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DOI： 10.1016/j.carres.2020.108129

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DOI： 10.1080/09168451.2019.1630257

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

endo--1,2-Glucanase (SGL) is an enzyme that hydrolyzes -1,2-glucans, which play important physiological roles in some bacteria as a cyclic form. To date, no eukaryotic SGL has been identified. We purified an SGL from Talaromyces funiculosus (TfSGL), a soil fungus, to homogeneity and then cloned the complementary DNA encoding the enzyme. TfSGL shows no significant sequence similarity to any known glycoside hydrolase (GH) families, but shows significant similarity to certain eukaryotic proteins with unknown functions. The recombinant TfSGL (TfSGLr) specifically hydrolyzed linear and cyclic -1,2-glucans to sophorose (Glc--1,2-Glc) as a main product. TfSGLr hydrolyzed reducing-end-modified -1,2-gluco-oligosaccharides to release a sophoroside with the modified moiety. These results indicate that TfSGL is an endo-type enzyme that preferably releases sophorose from the reducing end of substrates. Stereochemical analysis demonstrated that TfSGL is an inverting enzyme. The overall structure of TfSGLr includes an (/)(6) toroid fold. The substrate-binding mode was revealed by the structure of a Michaelis complex of an inactive TfSGLr mutant with a -1,2-glucoheptasaccharide. Mutational analysis and action pattern analysis of -1,2-gluco-oligosaccharide derivatives revealed an unprecedented catalytic mechanism for substrate hydrolysis. Glu-262 (general acid) indirectly protonates the anomeric oxygen at subsite -1 via the 3-hydroxy group of the Glc moiety at subsite +2, and Asp-446 (general base) activates the nucleophilic water via another water. TfSGLr is apparently different from a GH144 SGL in the reaction and substrate recognition mechanism based on structural comparison. Overall, we propose that TfSGL and closely-related enzymes can be classified into a new family, GH162.

DOI： 10.1074/jbc.RA118.007087

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DOI： 10.1016/j.ab.2018.08.021

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Language：English   Publisher：ELSEVIER SCI LTD  

Sophorose (Sop(2)) is known as a powerful inducer of cellulases in Trichoderma reesei, and in recent years 1,2-beta-D-oligoglucan phosphorylase (SOGP) has been found to use Sop(2) in synthetic reactions. From the structure of the complex of SOGP with Sop2, it was predicted that both the 3-hydroxy group at the reducing end glucose moiety of Sop(2) and the 3'-hydroxy group at the non-reducing end glucose moiety of Sop(2) were important for substrate recognition. In this study, three kinds of 3-and/or 3'-deoxy-Sop(2) derivatives were synthesized to evaluate this mechanism. The deoxygenation of the 3-hydroxy group of D-glucopyranose derivative was performed by radical reduction using a toluoyl group as a leaving group. The utilization of a toluoyl group that plays two roles (a leaving group for the deoxygenation and a protecting group for a hydroxy group) resulted in efficient syntheses of the three target compounds. The NMR spectra of the two final compounds (3-deoxy- and 3,3'-dideoxy-Sop(2)) suggested that the glucose moiety of the reducing end of Sop(2) can easily take on a furanose structure (five-membered ring structure) by deoxygenation of the 3-hydroxy group of Sop(2). In addition, the ratio of the five-and six-membered ring structures changed depending on the temperature. The SOGPs exhibited remarkably lower specific activity for 3'-deoxy- and 3,3'-dideoxy-Sop(2), indicating that the 3'-hydroxy group of Sop(2) is important for substrate recognition by SOGPs.

DOI： 10.1016/j.carres.2018.08.005

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DOI： 10.1021/acs.biochem.8b00385

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DOI： 10.1016/j.jdermsci.2018.02.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Biochemistry and Molecular Biology Inc.  

-1,2-Glucans are bacterial carbohydrates that exist in cyclic or linear forms and play an important role in infections and symbioses involving Gram-negative bacteria. Although several -1,2-glucan–associated enzymes have been characterized, little is known about how -1,2-glucan and its shorter oligosaccharides (Sopns) are captured and imported into the bacterial cell. Here, we report the biochemical and structural characteristics of the Sopn-binding protein (SO-BP, Lin1841) associated with the ATP-binding cassette (ABC) transporter from the Gram-positive bacterium Listeria innocua. Calorimetric analysis revealed that SO-BP specifically binds to Sopns with a degree of polymerization of 3 or more, with Kd values in the micromolar range. The crystal structures of SO-BP in an unliganded open form and in closed complexes with tri-, tetra-, and pentaoligosac-charides (Sop3–5) were determined to a maximum resolution of 1.6 Å. The binding site displayed shape complementarity to Sopn, which adopted a zigzag conformation. We noted that water-mediated hydrogen bonds and stacking interactions play a pivotal role in the recognition of Sop3–5 by SO-BP, consistent with its binding thermodynamics. Computational free-energy calculations and a mutational analysis confirmed that interactions with the third glucose moiety of Sopns are significantly responsible for ligand binding. A reduction in unfavorable changes in binding entropy that were in proportion to the lengths of the Sopns was explained by conformational entropy changes. Phylogenetic and sequence analyses indicated that SO-BP ABC transporter homologs, glycoside hydrolases, and other related proteins are co-localized in the genomes of several bacteria. This study may improve our understanding of bacterial -1,2-glucan metabolism and promote the discovery of unidentified -1,2-glucan–associated proteins.

DOI： 10.1074/jbc.RA117.001536

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Language：English   Publisher：Wiley Blackwell  

BT_3567 protein, a putative β-glucosidase from Bacteroides thetaiotaomicron, exhibits higher activity toward Sop3–5 (Sopn, n: degree of polymerization of β-1,2-glucooligosaccharides) than toward Sop2, unlike a known β-glucosidase from Listeria innocua which predominantly prefers Sop2. In the complex structure determined by soaking of a D286N mutant crystal with Sop4, a Sop3 moiety was observed at subsites −1 to +2. The glucose moiety at subsite +2 forms a hydrogen bond with Asn81, which is replaced with Gly in the L. innocua β-glucosidase. The Km values of the N81G mutant for Sop3–5 are much higher than those of the wild-type, suggesting that Asn81 contributes to the binding to substrates longer than Sop3.

DOI： 10.1002/1873-3468.12911

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

BT_3567 protein, a putative -glucosidase from Bacteroides thetaiotaomicron, exhibits higher activity toward Sop(3-5) (Sop(n), n: degree of polymerization of beta-1,2-glucooligosaccharides) than toward Sop(2), unlike a known beta-glucosidase from Listeria innocua which predominantly prefers Sop(2). In the complex structure determined by soaking of a D286N mutant crystal with Sop(4), a Sop(3) moiety was observed at subsites -1 to +2. The glucose moiety at subsite +2 forms a hydrogen bond with Asn81, which is replaced with Gly in the L. innocua beta-glucosidase. The K-m values of the N81G mutant for Sop(3-5) are much higher than those of the wild-type, suggesting that Asn81 contributes to the binding to substrates longer than Sop(3).

DOI： 10.1002/1873-3468.12911

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

-1,2-Glucan is an extracellular cyclic or linear polysaccharide from Gram-negative bacteria, with important roles in infection and symbiosis. Despite -1,2-glucan's importance in bacterial persistence and pathogenesis, only a few reports exist on enzymes acting on both cyclic and linear -1,2-glucan. To this end, we purified an endo--1,2-glucanase to homogeneity from cell extracts of the environmental species Chitinophaga arvensicola, and an endo--1,2-glucanase candidate gene (Cpin_6279) was cloned from the related species Chitinophaga pinensis. The Cpin_6279 protein specifically hydrolyzed linear -1,2-glucan with polymerization degrees of 5 and a cyclic counterpart, indicating that Cpin_6279 is an endo--1,2-glucananase. Stereochemical analysis demonstrated that the Cpin_6279-catalyzed reaction proceeds via an inverting mechanism. Cpin_6279 exhibited no significant sequence similarity with known glycoside hydrolases (GHs), and thus the enzyme defines a novel GH family, GH144. The crystal structures of the ligand-free and complex forms of Cpin_6279 with glucose (Glc) and sophorotriose (Glc--1,2-Glc--1,2-Glc) determined up to 1.7 angstrom revealed that it has a large cavity appropriate for polysaccharide degradation and adopts an (/)(6)-fold slightly similar to that of GH family 15 and 8 enzymes. Mutational analysis indicated that some of the highly conserved acidic residues in the active site are important for catalysis, and the Cpin_6279 active-site architecture provided insights into the substrate recognition by the enzyme. The biochemical characterization and crystal structure of this novel GH may enable discovery of other -1,2-glucanases and represent a critical advance toward elucidating structure-function relationships of GH enzymes.

DOI： 10.1074/jbc.M116.762724

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

Glycoside phosphorylases catalyze the phosphorolysis of oligosaccharides into sugar phosphates. Recently, we found a novel phosphorylase acting on beta-1,2-glucooligosaccharides with degrees of polymerization of 3 or more (1,2-beta-oligoglucan phosphorylase, SOGP) in glycoside hydrolase family (GH) 94. Here, we characterized SOGP from Lachnoclostridium phytofermentans (LpSOGP) and determined its crystal structure. LpSOGP is a monomeric enzyme that contains a unique beta-sandwich domain (Ndom1) at its N-terminus. Unlike the dimeric GH94 enzymes possessing catalytic pockets at their dimer interface, LpSOGP has a catalytic pocket between Ndom1 and the catalytic domain. In the complex structure of LpSOGP with sophorose, sophorose binds at subsites +1 to +2. Notably, the Glc moiety at subsite +1 is flipped compared with the corresponding ligands in other GH94 enzymes. This inversion suggests the great distortion of the glycosidic bond between subsites -1 and +1, which is likely unfavorable for substrate binding. Compensation for this disadvantage at subsite +2 can be accounted for by the small distortion of the glycosidic bond in the sophorose molecule. Therefore, the binding mode at subsites +1 and +2 defines the substrate specificity of LpSOGP, which provides mechanistic insights into the substrate specificity of a phosphorylase acting on beta-1,2-glucooligosaccharides.

DOI： 10.1038/srep42671

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

Autosomal recessive congenital ichthyosis (ARCI) is a heterogeneous group of hereditary skin disorder characterized by an aberrant cornification of the epidermis. ARCI is classified into a total of 11 subtypes (ARCI1-ARCI11) based on their causative genes or loci. Of these, the causative gene for only ARCI7 has not been identified, while it was previously mapped on chromosome 12p11.2-q13.1. In this study, we performed genetic analyses for three Lebanese families with ARCI, and successfully determined the linkage interval to 9.47Mb region on chromosome 12q13.13-q14.1, which was unexpectedly outside of the ARCI7 locus. Whole-exome sequencing and the subsequent Sanger sequencing led to the identification of missense mutations in short chain dehydrogenase/ reductase family 9C, member 7 (SDR9C7) gene on chromosome 12q13.3, i. e. two families shared an identical homozygous mutation c.599T> C (p.Ile200Thr) and one family had another homozygous mutation c.214C> T (p.Arg72Trp). In cultured cells, expression of both the mutant SDR9C7 proteins was markedly reduced as compared to wild-type protein, suggesting that the mutations severely affected a stability of the protein. In normal human skin, the SDR9C7 was abundantly expressed in granular and cornified layers of the epidermis. By contrast, in a patient's skin, its expression in the cornified layer was significantly decreased. It has previously been reported that SDR9C7 is an enzyme to convert retinal into retinol. Therefore, our study not only adds a new gene responsible for ARCI, but also further suggests a potential role of vitamin A metabolismin terminal differentiation of the epidermis in humans.

DOI： 10.1093/hmg/ddw277

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Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data.

DOI： 10.1371/journal.pone.0160112

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

An alpha-l-arabinofuranosidase of GH62 from Aspergillus nidulans FGSC A4 (AnAbf62A-m2,3) has an unusually high activity towards wheat arabinoxylan (WAX) (67 U/mg; k (cat) = 178/s, K (m) = 4.90 mg/ml) and arabinoxylooligosaccharides (AXOS) with degrees of polymerisation (DP) 3-5 (37-80 U/mg), but about 50 times lower activity for sugar beet arabinan and 4-nitrophenyl-alpha-l-arabinofuranoside. alpha-1,2- and alpha-1,3-linked arabinofuranoses are released from monosubstituted, but not from disubstituted, xylose in WAX and different AXOS as demonstrated by NMR and polysaccharide analysis by carbohydrate gel electrophoresis (PACE). Mutants of the predicted general acid (Glu(188)) and base (Asp(28)) catalysts, and the general acid pK (a) modulator (Asp(136)) lost 1700-, 165- and 130-fold activities for WAX. WAX, oat spelt xylan, birchwood xylan and barley beta-glucan retarded migration of AnAbf62A-m2,3 in affinity electrophoresis (AE) although the latter two are neither substrates nor inhibitors. Trp(23) and Tyr(44), situated about 30 from the catalytic site as seen in an AnAbf62A-m2,3 homology model generated using Streptomyces thermoviolaceus SthAbf62A as template, participate in carbohydrate binding. Compared to wild-type, W23A and W23A/Y44A mutants are less retarded in AE, maintain about 70 % activity towards WAX with K (i) of WAX substrate inhibition increasing 4-7-folds, but lost 77-96 % activity for the AXOS. The Y44A single mutant had less effect, suggesting Trp(23) is a key determinant. AnAbf62A-m2,3 seems to apply different polysaccharide-dependent binding modes, and Trp(23) and Tyr(44) belong to a putative surface binding site which is situated at a distance of the active site and has to be occupied to achieve full activity.

DOI： 10.1007/s00253-016-7417-8

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

Plant beta-glucosidases are usually members of the glucosyl hydrolase 1 (GH1) or 3 (GH3) families. Previously, a beta-glucosidase (torvosidase) was purified from Solanum torvum leaves that specifically catalyzed hydrolysis of two furostanol 26-O-beta-glucosides, torvosides A and H. Furostanol glycoside 26-O-beta-glucosides have been reported as natural substrates of some plant GH1 enzymes. However, torvosidase was classified as a GH3 beta-glucosidase, but could not hydrolyze beta-oligoglucosides, the natural substrates of GH3 enzymes. Here, the full-length cDNA encoding S. torvum beta-glucosidase (SBgl3) was isolated by the rapid amplification of cDNA ends method. The 1887 bp ORF encoded 629 amino acids and showed high homology to other plant GH3 beta-glucosidases. Internal peptide sequences of purified native Sbgl3 determined by LC-MS/MS matched the deduced amino acid sequence of the Sbgl3 cDNA, suggesting that it encoded the natural enzyme. Recombinant SBgl3 with a polyhistidine tag (SBgl3His) was successfully expressed in Pichia pastoris. The purified SBgl3His showed the same substrate specificity as natural SBgl3, hydrolyzing torvoside A with much higher catalytic efficiency than other substrates. It also had similar biochemical properties and kinetic parameters to the natural enzyme, with slight differences, possibly attributable to post-translational glycosylation. Quantitative real-time PCR (qRT-PCR) showed that SBgl3 was highly expressed in leaves and germinated seeds, suggesting a role in leaf and seedling development. To our knowledge, a recombinant GH3 beta-glucosidase that hydrolyzes furostanol 26-O-beta-glucosides, has not been previously reported in contrast to substrates of GH1 enzymes. (C) 2016 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.phytochem.2016.03.015

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

Marine glycoside hydrolases hold enormous potential due to their habitat-related characteristics such as salt tolerance, barophilicity, and cold tolerance. We purified an -glucosidase (PYG) from the midgut gland of the Japanese scallop (Patinopecten yessoensis) and found that this enzyme has unique characteristics. The use of acarbose affinity chromatography during the purification was particularly effective, increasing the specific activity 570-fold. PYG is an interesting chloride ion-dependent enzyme. Chloride ion causes distinctive changes in its enzymatic properties, increasing its hydrolysis rate, changing the pH profile of its enzyme activity, shifting the range of its pH stability to the alkaline region, and raising its optimal temperature from 37 to 55 degrees C. Furthermore, chloride ion altered PYG's substrate specificity. PYG exhibited the highest V-max/K-m value toward maltooctaose in the absence of chloride ion and toward maltotriose in the presence of chloride ion.

DOI： 10.1080/09168451.2015.1116926

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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   Publisher：WILEY-BLACKWELL  

DOI： 10.1111/bjd.14003

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

Cutaneous leiomyoma is a benign skin tumor that originates from the smooth muscle, such as the arrector pili muscle of the hair follicles. Familial cases with multiple cutaneous leiomyomas exist, which typically show an autosomal dominant inheritance trait. Most patients with the disease are known to carry heterozygous germ line mutations in the fumarate hydratase (FH) gene and can be complicated by tumors in internal organs, especially uterine leiomyoma and renal cell cancer in high frequency. In this study, we identified a Japanese male patient with multiple cutaneous leiomyomas and found a novel heterozygous splice site mutation, c. 738 + 2T> A, in the FH gene of the patient, which was unexpectedly inherited from his unaffected father. Further analysis demonstrated loss of heterozygosity in the tumor tissue, which resulted in a hemizygote state of the mutant allele. Expression studies with the tumor tissue showed that the mutation led to skipping of exon 5 at mRNA levels, which was predicted to cause an in-frame deletion of FH protein (p.Ser186_ Gln246del). The protein structure analysis strongly suggested that the deletion would severely disrupt the conformation of the FH protein including the substratebinding domain, and thus would severely affect the expression and the function. Our findings further disclose the molecular basis of multiple cutaneous leiomyomas and also provide precious information to the mutation carriers in the family for an early diagnosis of renal cell cancer in the future.

DOI： 10.1111/1346-8138.13019

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Authorship：Corresponding author   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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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

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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DOI： 10.5458/jag.jag.JAG-2015_006

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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   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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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 >= 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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DOI： 10.5458/jag.jag.JAG-2013_013

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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 >= 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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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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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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Authorship：Last author,　Corresponding author   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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All TGF-beta family members have a prodomain that is important for secretion. Lack of secretion of a TGF-beta family member GDF5 is known to underlie some skeletal abnormalities, such as brachydactyly type C that is characterized by a huge and unexplained phenotypic variability. To search for potential phenotypic modifiers regulating secretion of GDF5, we compared cells overexpressing wild type (Wt) GDF5 and GDF5 with a novel mutation in the prodomain identified in a large Pakistani family with Brachydactyly type C and mild Grebe type chondrodyslplasia (c527T > C; p.Leu176Pro). Initial in vitro expression studies revealed that the p.Leu176Pro mutant (Mut) GDF5 was not secreted outside the cells. We subsequently showed that GDF5 was capable of forming a complex with latent transforming growth factor binding proteins, LTBP1 and LTBP2. Furthermore, secretion of LTBP1 and LTBP2 was severely impaired in cells expressing the Mut-GDF5 compared to Wt-GDF5. Finally, we demonstrated that secretion of Wt-GDF5 was inhibited by the Mut-GDF5, but only when LTBP (LTBP1 or LTBP2) was co-expressed. Based on these findings, we suggest a novel model, where the dosage of secretory co-factors or stabilizing proteins like LTBP1 and LTBP2 in the microenvironment may affect the extent of GDF5 secretion and thereby function as modifiers in phenotypes caused by GDF5 mutations.

DOI： 10.1007/s00439-013-1330-3

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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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Language：English   Publisher：The Japanese Society of Applied Glycoscience  

<i>In vitro</i> and <i>in vivo</i> studies have demonstrated the prebiotic potential of isomalto-oligosaccharides (IMO), comprising α-(1,6)-gluco-oligosaccharides and panose, which selectively stimulate the growth of probiotic bifidobacteria and lactobacilli. The protein machinery conferring the utilization of IMO by probiotics, however, remains vaguely described. We have used genomic, transcriptomic, enzymatic, and biophysical analyses to explore IMO utilization routes in probiotic lactobacilli and bifidobacteria as re­presented by <i>Lactobacillus acidophilus</i> NCFM and <i>Bifidobacterium animalis</i> subsp. <i>lactis</i> Bl-04, respectively. Utilization of IMO and malto-oligosaccharide (α-(1,4)-glucosides) appears to be linked both at the genetic and transcriptomic level in the acidophilus group lactobacilli as suggested by reverse transcriptase PCR (RT-PCR) and gene landscape analysis. Canonical intracellular GH13_31 glucan 1,6-α-glucosidases active on IMO longer than isomaltose occur widely in acidophilus group lactobacilli. Interestingly, however, isomaltose, isomaltulose and panose seem to be internalized through a phosphoenoyl pyruvate transferase system (PTS) and subsequently hydrolyzed by a GH4 6-phosphate-α-glucosidases based on whole genome microarray analysis. This sub-specificity within GH4 seems to be unique for lactobacilli mainly from the gut niche, as the sequences from this group segregate from characterized GH4 maltose-6-phosphate-α-glucosidases in other organisms. By comparison, IMO utilization in bifidobacteria is linked to soybean oligosaccharide utilization loci harboring GH36 α-galactosidases, GH13_31 oligo 1,6-α-glucosidases and a dual specificity ATP-binding cassette (ABC) transport system active in the uptake of both classes of α-(1,6)-glycosides. These data bring novel insight to advance our understanding of the basis of selectivity of IMO metabolism by important gut microbiome members.

DOI： 10.5458/jag.jag.JAG-2012_022

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Authorship：Last author,　Corresponding author   Language：English   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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The present study aimed at examining oligosaccharides (OS) for potential stimulation of probiotic bacteria. Nineteen structurally well-defined candidate OS covering groups of β-glucosides, α-glucosides and α-galactosides with degree of polymerization 2-4 were prepared in &gt
100 mg amounts by chemoenzymatic synthesis (i.e. reverse phosphorolysis or transglycosylation). Fourteen of the OS are not naturally occurring and five (β-d-glucosyl-fructose, β-d-glucosyl-xylitol, α-glucosyl-(1,4)- d-mannose, α-glucosyl-(1,4)-d-xylose
α-glucosyl-(1,4)-l-fucose) have recently been synthesized for the first time. These OS have not been previously tested for effects of bacterial growth and here the ability of all 19 OS to support growth of four gastrointestinal bacteria: three probiotic bacteria Bifidobacterium lactis, Bifidobacterium longum, and Lactobacillus acidophilus, and one commensal bacterium, Bacteroides vulgatus has been evaluated in monocultures. The disaccharides β-d-glucosyl-xylitol and β-d-glucosyl-(1,4)-xylose noticeably stimulated growth yields of L. acidophilus NCFM, and additionally, β-d-glucosyl-(1,4)-xylose stimulated B. longum Bl-05. α-Glucosyl-(1,4)-glucosamine and α-glucosyl-(1,4)-N- acetyl-glucosamine enhanced the growth rate of B. animalis subsp. lactis and B. longum Bl-05, whereas L. acidophilus NCFM and Bac. vulgatus did not grow on these OS. α-Galactosyl-(1,6)-α-galactosyl-(1,6)-glucose advanced the growth rate of B. animalis subsp. lactis and L. acidophilus NCFM. Thus several of the structurally well-defined OS supported growth of beneficial gut bacteria. This reflects a broad specificity of their sugar transporters for OS, including specificity for non-naturally occurring OS, hence showing promise for design of novel prebiotics. © 2013 The Royal Society of Chemistry.

DOI： 10.1039/c3fo30357h

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Authorship：Lead author,　Corresponding author   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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Glycoside hydrolase family 31 alpha-glucosidases (31AGs) show various specificities for maltooligosaccharides according to chain length. Aspergillus niger alpha-glucosidase (ANG) is specific for short-chain substrates with the highest k(cat)/K-m for maltotriose, while sugar beet alpha-glucosidase (SBG) prefers long-chain substrates and soluble starch. Multiple sequence alignment of 31AGs indicated a high degree of diversity at the long loop (N-loop), which forms one wall of the active pocket. Mutations of Phe236 in the N-loop of SBG (F236A/S) decreased k(cat)/K-m values for substrates longer than maltose. Providing a phenylalanine residue at a similar position in ANG (T228F) altered the k(cat)/K-m values for maltooligosaccharides compared with wild-type ANG, i.e., the mutant enzyme showed the highest k(cat)/K-m value for maltotetraose. Subsite affinity analysis indicated that modification of subsite affinities at +2 and +3 caused alterations of substrate specificity in the mutant enzymes. These results indicated that the aromatic residue in the N-loop contributes to determining the chain-length specificity of 31AG5. (C) 2012 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbapap.2012.08.007

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DOI： 10.1016/j.bbapap.2012.08.007

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

Generalized pustular psoriasis (GPP) is a rare, potentially life threatening, and aggressive form of psoriasis, which is characterized by sudden onset with repeated episodic skin inflammation leading to pustule formation. Familial GPP is known to be caused by recessively inherited mutations in the IL36RN gene, which encodes interleukin 36 receptor antagonist (IL-36Ra). In this article, we performed mutation analysis of the IL36RN gene in 14 Japanese patients with GPP, and identified mutations in two of these patients analyzed. One patient was compound heterozygous for mutations c.115+6T&gt;C and c.368C&gt;G (p.Thr123Arg), whereas the other carried compound heterozygous mutations c.28C&gt;T (p.Arg10*) and c.115+6T&gt;C in the IL36RN gene. Expression studies using total RNA from the patients' skin revealed that the mutation c.115+6T&gt;C resulted in skipping of exon 3, leading to a frameshift and a premature termination codon (p.Arg10Argfs*1). The protein structure analysis suggested that the missensemutation p.Thr123Arg caused misfolding and instability of IL-36Ra protein. In vitro studies in cultured cells showed impaired expression of the p.Thr123Arg mutant IL-36Ra protein, which failed to antagonize the IL-36 signaling pathway. Our data further underscore the critical role of IL36RN in pathogenesis of GPP. Hum Mutat 34:176-183, 2013. (C) 2012 Wiley Periodicals, Inc.

DOI： 10.1002/humu.22203

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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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Honeybees, Apis mellifera, possess three alpha-glucosidase isozymes, HBG-I, HBG-II, and HBG-III, which belong to glycoside hydrolase family 13. They show high sequence similarity, but clearly different enzymatic properties. HBG-M preferred sucrose to maltose as substrate and formed only alpha-1,4-glucosidic linkages by transglucosylation, while HBG-II preferred maltose and formed the alpha-1,6-linkage. Mutation analysis of five amino acids in conserved region II revealed that Pro226-Tyr227 of HBG-III and the corresponding Asn226-His227 of HBG-II were crucial to the discriminating properties. By replacing these two amino acids, the substrate specificities and regioselectivity in transglucosylation were changed drastically toward the other. The HBG-III mutant, Y227H, and the HBG-II mutant, N226P, which harbor HBG-I-type Pro-His at the crucial positions, resembled HBG-I in enzymatic properties with marked increases in reaction velocities on maltose and transglucosylation ratios. These findings indicate that the two residues are determinants of the enzymatic properties of glycoside hydrolase family 13 (GH-13) alpha-glucosidases and related enzymes.

DOI： 10.1271/bbb.120473

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Bacteroides thetaiotaomicron VPI-5482 harbors a gene encoding a putative cycloisomaltooligosaccharide glucanotransferase (BT3087) belonging to glycoside hydrolase family 66. The goal of the present study was to characterize the catalytic properties of this enzyme. Therefore, we expressed BT3087 (recombinant endo-dextranase from Bacteroides thetaiotaomicron VPI-5482) in Escherichia coli and determined that recombinant endo-dextranase from Bacteroides thetaiotaomicron VPI-5482 preferentially synthesized isomaltotetraose and isomaltooligosaccharides (degree of polymerization &gt; 4) from dextran. The enzyme also generated large cyclic isomaltooligosaccharides early in the reaction. We conclude that members of the glycoside hydrolase 66 family may be classified into three types: (a) endo-dextranases, (b) dextranases possessing weak cycloisomaltooligosaccharide glucanotransferase activity, and (c) cycloisomaltooligosaccharide glucanotransferases.

DOI： 10.1111/j.1742-4658.2012.08698.x

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

Isomaltooligosaccharides (IMO) have been suggested as promising prebiotics that stimulate the growth of probiotic bacteria. Genomes of probiotic lactobacilli from the acidophilus group, as represented by Lactobacillus acidophilus NCFM, encode alpha-1,6 glucosidases of the family GH13_31 (glycoside hydrolase family 13 subfamily 31) that confer degradation of IMO. These genes reside frequently within maltooligosaccharide utilization operons, which include an ATP-binding cassette transporter and alpha-glucan active enzymes, e.g., maltogenic amylases and maltose phosphorylases, and they also occur separated from any carbohydrate transport or catabolism genes on the genomes of some acidophilus complex members, as in L. acidophilus NCFM. Besides the isolated locus encoding a GH13_31 enzyme, the ABC transporter and another GH13 in the maltooligosaccharide operon were induced in response to IMO or maltotetraose, as determined by reverse transcription-PCR (RT-PCR) transcriptional analysis, suggesting coregulation of alpha-1,6- and alpha-1,4-glucooligosaccharide utilization loci in L. acidophilus NCFM. The L. acidophilus NCFM GH13_31 (LaGH13_31) was produced recombinantly and shown to be a glucan 1,6-alpha-glucosidase active on IMO and dextran and product-inhibited by glucose. The catalytic efficiency of LaGH13_31 on dextran and the dextran/panose (trisaccharide) efficiency ratio were the highest reported for this class of enzymes, suggesting higher affinity at distal substrate binding sites. The crystal structure of LaGH13_31 was determined to a resolution of 2.05 angstrom and revealed additional substrate contacts at the +2 subsite in LaGH13_31 compared to the GH13_31 from Streptococcus mutans (SmGH13_31), providing a possible structural rationale to the relatively high affinity for dextran. A comprehensive phylogenetic and activity motif analysis mapped IMO utilization enzymes from gut microbiota to rationalize preferential utilization of IMO by gut residents.

DOI： 10.1128/JB.00622-12

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A novel endodextranase from Paenibacillus sp. (Paenibacillus sp. dextranase; PsDex) was found to mainly produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides (CIs) with a degree of polymerization of 7-14 from dextran. The 1,696-amino acid sequence belonging to the glycosyl hydrolase family 66 (GH-66) has a long insertion (632 residues; Thr(451)-Val(1082)), a portion of which shares identity (35% at Ala(39)-Ser(1304) of PsDex) with Pro(32)-Ala(755) of CI glucanotransferase (CITase), a GH-66 enzyme that catalyzes the formation of CIs from dextran. This homologous sequence (Val(837)-Met(932) for PsDex and Tyr(404)-Tyr(492) for CITase), similar to carbohydrate-binding module 35, was not found in other endodextranases (Dexs) devoid of CITase activity. These results support the classification of GH-66 enzymes into three types: (i) Dex showing only dextranolytic activity, (ii) Dex catalyzing hydrolysis with low cyclization activity, and (iii) CITase showing CI-forming activity with low dextranolytic activity. The fact that a C-terminal truncated enzyme (having Ala(39)-Ser(1304)) has 50% wild-type PsDex activity indicates that the C-terminal 392 residues are not involved in hydrolysis. GH-66 enzymes possess four conserved acidic residues (Asp(189), Asp(340), Glu(412), and Asp(1254) of PsDex) of catalytic candidates. Their amide mutants decreased activity (1/1,500 to 1/40,000 times), and D1254N had 36% activity. A chemical rescue approach was applied to D189A, D340G, and E412Q using alpha-isomaltotetraosyl fluoride with NaN3. D340G or E412Q formed a beta- or alpha-isomaltotetraosyl azide, respectively, strongly indicating Asp(340) and Glu(412) as a nucleophile and acid/base catalyst, respectively. Interestingly, D189A synthesized small sized dextran from alpha-isomaltotetraosyl fluoride in the presence of NaN3.

DOI： 10.1074/jbc.M111.339036

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alpha-Glucosidase is in the glycoside hydrolase family 13 (13AG) and 31 (31AG). Only 31AGs can hydrate the D-glucal double bond to form alpha-2-deoxyglucose. Because 1,5-anhydrofructose (AF), having a 2-OH group, mimics the oxocarbenium ion transition state, AF may be a substrate for alpha-glucosidases. alpha-Glucosidase-catalyzed hydration produced alpha-glucose from AF, which plateaued with time. Combined reaction with alpha-1,4-glucan lyase and 13AG eliminated the plateau. Aspergillus niger alpha-glucosidase (31AG), which is stable in organic solvent, produced ethyl alpha-glucoside from AF in 80% ethanol. The findings indicate that alpha-glucosidases catalyze trans-addition. This is the first report of alpha-glucosidase-associated glucose formation from AF, possibly contributing to the salvage pathway of unutilized AF.

DOI： 10.1074/jbc.C112.360909

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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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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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The glycoside hydrolase family 5 (GH5) endo-beta-1,4-mannanases ManA and ManC from Aspergillus nidulans FGSC A4 were produced in Pichia pastor-is X33 and purified in high yields of 120 and 145 mg/L, respectively, from the culture supernatants. Both enzymes showed increasing catalytic efficiency (k(cat)/K-M) towards beta-1,4 mannooligosaccharides with the degree of polymerisation (DP) from 4 to 6 and also hydrolysed konjac glucomannan, guar gum and locust bean gum galactomannans. ManC had up to two-fold higher catalytic efficiency for DP 5 and 6 manno-oligosaccharides and also higher activity than ManA towards mannans. Remarkably, ManC compared to ManA transglycosylated mannotetraose with formation of longer beta-1,4 manno-oligosaccharides 8-fold more efficiently and was able to use mannotriose, melezitose and isomaltotriose out of 36 tested acceptors resulting in novel penta- and hexasaccharides, whereas ManA used only mannotriose as acceptor. ManA and ManC share 39% sequence identity and homology modelling suggesting that they have very similar substrate interactions at subsites +1 and +2 except that ManC Trp283 at subsite +1 corresponded to Ser289 in ManA Site-directed mutagenesis to ManA S289W lowered K-M for manno-oligosaccharides by 30-45% and increased transglycosylation yield by 50% compared to wild-type. Conversely. K-M for ManC W283S was increased, the transglycosylation yield was reduced by 30-45% and furthermore activity towards mannans decreased below that of ManA. This first mutational analysis in subsite +1 of GH5 endo-beta-1,4-mannanases indicated that Trp283 in ManC participates in discriminating between mannan substrates with different extent of branching and has a role in transglycosylation and substrate affinity. (C) 2011 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbapap.2011.08.003

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DOI： 10.1016/j.bbapap.2011.08.003

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Language：English   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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Multiple forms of native and recombinant endo-dextranases (Dexs) of the glycoside hydrolase family (GH) 66 exist. The GH 66 Dex gene from Streptococcus mutans ATCC 25175 (SmDex) was expressed in Escherichia coli. The recombinant full-size (95.4 kDa) SmDex protein was digested to form an 89.8 kDa isoform (SmDex90). The purified SmDex90 was proteolytically degraded to more than seven polypeptides (23-70 kDa) during long storage. The protease-insensitive protein was desirable for the biochemical analysis and utilization of SmDex. GH 66 Dex was predicted to comprise four regions from the N- to C-termini: N-terminal variable region (N-VR), conserved region (CR), glucan-binding site (GBS), and C-terminal variable region (C-VR). Five truncated SmDexs were generated by deleting N-VR, GBS, and/or C-VR. Two truncation-mutant enzymes devoid of C-VR (TM-NCG Delta) or N-VR/C-VR (TM-Delta CG Delta) were catalytically active, thereby indicating that N-VR and C-VR were not essential for the catalytic activity. TM-Delta CG Delta did not accept any further protease-degradation during long storage. TM-NCG Delta and TM-Delta CG Delta enhanced substrate hydrolysis, suggesting that N-VR and C-VR induce hindered substrate binding to the active site.

DOI： 10.1007/s00253-011-3201-y

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Starch-binding domains are noncatalytic carbohydrate-binding modules that mediate binding to granular starch. The starch-binding domains from the carbohydrate-binding module family 45 (CBM45, http://www.cazy.org) are found as N-terminal tandem repeats in a small number of enzymes, primarily from photosynthesizing organisms. Isolated domains from representatives of each of the two classes of enzyme carrying CBM45-type domains, the Solanum tuberosum alpha-glucan, water dikinase and the Arabidopsis thaliana plastidial alpha-amylase 3, were expressed as recombinant proteins and characterized. Differential scanning calorimetry was used to verify the conformational integrity of an isolated CBM45 domain, revealing a surprisingly high thermal stability (T-m of 84.8 degrees C). The functionality of CBM45 was demonstrated in planta by yellow/green fluorescent protein fusions and transient expression in tobacco leaves. Affinities for starch and soluble cyclodextrin starch mimics were measured by adsorption assays, surface plasmon resonance and isothermal titration calorimetry analyses. The data indicate that CBM45 binds with an affinity of about two orders of magnitude lower than the classical starch-binding domains from extracellular microbial amylolytic enzymes. This suggests that low-affinity starch-binding domains are a recurring feature in plastidial starch metabolism, and supports the hypothesis that reversible binding, effectuated through low-affinity interaction with starch granules, facilitates dynamic regulation of enzyme activities and, hence, of starch metabolism.

DOI： 10.1111/j.1742-4658.2011.08043.x

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Two beta-xylosidases of glycoside hydrolase family 3 (GH 3) from Aspergillus nidulans FGSC A4, BxlA and BxlB were produced recombinantly in Pichia pastoris and secreted to the culture supernatants in yields of 16 and 118 mg/L, respectively. BxlA showed about sixfold higher catalytic efficiency (k(cat)/K-m) than BxlB towards para-nitrophenyl beta-o-xylopyranoside (pNPX) and beta-1,4-xylo-oligosaccharides (degree of polymerisation 2-6). For both enzymes k(cat)/K-m decreased with increasing beta-1,4-xylo-oligosaccharide chain length. Using pNPX as donor with 9 monosaccharides, 7 disaccharides and two sugar alcohols as acceptors 18 different p-xylosyl-oligosaccharides were synthesised in 2-36% (BxlA) and 6-66% (BxlB) yields by transxylosylation. BxlA utilised the monosaccharides D-mannose, D-lyxose, D-talose, D-xylose, D-arabinose, L-fucose, D-glucose, D-galactose and D-fructose as acceptors, whereas BxlB used the same except for D-lyxose, D-arabinose and L-fucose. BxlB transxylosylated the disaccharides xylobiose, lactulose, sucrose, lactose and turanose in upto 35% yield, while BxlA gave inferior yields on these acceptors. The regioselectivity was acceptor dependent and primarily involved beta-1,4 or 1,6 product linkage formation although minor products with different linkages were also obtained. Five of the 18 transxylosylation products obtained from D-lyxose, D-galactose, turanose and sucrose (two products) as acceptors were novel xylosyl-oligosaccharides, beta-D-Xylp-(1 -&gt; 6)-D-Galp, beta-D-Xylp-(1 -&gt; 4)-alpha-D-Glcp-(1 -&gt; 3)- beta-D-Fruf. beta-D-Xylp-(1 -&gt; 4)-alpha-D-Glcp-(1 -&gt; 2)-beta-D-Fruf, and beta-D-Xylp-(1 -&gt; 6)-beta-D-Fruf-(2 -&gt; 1)-alpha-D-Glcp, as structure-determined by 2D NMR, indicating that GH3 beta-xylosidases are able to transxylosylate a larger variety of carbohydrate acceptors than earlier reported. Furthermore, transxylosylation of certain acceptors resulted in mixtures. Some of these products are also novel, but the structures of the individual products could not be determined. (C) 2010 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.carres.2010.12.010

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Inverting cellobiose phosphorylase (CtCBP) and cellodextrin phosphorylase (CtCDP) from Clostridium thermocellum ATCC27405 of glycoside hydrolase family 94 catalysed reverse phosphorolysis to produce cellobiose and cellodextrins in 57% and 48% yield from alpha-D-glucose 1-phosphate as donor with glucose and cellobiose as acceptor, respectively. Use of alpha-D-glucosyl 1-fluoride as donor increased product yields to 98% for CtCBP and 68% for CtCDP. CtCBP showed broad acceptor specificity forming beta-glucosyl disaccharides with beta-(1--&gt;4)- regioselectivity from five monosaccharides as well as branched beta-glucosyl trisaccharides with beta-(1--&gt;4)-regioselectivity from three (1--&gt;6)-linked disaccharides. CtCDP showed strict beta-(1--&gt;4)-regioselectivity and catalysed linear chain extension of the three beta-linked glucosyl disaccharides, cellobiose, sophorose, and laminaribiose, whereas 12 tested monosaccharides were not acceptors. Structure analysis by NMR and ESI-MS confirmed two beta-glucosyl oligosaccharide product series to represent novel compounds, i.e. beta-D-glucopyranosyl-[(1--&gt;4)-beta-D-glucopyranosyl](n)-(1--&gt;2)-D-glucopyranose, and beta-D-glucopyranosyl-(1--&gt;4)-beta-D-glucopyranosyl](n)-(1--&gt;3)-D-glucopyranose (n = 1-7). Multiple sequence alignment together with a modelled CtCBP structure, obtained using the crystal structure of Cellvibrio gilvus CBP in complex with glucose as a template, indicated differences in the subsite +1 region that elicit the distinct acceptor specificities of CtCBP and CtCDP. Thus Glu636 of CtCBP recognized the Cl hydroxyl of beta-glucose at subsite +1, while in CtCDP the presence of Ala800 conferred more space, which allowed accommodation of Cl substituted disaccharide acceptors at the corresponding subsites +1 and +2. Furthermore, CtCBP has a short Glu496-Thr500 loop that permitted the C6 hydroxyl of glucose at subsite +1 to be exposed to solvent, whereas the corresponding longer loop Thr637-Lys648 in CtCDP blocks binding of C6-linked disaccharides as acceptors at subsite +1. High yields in chemoenzymatic synthesis, a novel regioselectivity, and novel oligosaccharides including products of CtCDP catalysed oligosaccharide oligomerisation using alpha-D-glucosyl 1-fluoride, all together contribute to the formation of an excellent basis for rational engineering of CBP and CDP to produce desired oligosaccharides. (C) 2010 Elsevier Masson SAS. All rights reserved.

DOI： 10.1016/j.biochi.2010.07.013

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Three kinds of ω-epoxyalkyl α-glucopyranosides (3′,4′-epoxybutyl α-<small>D</small>-glucopyranoside (E4G), 4′,5′-epoxypentyl α-<small>D</small>-glucopyranoside (E5G) and 5′,6′-epoxyhexyl α-<small>D</small>-glucopyranoside (E6G)), having alkyl chains of different lengths at their aglycone moieties, inactivated the endodextranase from <i>Streptococcus mutans</i> ATCC 25175 (SmDex) irreversibly with the pseudo-first order kinetics. Alkyl chain length-dependent inactivation was observed and the degree of activity loss was E5G, E6G and E4G, in that order, implying that the distance between epoxide group and glucosyl residue of ω-epoxyalkyl α-glucopyranoside was important in the modification of endodextranase. Inactivation by E5G followed the model of reversible intermediate-complex formation mechanism (suicide inhibitor-based mechanism). The rate constant of irreversible inactivation (<i>k</i>) and the dissociation constant of intermediate-complex (<i>K</i><small>R</small>) of SmDex and E5G were 0.44 min^-1 and 1.45 m<small>M</small>, respectively. Hydrolytic reaction product (isomaltose) protected SmDex from E5G-inactivation, suggesting that E5G bound to the catalytic site of SmDex. This is the first report that ω-epoxyalkyl α-glucopyranoside becomes a suicide substrate for endodextranase.

DOI： 10.5458/jag.57.269

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Lactobacillus acidophilus NCFM maltose phosphorylase (LaMP) of the (alpha/alpha)(6)-barrel glycoside hydrolase family 65 (GH65) catalyses both phosphorolysis of maltose and formation of maltose by reverse phosphorolysis with beta-glucose 1-phosphate and glucose as donor and acceptor, respectively. LaMP has about 35 and 26% amino acid sequence identity with GH65 trehalose phosphorylase (TP) and kojibiose phosphorylase (KP) from Thermoanaerobacter brockii ATCC35047. The structure of L. brevis MP and multiple sequence alignment identified (alpha/alpha)(6)-barrel loop 3 that forms the rim of the active site pocket as a target for specificity engineering since it contains distinct sequences for different GH65 disaccharide phosphorylases. Substitution of LaMP His413-Glu421, His413-Ile418 and His413-Glu415 from loop 3, that include His413 and Glu415 presumably recognising the alpha-anomeric O-1 group of the glucose moiety at subsite +1, by corresponding segments from Ser426-Ala431 in TP and Thr419-Phe427 in KP, thus conferred LaMP with phosphorolytic activity towards trehalose and kojibiose, respectively. Two different loop 3 LaMP variants catalysed the formation of trehalose and kojibiose in yields superior of maltose by reverse phosphorolysis with (alpha 1, alpha 1)- and alpha-(1,2)- regioselectivity, respectively, as analysed by nuclear magnetic resonance. The loop 3 in GH65 disaccharide phosphorylase is thus a key determinant for specificity both in phosphorolysis and in regiospecific reverse phosphorolysis.

DOI： 10.1093/protein/gzq055

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The alpha-galactosidase from Aspergillus nidulans (AglC) belongs to a phylogenetic cluster containing eukaryotic alpha-galactosidases and alpha-galacto-oligosaccharide synthases of glycoside hydrolase family 36 (GH36). The recombinant AglC, produced in high yield (0.65 g center dot L-1 culture) as His-tag fusion in Escherichia coli, catalysed efficient transglycosylation with alpha-(1 -&gt; 6) regioselectivity from 40 mm 4-nitrophenol alpha-d-galactopyranoside, melibiose or raffinose, resulting in a 37-74% yield of 4-nitrophenol alpha-d-Galp-(1 -&gt; 6)-d-Galp, alpha-d-Galp-(1 -&gt; 6)-alpha-d-Galp-(1 -&gt; 6)-d-Glcp and alpha-d-Galp-(1 -&gt; 6)-alpha-d-Galp-(1 -&gt; 6)-d-Glcp-(alpha 1 -&gt;beta 2)-d-Fruf (stachyose), respectively. Furthermore, among 10 monosaccharide acceptor candidates (400 mm) and the donor 4-nitrophenol alpha-d-galactopyranoside (40 mm), alpha-(1 -&gt; 6) linked galactodisaccharides were also obtained with galactose, glucose and mannose in high yields of 39-58%. AglC did not transglycosylate monosaccharides without the 6-hydroxymethyl group, i.e. xylose, l-arabinose, l-fucose and l-rhamnose, or with axial 3-OH, i.e. gulose, allose, altrose and l-rhamnose. Structural modelling using Thermotoga maritima GH36 alpha-galactosidase as the template and superimposition of melibiose from the complex with human GH27 alpha-galactosidase supported that recognition at subsite +1 in AglC presumably requires a hydrogen bond between 3-OH and Trp358 and a hydrophobic environment around the C-6 hydroxymethyl group. In addition, successful transglycosylation of eight of 10 disaccharides (400 mm), except xylobiose and arabinobiose, indicated broad specificity for interaction with the +2 subsite. AglC thus transferred alpha-galactosyl to 6-OH of the terminal residue in the alpha-linked melibiose, maltose, trehalose, sucrose and turanose in 6-46% yield and the beta-linked lactose, lactulose and cellobiose in 28-38% yield. The product structures were identified using NMR and ESI-MS and five of the 13 identified products were novel, i.e. alpha-d-Galp-(1 -&gt; 6)-d-Manp; alpha-d-Galp-(1 -&gt; 6)-beta-d-Glcp-(1 -&gt; 4)-d-Glcp; alpha-d-Galp-(1 -&gt; 6)-beta-d-Galp-(1 -&gt; 4)-d-Fruf; alpha-d-Galp-(1 -&gt; 6)-d-Glcp-(alpha 1 -&gt;alpha 1)-d-Glcp; and alpha-d-Galp-(1 -&gt; 6)-alpha-d-Glcp-(1 -&gt; 3)-d-Fruf.

DOI： 10.1111/j.1742-4658.2010.07763.x

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Lactobacillus acidophilus NCFM maltose phosphorylase (LaMalP) of glycoside hydrolase family 65 catalysed enzymatic synthesis of alpha-(1 -&gt; 4)-glucostdic disacchandes from maltose and five monosacchandes in a coupled phosphorolysis/reverse phosphorolysis one-pot reaction Thus phosphorolysis of maltose to 0-glucose I -phosphate circumvented addition of costly 0-glucose 1-phosphate for reverse phosphorolysis with different monosacchande acceptors, resulting in 91%, 89%, 88%, 86% and 84% yield of alpha-a-glucopyranosyl-(1 4)-N-acetyl-a-glucosain inopyranose IN-acetyl-maltosamine I, alpha-b-glucopyranosyl( 1 -&gt; 4)-o-glucosaminopyranose I maltosaminej, a-a-glucopyranosyl-(1 -&gt; 4)-b-mannopyranose, alpha-n-glucopyranosyl-(1 -&gt; 4)-t-fucopyranose and alpha-b-glucopyranosyl-(1 -&gt; 4)-D-xylopyranose, respectively, from 0 1 M maltose, 0.5 M N-acetyl glucosamine, 0.1 M glucosamine. 0.1 M mannose, 1 M t-fucose and 0.5 M xylose in 02 M phosphate-citrate p1-I 62 These current yields of 0.27-0.34 g of disaccharide products from 10 mL reaction mixtures are easy to scale up and moreover the strategy can be applied to large-scale production of other oligosacchandes from low-cost disacchandes as catalysed by phosphorylases with different substrate specificities 2010 Elsevier Ltd All rights reserved.

DOI： 10.1016/j.carres.2010.03.021

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A gene cluster involved in maltodextrin transport and metabolism was identified in the genome of Lactobacillus acidophilus NCFM, which encoded a maltodextrin-binding protein, three maltodextrin ATP-binding cassette transporters and five glycosidases, all under the control of a transcriptional regulator of the LacI-GalR family. Enzymatic properties are described for recombinant maltose phosphorylase (MalP) of glycoside hydrolase family 65 (GH65), which is encoded by malP (GenBank: AAV43670.1) of this gene cluster and produced in Escherichia coli. MalP catalyses phosphorolysis of maltose with inversion of the anomeric configuration releasing beta-glucose 1-phosphate (beta-Glc 1-P) and glucose. The broad specificity of the aglycone binding site was demonstrated by products formed in reverse phosphorolysis using various carbohydrate acceptor substrates and beta-Glc 1-P as the donor. MalP showed strong preference for monosaccharide acceptors with equatorial 3-OH and 4-OH, such as glucose and mannose, and also reacted with 2-deoxy glucosamine and 2-deoxy N-acetyl glucosamine. By contrast, none of the tested di- and trisaccharides served as acceptors. Disaccharide yields obtained from 50 mm beta-Glc 1-P and 50 mm glucose, glucosamine, N-acetyl glucosamine, mannose, xylose or l-fucose were 99, 80, 53, 93, 81 and 13%, respectively. Product structures were determined by NMR and ESI-MS to be alpha-Glcp-(1 -&gt; 4)-Glcp (maltose), alpha-Glcp-(1 -&gt; 4)-GlcNp (maltosamine), alpha-Glcp-(1 -&gt; 4)-GlcNAcp (N-acetyl maltosamine), alpha-Glcp-(1 -&gt; 4)-Manp, alpha-Glcp-(1 -&gt; 4)-Xylp and alpha-Glcp-(1 -&gt; 4)- l-Fucp, the three latter being novel compounds. Modelling using L. brevis GH65 as the template and superimposition of acarbose from a complex with Thermoanaerobacterium thermosaccharolyticum GH15 glucoamylase suggested that loop 3 of MalP involved in substrate recognition blocked the binding of candidate acceptors larger than monosaccharides.

DOI： 10.1111/j.1742-4658.2009.07445.x

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α-Glucosidase (EC 3.2.1.20), an exo-glycosylase to hydrolyze α-glucosidic linkage, is characterized by the variety of substrate specificity. Enzyme also catalyzes the transglucosylation, on which industrial interests focus due to the production of valuable glucooligosaccharides. α-Glucosidase is a physiologically important enzyme in most of organisms (microorganisms, insects, plants and animals including human). Therefore, there are many types of α-glucosidases to display unique functions, in which we are interested. This report describes the recently analyzed unique functions of α-glucosidases by mainly focusing on honeybee α-glucosidase isoenzymes, dextran glucosidase, multiple forms of rice α-glucosidases, and Escherichia coli α-xylosidase. © 2008 Woodhead Publishing Limited. All rights reserved.

DOI： 10.1533/9781845695750.1.64

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Insoluble starch granules stored in plant seeds have generally been considered to be degraded effectively by the combination of amylolytic enzymes following initial attack by de novo synthesized alpha-amylase at germination. We have shown that rice (Oryza sativa L., var Nipponbare) alpha-glucosidase isozymes (ONG1, ONG2, and ONG3) are also capable of binding to and degrading starch granules directly, indicating the direct liberation of glucose from starch granules by alpha-glucosidase at germination. ONG1 and ONG2 are encoded in a distinct locus of the rice genome, while ONG2 and ONG3 are generated by alternative splicing. Interestingly, each of the alpha-glucosidase isozymes showed different action toward starch granules. In addition, two ONG2 isoforms were found to be produced by post-translational proteolysis. The proteolysis induced changes in binding to and degradation of starch granules.

DOI： 10.1080/10242420701788736

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In rice (Oryza sativa L., var Nipponbare) seeds, there were three mRNAs encoding for function-unknown hydrolase family 31 homologous proteins (ONGX-H1, ONGX-H3 and ONGX-H4): ONGX-H1 mRNA was expressed in ripening stage and mRNAs of ONGX-H3 and ONGX-H4 were found in both the ripening and germinating stages [Nakai et al., (2007) Biochimie 89, 49-62]. This article describes that the recombinant proteins of ONGX-H1 (rONGXG-H1), ONGX-H3 (rONGXG-H3) and ONG-H4 (rONGXG-H4) were overproduced in Pichia pastoris as fusion protein with the alpha-factor signal peptide of Saccharomyces cerevisiae. Purified rONGXG-H1 and rONGXG-H3 efficiently hydrolysed malto-oligosaccharides, kojibiose, nigerose and soluble starch, indicating that ONGX-H1 and ONGX-H3 are alpha-glucosidases. Their substrate specificities were similar to that of ONG2, a main alpha-glucosidase in the dry and germinating seeds. The rONGXG-H1 and rONGX-H3 demonstrated the lower ability to adsorb to and degradation of starch granules than ONG2 did, suggesting that three a-glucosidases, different in action to starch granules, were expressed in ripening stage. Additionally, purified rONGXG-H4 showed the high activity towards alpha-xylosides, in particular, xyloglucan oligosaccharides. The enzyme hardly hydrolysed alpha-glucosidic linkage, so that ONGX-H4 was an alpha-xylosidase. alpha-Xylosidase encoded in rice genome was found for the first time.

DOI： 10.1093/jb/mvm174

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Two isoforms of alpha-glucosidases (ONG2-I and ONG2-II) were purified from dry rice seeds (Oryza sativa L., var Nipponbare). Both ONG2-I and ONG2-II were the gene products of ONG2 mRNA expressed in ripening seeds. Each enzyme consisted of two components of 6 kDa-peptide and 88 kDa-peptide encoded by this order in ONG2 cDNA (ong2), and generated by post-translational proteolysis. The 88 kDa-peptide of ONG2-II had 10 additional N-terminal amino acids compared with the 88 kDa-peptide of ONG2-I. The peptides between 6 kDa and 88 kDa components (26 amino acids for ONG2-I and 16 for ONG2-II) were removed by post-translational proteolysis. Proteolysis induced changes in adsorption and degradation of insoluble starch granules. We also obtained three alpha-glucosidase cDNAs (ong1, ong3, and ong4) from ripening seeds. The ONG1, ONG2, and ONG4 genes were situated in distinct locus of rice genome. The transcripts encoding ONG2 and ONG3 were generated by alternative splicing. Members of alpha-glucosidase multigene family are differentially expressed during ripening and germinating stages in rice. (c) 2006 Elsevier Masson SAS. All rights reserved.

DOI： 10.1016/j.biochi.2006.09.014

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a-Glucosidase (JHGase I) was purified from a Japanese subspecies of eastern honeybee (Apis cerana japonica) as an electrophoretically homogeneous protein. Enzyme activity of the crude extract was mainly separated into two fractions (component I and II) by salting-out chromatography. JHGase I was isolated from component I by further purification procedure using CM-Toyopearl 650M and Sephacryl S-100. JHGase I was a monomeric glycoprotein (containing 15% carbohydrate), of which the molecular weight was 82,000. Enzyme displayed the highest activity at pH 5.0, and was stable up to 40 degrees C and in a pH-range of 4.5-10.5. JHGase I showed unusual kinetic features: the negative cooperative behavior on the intrinsic reaction on cleavage of sucrose, maltose, and p-nitrophenyl alpha-glucoside, and the positive cooperative behavior on turanose. We isolated cDNA (1,930bp) of JHGase I, of which the deduced amino-acid sequence (577 residues) confirmed that JHGase I was a member of alpha-amylase family enzymes. Western honeybees (Apis mellifera) had three alpha-glucosidase isoenzymes (WHGase I, II, and III), in which JHGase I was considered to correspond to WHGase I.

DOI： 10.1271/bbb.60302

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In germination of plant seeds, storage starch is principally degraded by the combination of amylolytic enzymes. As starch is an insoluble granule, a conventional view of the degradation pathway is that the initial attack is performed by α-amylase having the starch granule-binding ability. Plant α-glucosidase was also capable of adsorbing and hydrolyzing starch granules directly, indicating a possible second pathway: the direct liberation of glucose from starch granules by plant α-glucosidase rather than the α-amylase-mediated system. We found that the starch-binding site of plant α-glucosidase was situated in its C-terminal region, of which function was independent of the catalytic domain. Site-directed mutagenesis analysis on the aromatic amino acid residues conserved in this region revealed that Trp803 and Phe895 of rice α-glucosidase were responsible for binding to starch granules. Mold α-glucosidases were devoid of the ability to attack starch granules. In plant seeds, multiple α-glucosidases have been observed. Two types of α-glucosidases, insoluble and soluble enzymes, were found in the germinating stage of rice. Expression patterns of their activities classified 14 rice varieties into two groups (Groups 1 and 2). In Group 1 varieties, insoluble enzyme decreased immediately after germination. The soluble enzyme increased by <i>de novo</i> synthesis. Group 2 maintained a constant activity level of insoluble and soluble α-glucosidases in germination. From Groups 1 and 2, we selected varieties of Akamai and Nipponbare, respectively, of which analysis elucidated interesting molecular mechanisms of insoluble and soluble enzymes: i) isoform and isozyme formations by post-translational proteolysis as well as by chromosomal gene expression; ii) characterization of purified enzymes exhibiting different activities to starch granules.

DOI： 10.5458/jag.53.137

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A starch-hydrolyzing enzyme from Schwanniomyces occidentalis has been reported to be a novel glucoamylase, but there is no conclusive proof that it is glucoamylase. An enzyme having the hydrolytic activity toward soluble starch was purified from a strain of S. occidentalis. The enzyme showed high catalytic efficiency (k(cat)/K-m) for maltooligosaccharides, compared with that for soluble starch. The product anomer was alpha-glucose, differing from glucoamylase as a beta-glucose producing enzyme. These findings are striking characteristics of alpha-glucosidase. The DNA encoding the enzyme was cloned and sequenced. The primary structure deduced from the nucleotide sequence was highly similar to mold, plant, and mammalian alpha-glucosidases of alpha-glucosidase family II and other glucoside hydrolase family 31 enzymes, and the two regions involved in the catalytic reaction of alpha-glucosidases were conserved. These were no similarities to the so-called glucoamylases. It was concluded that the enzyme and also S. occidentalis glucoamylase, had been already reported, were typical alpha-glucosidases, and not glucoamylase.

DOI： 10.1271/bbb.69.1905

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

Aspergillus niger alpha-glucosidase (ANGase) was used for an efficient syntheses of alkyl alpha-D-2-deoxyglucosides (A2DGs) and for regioselectivity studies of alkoxy-hydro additions Of D-glucal in the presence of alkyl alcohols. ANGase showed a high stability with respect to the high concentration of alkyl alcohols. The reaction conditions were optimized for pH, temperature, alkyl alcohol concentration, and D-glucal concentration. On the basis of MS and NMR analyses, A2DGs were confirmed to have only an alpha-2deoxyglucosidic bond and the two-dimensional NMR (HMBC) spectra showed to be made up of 2-deoxyglucosyl and alkyl moieties. (C) 2004 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.tetasy.2004.11.046

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：VERSITA  

On the basis of amino-acid sequence similarity, alpha-glucosidases are mainly divided into two groups, GH-family 13 and 31 enzymes. The former enzyme group strongly recognizes the alpha-glucosyl-moiety in the heterogeneous substrates, such as synthetic alpha-glucoside and sucrose. The latter family member recognizes the maltosyl-structure of maltooligosaccharide more than the alpha-glucosyl-structure of heterogeneous substrate, and some enzymes show a higher activity towards polymer-substrate. The hydration of D-glucal as well as the hydrolysis of 2-deoxy-substrate was only catalyzed by the latter group, meaning less recognition of C2 OH-group in the non-reducing terminal glucose unit of the substrate. Inhibitory effects of acarviosine-derivatives, competitive inhibitors, were also different in two enzyme groups. Inhibition of isoacaxbose on GH-family 31 alpha-glucosidases were 20- to 450-times stronger than that on GH-family 13 alpha-glucosidases, whereas acarviosine-glucose inhibited both groups by almost the same level. Series of one point replacement works of the conserved acidic amino acids, Asp and Glu, of GH-family 31 alpha-glucosidase from fission yeast revealed that Asp481 and Asp647 were the catalytic residues. Asp481 and Asp647 are observed in the conservative sequences, region-A and region-B, respectively. The D481G mutant catalyzed the "alpha-glycosynthase-reaction" to form alpha-configurated products from beta-glucosyl fluoride and p-nitrophenyl monosacchaxides. Plant alpha-glucosidases showed starch granule-binding and -degrading abilities, in which their C-terminal region was responsible for the binding to raw starch.

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Language：English   Publisher：The Japanese Society of Applied Glycoscience  

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DOI： 10.5458/bag.4.2_147

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：WILEY-BLACKWELL  

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DOI： 10.1016/j.bbapap.2012.08.007

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DOI： 10.5458/bag.2.4_223

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Other Link： http://hdl.handle.net/10191/22583

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DOI： 10.5458/bag.2.2_117

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糖質リン酸分解酵素(ホスホリラーゼ)は、その厳密な基質特異性と反応の可逆性からオリゴ糖合成に利用可能である。しかし既知のホスホリラーゼは15種類のみで、新たな基質特異性を有する新規ホスホリラーゼの発見が強く望まれている。そこで、これまで数種のホスホリラーゼが単離されているClostridium phytofermentansが有するGlycoside Hydrolase Family65に属する遺伝子(cphy1874)をクローニングし、組換え酵素の酵素化学的性質を調査した。結果、Cphy1874はリン酸存在下でニゲロースに対し高い加リン酸分解活性を示すことが明らかになった。β-グルコース1リン酸と各種糖を作用させたところ,グルコースをアクセプターとした際に高い合成活性を示した。キシロース、1,5-アンヒドログルシトール、ガラクトース、メチル-α-グルコシドにおいてもアクセプターとなり、主生成物はいずれもα-1,3結合であった。このことから本酵素は,これまで報告の無かった新規加リン酸分解酵素ニゲロースホスホリラーゼであることが判明した。

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Language：English   Publisher：新潟大学農学部  

乳酸菌Lactobacillus acidophilus NCFMでのフルクトオリゴ糖代謝に関与する2つの糖質加水分解酵素(BfrA. β-フルクトシダーゼ;ScrB,スクロース6-リン酸ハイドロラーゼ)基質認識機構を明らかにすることを目的とし、各種組み換え酵素を用いてフルクトオリゴ糖に対する加水分解反応の速度論的解析を行った。ScrBはフルクトオリゴ糖よりスクロースに対して高い加水分解活性を示し、BfrAは短鎖なフルクトオリゴ糖に対して特異性を示すことを明らかにした。加えて、両酵素の基質特異性に関与する領域(BfrAループ1)およびアミノ酸残基(ScrB His71)を、ホモロジーモデリング法により推定した。

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DOI： 10.1271/kagakutoseibutsu.49.669

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Other Link： http://hdl.handle.net/10191/18620

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Other Link： http://agriknowledge.affrc.go.jp/RN/2010814991

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