, contributing to the specific recognition of anomers at the reducing end.

DOI： 10.1074/jbc.M413693200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We describe a novel method of PCR-mediated mutagenesis employing DNA containing a natural abasic site and translesional Taq DNA polymerase. This method incorporated an adenine (80.8%) or guanine (7.7%) residue or led to a base deletion mutation (11.2%) opposite the abasic site. We conclude that the combination of DNA containing an abasic site and translesional Taq DNA polymerase is an easy and useful technique for PCR-mediated mutagenesis, having advantages different from those of conventional error-prone PCR. (C) 2004 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.biotec.2004.10.016

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

The reducing-end xylose-releasing wxo-oligoxylanase (Rex) from Bacillus halodurans C-125, a novel family GH8 glycoside hydrolase, was crystallized by the hanging-drop vapour-diffusion method using 13.6 mg ml(-1) purified Rex, 5.6% (v/v) polyethylene glycol 4000, 70 mM sodium acetate pH 4.6 and 30% (v/v) glycerol. Suitable crystals grew after incubation for 5 d at 293 K. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 52.69, b = 86.02, c = 87.92 angstrom. X-ray diffraction data were collected at a resolution of 1.35 angstrom.

DOI： 10.1107/S1744309105003635

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The gene encoding family 8 glycoside hydrolases from Bacillus halodurans C-125 (BH2105), an alkalophilic bacterium with a known genomic sequence, was expressed in Escherichia coli. The protein was expressed with the intact N-terminal sequence, suggesting that it did not possess a signal peptide and that it was an intracellular enzyme. The recombinant enzyme showed no hydrolytic activity on xylan, whereas it had been annotated as xylanase Y. It hydrolyzed xylooligosaccharide whose degree of polymerization is greater than or equal to 3 in an exosplitting manner with anomeric inversion, releasing the xylose unit at the reducing end. Judging from its substrate specificity and reaction mechanism, we named the enzyme reducing end xylose-releasing exo-oligoxylanase (Rex). Rex was found to utilize only the beta-anomer of the substrate to form beta-xylose and alpha-xylooligosaccharide. The optimum pH of the enzymatic reaction (6.2-7.3) was found in the neutral range, a range beneficial for intracellular enzymes. The genomic sequence suggests that B. halodurans secretes two endoxylanases and possesses two alpha-arabinofuranosidases, one alpha-glucuronidase, and three beta-xylosidases intracellularly in addition to Rex. The extracellular enzymes supposedly hydrolyze xylan into arabino/glucurono-xylooligosaccharides that are then transported into the cells. Rex may play a role as a key enzyme in intracellular xylan metabolism in B. halodurans by cleaving xylooligosaccharides that were produced by the action of other intracellular enzymes from the arabino/glucurono-xylooligosaccharides.

DOI： 10.1074/jbc.M409832200

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To elucidate the interaction between substrate inhibition and substrate transglycosylation of retaining glycoside hydrolases (GHs), a steady-state kinetic study was performed for the GH family 3 glucan (1--&gt;3)-beta-glucosidase from the white-rot fungus Phanerochaete chrysosporium, using laminarioligosaccharides as substrates. When laminaribiose was incubated with the enzyme, a transglycosylation product was detected by thin-layer chromatography. The product was purified by size-exclusion chromatography, and was identified as a 6-O-glucosyl-laminaribiose (beta-D-Glcp-(1--&gt;6)-beta-D-Glcp-(1 --&gt; 3)-D-Glc) by H-1 NMR spectroscopy and electrospray ionization mass spectrometry analysis. In steady-state kinetic studies, an apparent decrease of laminaribiose hydrolysis was observed at high concentrations of the substrate, and the plots of glucose production versus substrate concentration were thus fitted to a modified Michaelis-Menten equation including hydrolytic and transglycosylation parameters (K-m, K-m2, k(cat), k(cat2)). The rate of 6-0-glucosyl-laminaribiose production estimated by high-performance anion-exchange chromatography coincided with the theoretical rate calculated using these parameters, clearly indicating that substrate inhibition of this enzyme is fully explained by substrate transglycosylation. Moreover, when K-m k(cat), and affinity for glucosyl-enzyme intermediates (K-m2) were estimated for laminarioligosaccharides (DP = 3-5), the K-m value of laminaribiose was approximately 5 9 times higher than those of the other oligosaccharides (DP = 3-5), whereas the Km2 values were independent of the DP of the substrates. The kinetics of transglycosylation by the enzyme could be well interpreted in terms of the subsite affinities estimated from the hydrolytic parameters (K-m and k(cat)), and a possible mechanism of transglycosylation is proposed. (C) 2004 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.carres.2004.09.019

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL MUNKSGAARD  

A recombinant cellobiose phosphorylase from Cellvibrio gilvus has been prepared and crystallized by the sitting-drop vapour-diffusion method using 10 mg ml(-1) purified enzyme, 1.5 M ammonium sulfate, 0.1 M MES buffer pH 7.0 and 5 mM glucose. A suitable crystal was obtained after 10 d incubation at 298 K. The crystal belongs to space group P2(1), with unit-cell parameters a = 84.77, b = 98.31, c = 104.04 Angstrom, beta = 102.73degrees. X-ray diffraction data to 2.1 Angstrom resolution have been collected at KEK-PF BL-5A.

DOI： 10.1107/S0907444904017767

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The hydrolytic reaction of family 18 chitinase has been considered to occur via substrate assisted catalysis. To kinetically investigate the enzyme reaction mechanism, we synthesized compounds designed to reduce the polarization of the carbonyl in N acetyl group, GlcNAc-GlcN(TFA)-UMB (2) and GlcNAc-GlcN(TAc)-UMB (3). Kinetic parameters in the hydrolysis of these compounds by chitinase A from Serratia marcescens (ChiA) were compared with those from the hydrolysis of (GlcNAC)(2)-UMB (1). The k(cat) of 2 was 3.4% of 1, but the K-m of 2 was 10-fold that of 1. In contrast, the k(cat) of 3 was only 0.3%, of that of 1, and the two reactions had an identical K-m. The drastic decreases in k(cat) were probably due to the weak nucleophilic activity of the C2-N-trifluoroacetamide and N-thioacetamide groups at reducing ends of compounds 2 and 3, respectively. These results indicate that the anchimeric assistance of the C2 N-acetamide group at GlcNAc plays a key role in the hydrolytic reactions catalyzed by family 18 chitinases. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2004.05.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Vibrio proteolyticus chitobiose phosphorylase (ChBP) belongs to glycosyl transferase family 36 (GT-36), and catalyzes the reversible phosphorolysis of chitobiose into alpha-GlcNAc-1-phosphate and GlcNAc with inversion of the anomeric configuration. As the first known structures of a GT-36 enzyme, we determined the crystal structure of ChBP in a ternary complex with GlcNAc and SO(4). It is also the first structures of an inverting phosphorolytic enzyme in a complex with a sugar and a sulfate ion, and reveals a pseudo-ternary complex structure of enzyme-sugar-phosphate. ChBP comprises a beta sandwich domain and an (alpha/alpha)(6) barrel domain, constituting a distinctive structure among GT families. Instead, it shows significant structural similarity with glycoside hydrolase (GH) enzymes, glucoamylases (GH-15), and maltose phosphorylase (GH-65) in clan GH-L. The structural similarity reported here, together with distant sequence similarities between ChBP and GHs, led to the reclassification of family GT-36 into a novel GH family, namely GH-94.

DOI： 10.1016/j.str.2004.03.027

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

The function of the non-homologous region of a family 3 beta-glucosidase from Agrobacterium tumefaciens (Cbg1) was studied by analyzing the properties of mutant enzymes that have internal truncated amino acid sequences in the region. Five truncated mutants named Cbg1-d4, Cbg1-d31, Cbg1-d62, Cbg1-d89, and Cbg1-d119 having deletions of 4, 31, 62, 89, and 119 amino acid residues starting from Phe417, respectively, were expressed in Escherichia coli and purified. All the mutants exhibited beta-glucosidase activity, indicating that the non-homologous region was not essential for the activity. The truncation caused thermal instability, decrease in pK(a) of the proton donor residue (Glu616), and deficient transglycosylation activity. The thermal stability and the pKa of Glu616 were partially recovered with longer truncation, suggesting that the truncation perturbed the structure and that their presence in the region was not essential. The main role of the non-homologous region could be formation of a hydrophobic atmosphere at the acceptor site to make the enzyme suitable for hydrolyzing hydrophobic glucosides.

DOI： 10.1271/bbb.68.1113

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

A novel intracellular cycloalternan-degrading enzyme (CADE) was purified to homogeneity from the cell pellet of Bacillus sp. NRRL B-21195. The enzyme has a molecular mass of 125 kDa on SDS-PAGE. The pH optimum was 7.0. and the enzyme was stable from pH 6.0 to 9.2. The temperature optimum was 35degreesC and the enzyme exhibited stability up to 50degreesC. The enzyme hydrolyzed cycloalternan [CA; cyclo{--&gt; 6)-alpha-D-Glcp-(1 --&gt; 3)-alpha-D-Glcp-(1--&gt;6)-alpha-D-Glcp-(--&gt;3)alpha-D-Glcp-(1--&gt;}] as the best substrate. to produce only isomaltose via an intermediate, alpha-isomaltosyl-(l --&gt; 3)-isomaltose. This enzyme also hydrolyzed isomaltosyl substrates. such as panose, alpha-isomaltosyl-(1--&gt;4)-maltooligosaccharides, alpha-isomaltosyl-(1--&gt;3)-glucose, and alpha-isomaltosyl-(1 --&gt; 3)-isomaltose to liberate isomaltose. Neither maltooligosaccharides nor isomaltooligosaccharides were hydrolyzed by the enzyme, indicating that CADE requires alpha-isomaltosyl residues connected with (1--&gt;4)- or (1--&gt;3)-linkages. The K-m value of cycloalternan (1.68 mM) was 20% of that of panose (8.23 mM). The k(cat) value on panose (14.4 s(-1)) was not significantly different from that of cycloalternan (10.8s(-1)). Judging from its specificity, the systematic name of the enzyme should be cycloalternan isomaltosylhydrolase. This intracellular enzyme is apparently involved in the metabolism of starch via cycloalternan in Bacillus sp. NRRL B-21195. its role being to hydrolyze cycloalternan inside the cells. (C) 2004 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.carres.2004.02.008

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

A new hydroxy protecting group for convenient preparation of oligosaccharide was developed using uni-chemo Protection (UCP) concept. The UCP group was comprised of polymerized amino acid derivatives and protecting each hydroxyl groups by ester linkage. Depending on the polymerization degree, the hydroxyl groups were characterized and controlled. Using this protecting group, two kinds of sialyl-T analogues were Successfully synthesized from same sugar parts merely by repeating Edman degradation and coupling. (C) 2004 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.tetlet.2004.02.032

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL MUNKSGAARD  

Recombinant mature xylanase B from Clostridium stercorarium has been prepared and crystallized by the sitting-drop vapour-diffusion method using 4 mg ml(-1) purified enzyme, 10.3%(w/v) polyethylene glycol 1500, 8.6%(v/v) glycerol and 0.34 M non-detergent sulfobetaine 195. A suitable crystal grew after incubation for ten weeks at 293 K. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 64.76, b = 96.60, c = 138.44 Angstrom. X-ray diffraction data were collected to 1.80 Angstrom resolution.

DOI： 10.1107/S090744490302715X

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A family 36 glycosyltransferase gene was cloned from Vibrio proteolyticus. The deduced amino acid sequence showed a high degree of identity with ChBP (chitobiose phosphorylase) from another species, Vibrio furnissii. The recombinant enzyme catalysed the reversible phosphorolysis of (GlcNAc) 2 (chitobiose) to form 2-acetamide-2-deoxy-α-D-glucose 1-phosphate [GlcNAc-1-P] and GlcNAc, but showed no activity on cellobiose, indicating that the enzyme was ChBP, not cellobiose phosphorylase. In the synthetic reaction, the ChBP was active with α-D-glucose 1-phosphate as the donor substrate as well as GlcNAc-1-P to produce β-D-glucosyl-(1 → 4)-2-acetamide-2-deoxy-D-glucose with GlcNAc as the acceptor substrate. The enzyme allowed aryl-β-glycosides of GlcNAc as the acceptor substrate with 10-20% activities of GlcNAc. Kinetic parameters of (GlcNAc)2 in the phosphorolysis and GlcNAc-1-P in the synthetic reaction were determined as follows: phosphorolysis, k0 = 5.5 s-1, Km = 2. 0 mM
synthetic reaction, k0 = 10 s-1, Km = 14 mM, respectively. The mechanism of the phosphorolytic reaction followed a sequential Bi Bi mechanism, as frequently observed with cellobiose phosphorylases. Substrate inhibition by GlcNAc was observed in the synthetic reaction. The enzyme was considered a unique biocatalyst for glycosidation.

DOI： 10.1042/BJ20031171

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DOI： 10.1093/nass/48.1.225

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

In vitro random mutagenesis is a powerful tool for altering properties of enzymes. We describe here a novel random mutagenesis method using rolling circle amplification, named error-prone RCA. This method consists of only one DNA amplification step followed by transformation of the host strain, without treatment with any restriction enzymes or DNA ligases, and results in a randomly mutated plasmid library with 3-4 mutations per kilobase. Specific primers or special equipment, such as a thermal-cycler, are not required. This method permits rapid preparation of randomly mutated plasmid libraries, enabling random mutagenesis to become a more commonly used technique.

DOI： 10.1093/nar/gnh147

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A putative alpha-glucosidase belonging to glycosyl hydrolase family 4 of Thermotoga maritima (TM0752) was expressed in Escherichia coli and it was found that the recombinant protein (Agu4B) was a p-nitrophenyl alpha-D-glucuronopyranoside hydrolyzing alpha-glucuronidase, not alpha-glucosidase. It did not hydrolyze 4-O-methyl-D-glucuronoxylan or its fragment oligosaccharides. Agu4B was thermostable with an optimum temperature of 80degreesC. It strictly required Mn2+ and thiol compounds for its activity. The presence of NAD(+) slightly activated the enzyme. The amino acid sequence of Agu4B showed higher identity with Agu4A (another alpha-glucuronidase of T. maritima, 61%) than with AglA (alpha-glucosidase of T. maritima, 48%).

DOI： 10.1271/bbb.67.2359

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Cycloalternan-forming enzyme (CAFE) was first described as the enzyme that produced cycloalternan from alternan. In this study, we found that a partially purified preparation of CAFE containing two proteins catalyzed the synthesis of cycloalternan from maltooligosaccharides, whereas the purified CAFE alone was unable to do so. In addition to the 117 kDa CAFE itself, the mixture also contained a 140 kDa protein. The latter was found to be a disproportionating enzyme (DE) that catalyzes transfer of a D-glucopyranosyl residue from the non-reducing end of one maltooligosaccharide to the non-reducing end of another, forming an isomaltosyl residue at the non-reducing end. CAFE then transfers the isomaltosyl residue to the non-reducing end of another isomaltosyl maltooligosaccharide, to form an alpha-isomaltosyl-(1--&gt;3)-alpha-isomaltosyl-(1--&gt;4)-maltooligosaccharide, and subsequently catalyzes a cyclization to produce cycloalternan. Thus, DE and CAFE act synergistically to produce cycloalternan directly from maltodextrin or starch. (C) 2003 Elsevier Ltd. All rights reserved.

DOI： 10.1016/S0008-6215(03)00375-6

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Enzymatic synthesis was attempted of six trisaccharides and 14 tetrasaccharides comprising beta-(1--&gt;4)-linked D-glucose and D-xylose residues, using cellodextrin phosphorylase (CDP, EC 2.4.1.49) as the enzyme catalyst, with alpha-D-glucose 1-phosphate (1) alpha-D-xylose 1-phosphate (2) as the donor substrates, and cellobiose (3), xylobiose (4), betaGlc-(l--&gt;4)-Xyl (5), or betaXyl-(1--&gt;4)-Glc (6) as the acceptor substrates. All enzymatic reactions were performed at pH 7.0 and the products purified by gel-filtration chromatography. We successfully synthesized all six hetero-trisaccharides and 10 of the 14 possible hetero-tetrasaccharides. It was not found possible to synthesize the four tetrasaccharides with a Xyl--&gt;Glc sequence at their non-reducing ends employing this method. The stereochemistries of the isolated products were assessed by analysis of their 2D NMR spectra (DQF-COSY, TOCSY, HSQC, HMBC). confirming that all of the glycosidic bonds in the products were beta-(1--&gt;4) linkages. (C) 2003 Elsevier Ltd. All rights reserved.

DOI： 10.1016/S0008-6215(03)00314-8

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A family 2b carbohydrate-binding module from Streptomyces thermoviolaceus STX-II was fused at the carboxyl-terminus of XynB, a thermostable and single domain family 10 xylanase from Thermotoga maritima, to create a chimeric xylanase. The chimeric enzyme (XynB-CBM2b) was purified and characterized. It displayed a pH-activity profile similar to that of XynB and was stable up to 90degreesC. XynB-CBM2b bound to insoluble birchwood and oatspelt xylan. Whereas its hydrolytic activities toward insoluble xylan and p-nitrophenyl-p-xylopyranoside were similar to those of XynB, its activity toward soluble xylan was moderately higher than that of XynB. (C) 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793(03)00803-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

To examine the accessibility of the enzyme cellodextrin phosphorylase (CDP) towards multivalent cluster carbohydrates, the cellobiosylated dimer 10 and trimer 12, as well as the cellobiose-coated PAMAM dendrimers 14, 16, 18, 20 and 22, with four, eight, sixteen, thirty-two and sixty-four cellobiose units at the outer surface of PAMAM dendrimers, respectively, have been synthesized for the first time and used as acceptor substrates for the enzyme CDP. It was found that CDP was able to transfer a glucosyl moiety from glucose-1-phosphate (Glc-1-P) into these synthesized cluster cellobiosylated glycoconjugates and cellobiose-coated PAMAM dendrimers, which were thus acceptor substrates for CDP. It was found that the ability of CDP to interact with smaller cellobiosylated glyconjugates and with PAMAM dendrimers containing up to eight cellobiose units was similar to that seen with cellobiose. However, this capability of CDP was somewhat lessened with the PAMAM dendrimer containing sixteen cellobiose moieties and dramatically decreased towards PAMAM dendrimers with thirty-two and sixty-four cellobiose units. This might be due to their steric bulk, CDP enzyme no longer being able to hold them properly on its active site. ((C) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003).

DOI： 10.1002/ejoc.200300018

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

We found that the N,6-O-diacetylmuramidase from Streptomyces globisporus (M-1) hydrolyzed the cell walls from Micrococcus lysodeikticus and Staphylococcus aureus. In contrast, hen egg white lysozyme (HEWL) was only able to hydrolyze the cell walls from M. lysodeikticus. 6-O-Acetylation of the muramoyl moieties, as found in the S. aureus cell walls, did not inhibit the activity of the M-1 enzyme whereas it was sufficient to inhibit HEWL. The disaccharide GlcNAc-MurNAc was not observed in the M. lysodeikticus cell wall hydrolyzate produced by the M-1, indicating that M-1 acts on the MurNAc moiety which are linked by peptides at the lactyl groups of the MurNAc moiety. M-1 displays both N-acetylmuramidase and N,6-O-diacetylmuramidase activity and has a different substrate specificity from HEWL.

DOI： 10.1263/jbb.95.313

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Pro-aminopeptidase processing protease (PA protease) is a thermolysin-like metalloprotease produced by Aeromonas caviae T64. The N-terminal propeptide acts as an intramolecular chaperone to assist the folding of PA protease and shows inhibitory activity toward its cognate mature enzyme.. Moreover, the N-terminal propeptide strongly inhibits the autoprocessing of the C-terminal propeptide by forming a complex with the folded intermediate pro-PA protease containing the C-terminal propeptide (MC). In order to investigate the structural determinants within the N-terminal propeptide that play a role in the folding, processing, and enzyme inhibition of PA protease, we constructed a chimeric pro-PA protease by replacing the N-terminal propeptide with that of vibriolysin, a homologue of PA protease. Our results indicated that, although the N-terminal propeptide of vibriolysin shares only 36% identity with that of PA protease, it assists the refolding of MC, inhibits the folded MC to process its C-terminal propeptide, and shows a stronger inhibitory activity toward the mature PA protease than that of PA protease. These results suggest that the N-terminal propeptide domains in these thermolysin-like proteases may have similar functions, in spite of their primary sequence diversity. In addition, the conserved regions in the N-terminal propeptides of PA protease and vibriolysin may be essential for the functions of the N-terminal propeptide. (C) 2003 Elsevier Science (USA). All rights reserved.

DOI： 10.1016/S0006-291X(03)00084-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

Kinetic analyses of the hydrolysis reactions of N-acetylated and N-deacetylated derivatives of 4-methylumbelliferyl chitobioside [(GlcNAc)(2)-UMB (1), GlcN-GlcNAc-UMB (2), GlcNAc-GlcN-UMB (3), and (GlcN)(2)-UMB (4)] by ChiA and ChiB from Serratia marcescens were performed. Both enzymes released UMB from all compounds apart from 4. The S-v curves of the hydrolyses of I by ChiA and ChiB both exhibited atypical kinetic patterns, and the shapes of the two S-v curves were different from one another. However, both curve shapes were explained by assuming some of the enzyme present formed complexes with multiple molecules of the substrate. Conversely, the S-v curves generated in the cleavage of 2 and 3 by ChiA exhibited typical Michaelis-Menten profiles. Both enzymes hydrolysed 2 with an approximately 14-fold higher K-m value relative to 1, indicating that the N-acetyl group was recognised at the -2 subsite. The k(cat) value obtained with ChiA was identical to the k(cat) value observed for 1. However, the k(cat) value for ChiB was one-fourth that of 1, suggesting that the removal of the N-acetyl group caused an increase in the formation of a non-productive ES-complex. ChiA and ChiB hydrolysed 3 with 5- and 20-fold greater K-m values relative to 1, respectively, and 60- and 30-fold smaller k(cat) values relative to 1, respectively. The reaction mechanism of family 18 chitinases is discussed based upon the results obtained from the hydrolysis of these compounds.

DOI： 10.1093/jb/mvg031

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The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80degreesC. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70degreesC in the pH range of 6-8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and alpha-D-glucose-1-phosphate. The K-m for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the k(cat) was 5.4 s(-1). In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-beta-D-glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s(-1)) k(cat) followed by 6-deoxy-D-glucose (17 s(-1)) and 2-deoxy-D-glucose (16 s(-1)). The natural substrate, D-glucose with the k(cat) of 8.0 s(-1) had the highest (1.1x10(4) M-1 s(-1)) k(cat)/K-m compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, alpha-D-glucose-1-phosphate, at higher concentrations.

DOI： 10.1271/bbb.66.2578

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The family 3 beta-glucosidase from Thermotoga maritima is a highly thermostable enzyme (85degreesC) that displays transglycosylation activity. In contrast, the beta-glucosidase from Cellvibrio gilvus is mesophilic (35degreesC) and displays no such transglycosylation activity. Both enzymes consist of two domains, an N-terminal and a C-terminal domain, and the amino acid identities between the two enzymes in these domains are 32.4 and 36.4%, respectively. In an attempt to identify the molecular basis underpinning the display of transglycosylation activity and the requirements for thermal stability, eight chimeric genes were constructed by shuffling the two parental beta-glucosidase genes at four selected borders, two in the N-terminal domain and two in the C-terminal domain. Of the eight chimeric genes constructed, only two chimeric enzymes (Tm578/606Cg and Tm638/666Cg) gave catalytically active forms and these were the ones shuffled in the C-terminal domain. For these active chimeric enzymes, 80%, (Tm578/606Cg) and 88% (Tm638/666Cg) of their amino acid sequences originated from T. maritima. With regard to their thermal profiles, the two active chimeric enzymes, Tm578/606Cg and Tm638/666Cg, displayed profiles intermediate to those of the two parental enzymes as they were optimally active at 65 and 70degreesC, respectively. These two chimeric enzymes were optimally active at pH 4.1 and 3.9, which is closer to that observed for the T. maritima enzyme (pH 3.2-3.5) than that for the C. gilvus enzyme (pH 6.2-6.5). Kinetic parameters for the chimeric enzymes were investigated with five different substrates including pNP-beta-D-glucopyranoside. The kinetic parameters obtained for the chimeric enzymes were closer to those of the T. maritima enzyme than to those of the C. gilvus enzyme. Transglycosylation activity was observed for both chimeric enzymes and the activity of the Tm578/606Cg chimera was at a level twice that observed with the T. maritima enzyme. This study is an effective demonstration of the usefulness of chimeric enzymes in altering the characteristics of an enzyme. (C) 2002 Elsevier Science (USA). All rights reserved.

DOI： 10.1016/S0003-9861(02)00470-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

The xylanase A (XynA) from the alkaliphilic Bacillus halodurans C-125 and the xylanase B (XynB) from Clostridium stercorarium F9 were subdivided into four fragments at highly homologous regions present in their primary structures: an amino-terminal region (A or a), a region containing the putative proton donor (P or p), a region containing the putative catalytic nucleophile (N or n), and a carboxyl-terminal region (C or c). Six chimeric xylanases were constructed by the selective substitution of the four fragments using an overlapping PCR technique. Two of the six xylanases, APnc and Apnc (regions originating from XynA are denoted by upper case letters and those from XynB are denoted by lower case letters), were produced in Escherichia coli while the other four xylanases were obtained only as inclusion bodies. The APnc and Apnc chimeric enzymes were purified by column chromatography using Ni-NTA agarose and DEAE-Toyopearl. The respective pH and temperature stabilities of the purified enzymes were observed from pH 5.6 to 11.6 and up to 45degreesC for APnc, and from pH 5.6 to 11.2 and up to 45degreesC for Apnc. Thus, these enzymes were slightly less stable than the parental xylanases. An assessment of the pH-activity relationships for the chimeric xylanases employed p-nitrophenyl-beta-D-xylobioside as the substrate in determinations of the k(cat) values. The pK(a1) values for the APnc and Apnc chimeric enzymes were 4.3 and 4.2, respectively, which were almost identical to those for the parental xylanases. In contrast, the pK(a2) values obtained for APnc and Apnc were 9.1 and 8.5, respectively; these values fall between those for the parental xylanases, XynA (9.4) and XynB (7.8). These results indicate that the main regions necessary to maintain the high pK(a2) value of XynA locate in the A and P sections.

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A cellobiose phosphorylase (CBP) cloned from the Clostridium thermocellum YM4 strain was purified to homogeneity, and the reaction mechanisms of both the phosphorolytic and synthetic reactions were studied in detail. The enzyme reaction proceeded via an ordered bi bi mechanism, in which P-i bound to the enzyme prior to D-cellobiose and then G 1-P was released after D-glucose. The order of substrate binding was different from that of CBP from Cellvibrio gilvus, which bound to cellobiose prior to P-i. In the synthetic reaction, the enzyme showed three times higher activity with beta-D-glucose than with alpha-D-glucose, and also showed weak activity with 1,5-anhydro-D-glucitol, indicating that the beta-anomeric hydroxyl group of D-glucose is highly required. However, even when it is removed enzyme activity remains. The substrate specificity and kinetic studies revealed that the configurations of the C3 and C4 hydroxyl groups were strictly required for the enzyme activity, whereas those of C2 and C6 could be substituted or deleted. The mechanism of substrate inhibition by D-glucose was studied in detail and it was concluded that D-glucose competed with G 1-P for its binding site in the synthetic reaction.

DOI： 10.1093/oxfordjournals.jbchem.a003210

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Pro-aminopeptidase processing protease (PA protease) is an extracellular zinc metalloprotease produced by Aeromonas caviae T-64 and it is classified as M04.016 according to the MEROPS database. The precursor of PA protease consists of four regions; a signal peptide, an N-terminal propeptide, a C-terminal propeptide, and the mature PA protease. The in vitro refolding of the intermediate pro-PA protease containing the C-terminal propeptide (MC) was investigated in the presence and absence of the N-terminal propeptide. The results indicate that the noncovalently linked N-terminal propeptide is able to assist in the refolding of MC. In the absence of the N-terminal propeptide, MC is trapped into a folding competent state that is converted into the active form by the addition of the N-terminal propeptide. Moreover, the N-terminal propeptide was found to form a complex with the folded MC and inhibit further processing of MC into the mature PA protease. Inhibitory activity of the purified N-terminal propeptide toward mature PA protease was also observed, and the mode of this inhibition was determined to be a mixed, noncompetitive inhibition with an associated allosteric effect. (C) 2002 Elsevier Science (USA). All rights reserved.

DOI： 10.1016/S0006-291X(02)00838-0

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Language：English   Publisher：The Japanese Society of Applied Glycoscience  

Two intracellular enzymes, cellobiose phosphorylase (CBP) and cellodextrin phosphorylase (CDP) are involved in the phosphorolytic pathway in cellulose degradation. Those enzymes are considered to be useful in syntheses of oligosaccharides because the reactions are reversible. CBP from Cellvibrio gilvus and Clostridium thermocellum YM4, and CDP from C. thermocellum YM4 were cloned and over-expressed in Escherichia coli. All the three enzymes showed ordered bi bi mechanism. However the orders of the substrate binding of the CBPs were different. It was found that CBP from C. gilvus strictly recognized the hydroxyl groups at positions β-1, 3, and 4 of the acceptor molecule in the reverse reaction. On the other hand, the recognition of the hydroxyl groups at positions 2 and 6 was not so strict. Three branched β-1, 4-glucosyl trisaccharides were synthesized by using the reverse reaction of C. gilvus CBP. A new substrate inhibition pattern, competitive substrate inhibition, was also found in the reverse reaction of CBP using glucose as the acceptor. Specific colorimetric quantification of cellobiose was designed by using the reaction of CBP. Cellobiose was produced from sucrose at 90% yield by a combined action of three enzymes including CBP.

DOI： 10.5458/jag.49.221

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The gene (agu) encoding p-nitrophenyl alpha-D-glucuronopyranoside (pNP-GUA) hydrolyzing alpha-glucuronidase of the hyperthermophilic bacterium Thermotoga maritima was cloned and expressed in Escherichia coli. The recombinant enzyme was purified and characterized. The gene previously designated as putative alpha-glucosidase was found to code for a protein that had no alpha-glucosidase activity. It showed a rare activity profile with its ability to hydrolyze pNP-GUA, an activity not known in the alpha-glucuronidases from microbial sources. This is the first report on the occurrence of an alpha-glucuronidase which belongs to the family 4 of glycosyl hydrolases. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0014-5793(02)02611-X

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DOI： 10.1016/S0167-4838(01)00315-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

Xylanase A from alkaliphilic Bacillus halodurans C-125 was expressed in Escherichia coli and purified by affinity and anion exchange chromatographies. It exhibited a strong substrate inhibition using xylan as the substrate. Its k(i) value increased with an increase in pH. The effect of pH on the enzyme activity was determined using two aryl-xylobiosides as substrates, and it was found that the enzyme had a flat k(cat)-pH curve in the pH range of 5.8-8.8. This range was different from that obtained with 0.45% xylan as previously reported (Honda, H. et. al., Agric. Biol. Chem., 49, 3165-3169, 1985). The substrate inhibition was presumed to cause the difference. It has been clarified that the use of aryl-xylobiosides as substrates yields more accurate kinetic results than that of xylan.

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An insolubilized β-cyclodextrin derivative (polyCD) was prepared by polymerizing cyclodextrin with epichlrohydrine. By stirring 5 mg/ml of polyCD in 0.2 mM bisphenol A (BPA) solution at pH 7.0 for 2 h, more than 98% of BPA was adsorbed on polyCD. The capacity of the adsorption was determined to be 84 mg-BPA/g-polyCD, meaning 0.65 mol-BPA/mol-CD (in polyCD). The effect of pH on the adsorption was studied and found that BPA was effectively adsorbed on polyCD at pH between pH 2.2-9.1, but the efficiency decreased at pH 10.8, suggesting that it did not adsorb the BPA anion. The polyCD did not adsorb aromatic amino acids, indicating that BPA could selectively be removed from a solution containing amino acids.

DOI： 10.1023/A:1023024004103

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4-Methylumbelliferyl-β-D-xylobioside (MU-X2) and 5-bromo-3-indolyl-β-D-xylobioside (BI-X2) were synthesized as substrates for the detection of xylanase activity on agar plates. A family F/10 xylanase from Streptomyces olivaceoviridis E-86 (FXYN) was able to be more sensitively detected than RBB-xylan by using MU-X2 as a substrate. A mutant xylanase E128H/FXYN having only 1/1000 of the activity of FXYN was also able to be detected on the MU-X2 plate but was not detected on the RBB-xylan plate. A family G/11 xylanase from Streptomyces lividans 66 (Xyn B) was not detected on the MU-X2 plate, but it was able to be detected on the RBB-xylan plate, suggesting that the MU-X2 substrate is specific to family F/10 xylanases. However, none of the xylanases were detected effectively by using BI-X2 as a substrate. © 2000 by Japan Society for Bioscience, Biotechnology, and Agrochemistry.

DOI： 10.1271/bbb.64.741

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Reactions of the p-nitrophenyl-cellobioside (G2-pNP) hydrolyzing xylanase from Cellvibrio gilvus (XCEL) were investigated kinetically in detail. XCEL hydrolyzed only aglyconic bonds in various arylcellobiosides with close kinetic paramters together. Its kinetic parameters toward various p-nitrophenyl cellooligosaccharides were also close. Two more xylanases, from Streptomyces sp. E86 (XSTR) and from Aspergillus japonicus (XASP), were found to hydrolyze G2-pNP at a lower rate compared with XCEL. The V(max) of XSTR and XASP were comparable to that of XCEL, suggesting that the high G2-pNP-hydrolyzing activity of XCEL was due to its small K(m). A xylanse from Robillarda sp. Y-20 (XROB) did not have any activity on G2-pNP. p-Nitrophenyl-xylobioside (X2-pNP) and p-nitrophenyl-glucosyl-xyloside (GX-pNP) were examined as substrates to the four xylanases. Three of the four xylanases hydrolyzed these substrates, only at their aglyconic bonds, rather faster than xylan, but XROB hydrolyzed them with a very small rate. Classification of xylanases based on their activity on the aryl-glycosides is discussed. The advantage of using X2-pNP or GX-pNP for xylanase assay is also discussed.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPANESE BIOCHEMICAL SOC  

The synthetic reactions of the cellobiose phosphorylase from Cellvibrio gilvus were investigated in detail.It was found that, besides D-glucose, some sugars having substitution or deletion of the hydroxyl group at C2 or C6 of the D-glucose molecule could serve as a glucosyl acceptor, though less effectively than D-glucose. The enzyme showed higher activity with beta-D-glucose than with the alpha-anomer as an acceptor. This result indicates that it recognizes the anomeric hydroxyl group not involved directly in the reaction. beta-D-Cellobiose was also phosphorolyzed faster than the alpha-anomer. Substrate inhibition was observed with D-glucose, 6-deoxy-D-glucose, or D-glucosamine as an acceptor, with D-glucose being most inhibiting. This inhibition was studied in detail and it was found that D-glucose competes with alpha-D-glucose-1-phosphate for its binding site. A model of competitive substrate inhibition was proposed, and the experimental data fit well to the theoretical values that were calculated in accordance with this model.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Cellobiose phosphorylase was purified from Cellvibrio gilvus cells by the method reported previously with some modifications, and its kinetic properties were studied in detail. The initial velocity of the synthetic reaction was 1.4 times as fast as that of the phosphorolytic one. The equilibrium constant of the phosphorolysis was 0.32 at 37-degrees-C and pH 7.0. NO D-[U-C-14]glucose exchange reaction was observed in the absence of Pi. Kinetic studies on the phosphorolytic reaction showed that the reaction follows an ordered bi bi mechanism. These results make a sharp contrast to those of sucrose phosphorylase, which catalyzes fructose exchange reaction and follows a ping pong bi bi mechanism. Kinetic parameters were calculated as K(mA) = 2.6mM, K(mB) = 0.61 mM, and K(iA) = 6.8 mM (A, D-cellobiose; B, Pi).

DOI： 10.1271/bbb.56.652

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1080/00021369.1991.10870772

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An enzyme component that hydrolyzes pNP-G2but not CMC has been isolated from a culture broth of Cellvibrio gilvus by a multi-step procedure involving Butyl-Toyopearl, DEAE-Toyopearl, and CM-Toyopearl chromatographies. The purified enzyme gave a single protein band on native, SDS-, and IEF-PAGE. The enzyme had a molecular weight of 40, 000, an isoelectric point of 5.0, an optimum pH of 6.5, and an optimum temperature of 55°C. It was stable from pH 4.0 to 9.0 at 37°C for 1 hr and below 50°C for 30min. It hydrolyzed agluconic bonds not only of pNP-G2but also of pNP-G3, pNP-G4, and pNP-G5. Cellooligosaccharides with D.P. of 3 to 5 were not hydrolyzed at all. Instead, the enzyme hydrolyzed xylan 4 times as fast as pNP-G2. Both HgCl2and p-chloromercuribenzoic acid inhibited the two activities completely. Xylan inhibited the hydrolysis of pNP-G2competitively. From these results, the purified enzyme was considered to be a unique xylanase that hydrolyzed the agluconic bonds of pNP-Gn. © 1991 by the Japan Society for Bioscience, Biotechnology, and Agrochemistry.

DOI： 10.1080/00021369.1991.10870897

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

4-O-\-d-Glucopyranosyl-d-xylose (GX) was synthesized from equimolar amounts of d-xylose and α-d-glucose-1-phosphate (G-1-P) using acetone-treated cells of Cellvibrio gilvus. It was found that ethanol treatment of acetone-treated cells selectively removed phosphoglucomutase activity, which competes with cellobiose phosphorylase for G-1-P in the synthetic reaction. The yield of synthesis was 60%, based on d-xylose used. GX was purified by charcoal column chromatography with a 32% yield based on d-xylose. Nuclear magnetic resonance and fast atm bombardment mass data of GX are presented. The possibility for this saccharide to be used as a new foodstuff is also discussed. © 1990 Springer-Verlag.

DOI： 10.1007/BF00166776

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC FERMENTATION BIOENGINEERING, JAPAN  

Two types of cellulase (Robillarda sp. Y-20 and Trichoderma reesei) were immobilized on Aminosilica-1 by physical adsorption. Enzymes were quickly immobilized and stable on the support. Specific activities of two types of immobilized cellulase were 0.24 U/mg support with Robillarda cellulase and 0.15 U/mg support with the Trichoderma one as CMCase. The pH-activity curves of both cellulase shifted slightly to lower pH on immobilization. Both immobilized cellulases showed essentially the same pH stabilities as their free forms. However, the immobilized enzymes were less stable than the free forms at temperatures higher than 50°C. © 1989.

DOI： 10.1016/0922-338X(89)90119-0

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Language：Japanese  

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Language：English   Publisher：OXFORD UNIV PRESS INC  

CAZypedia was initiated in 2007 to create a comprehensive, living encyclopedia of the carbohydrate active enzymes (CAZymes) and associated carbohydrate-binding modules involved in the synthesis, modification and degradation of complex carbohydrates. CAZypedia is closely connected with the actively curated CAZy database, which provides a sequence-based foundation for the biochemical, mechanistic and structural characterization of these diverse proteins. Now celebrating its 10th anniversary online, CAZypedia is a successful example of dynamic, community-driven and expert-based biocuration. CAZypedia is an open-access resource available at URL http://www.cazypedia.org.

DOI： 10.1093/glycob/cwx089

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Language：Japanese   Publisher：The Japanese Society of Applied Glycoscience  

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