掲載種別：研究論文（学術雑誌）   出版者・発行元：Elsevier BV  

DOI： 10.1016/j.carres.2020.108129

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1080/09168451.2019.1630257

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1074/jbc.RA118.007087

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DOI： 10.1016/j.ab.2018.08.021

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記述言語：英語  

DOI： 10.1016/j.carres.2018.08.005

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DOI： 10.1021/acs.biochem.8b00385

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記述言語：英語  

DOI： 10.1016/j.jdermsci.2018.02.006

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1074/jbc.RA117.001536

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記述言語：英語  

DOI： 10.1002/1873-3468.12911

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1074/jbc.M116.762724

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/srep42671

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PUBLIC LIBRARY SCIENCE  

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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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1007/s00253-016-7417-8

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.phytochem.2016.03.015

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1080/09168451.2015.1116926

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1371/journal.pone.0148870

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記述言語：英語   出版者・発行元：WILEY-BLACKWELL  

DOI： 10.1111/bjd.14003

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.febslet.2015.11.034

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.febslet.2015.10.008

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記述言語：英語  

DOI： 10.5458/jag.jag.JAG-2015_006

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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記述言語：英語   出版者・発行元：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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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1371/journal.pone.0114882

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担当区分：最終著者,　責任著者   記述言語：英語  

DOI： 10.5458/jag.jag.JAG-2013_012

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1074/jbc.M114.573212

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記述言語：英語  

DOI： 10.5458/jag.jag.JAG-2013_013

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1371/journal.pone.0092353

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担当区分：責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1371/journal.pone.0086548

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY  

A novel phosphorylase was characterized as new member of glycoside hydrolase family 94 from the cellulolytic bacterium Xanthomonas campestris and the fungus Neurospora crassa. The enzyme catalyzed reversible phosphorolysis of cellobionic acid. We propose 4-O-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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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

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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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1074/jbc.M113.469080

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記述言語：英語   出版者・発行元：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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担当区分：最終著者,　責任著者   記述言語：英語   出版者・発行元：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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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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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担当区分：筆頭著者,　責任著者   記述言語：英語   出版者・発行元：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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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

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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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.carres.2012.08.006

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.carres.2012.07.014

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1271/bbb.120473

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

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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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1128/JB.00622-12

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

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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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1074/jbc.C112.360909

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.carres.2011.12.019

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

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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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

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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記述言語：英語   出版者・発行元：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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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：SPRINGER  

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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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1111/j.1742-4658.2011.08043.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.carres.2010.12.010

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER  

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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記述言語：英語   出版者・発行元：日本応用糖質科学会  

<i>Streptococcus mutans</i> ATCC 25175由来のエンド型デキストラナーゼ(SmDex)に対し，3種類のω-エポキシアルキルα-<small>D</small>-グルコピラノシド(3′,4′-エポキシブチルα-<small>D</small>-グルコピラノシド(E4G)，4′,5′-エポキシペンチルα-<small>D</small>-グルコピラノシド(E5G)および5′,6′-エポキシヘキシルα-<small>D</small>-グルコピラノシド(E6G)：アグリコンのアルキル鎖長が異なる)を作用させると，SmDexは擬一次的な活性低下を示した．アルキル鎖長に依存した失活が認められ，失活の度合いはE5G > E6G > E4Gであった．したがってω-エポキシアルキルα-<small>D</small>-グルコピラノシドのグルコース残基とエポキシ基の距離が，SmDexの失活に対し重要であることが判明した．E5Gは可逆的な中間体を形成する失活機構(自殺基質型の失活機構)を与え，不活性化の一次定数(<i>k</i>)と中間体の解離定数(<i>K</i><small>R</small>)はそれぞれ0.44 min^-1および1.45 m<small>M</small>と算出された．SmDexの加水分解反応の生成物であるイソマルトースの存在によりE5Gの失活が防御されたため，E5GはSmDexの触媒部位に結合すると示唆された．本論文は，ω-エポキシアルキルα-<small>D</small>-グルコピラノシドがエンド型デキストラナーゼの自殺基質になることを示す初めての報告である．

DOI： 10.5458/jag.57.269

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS  

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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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

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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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCI LTD  

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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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC  

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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掲載種別：論文集(書籍)内論文  

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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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1080/10242420701788736

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：OXFORD UNIV PRESS  

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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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER  

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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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

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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担当区分：筆頭著者   記述言語：英語   出版者・発行元：The Japanese Society of Applied Glycoscience  

植物種子が発芽する際に貯蔵物質である澱粉は，各種加水分解酵素により酵素分解され，生長に必要な物質・エネルギーの供給源となる．澱粉は植物種子中に不溶性の澱粉粒として存在するため，現在までα-アミラーゼが唯一澱粉粒に直接作用できる酵素であると考えられてきた．しかし，我々は植物α-グルコシダーゼが澱粉粒への吸着・分解能をもつことを明らかにし，第2の澱粉分解経路 (澱粉粒にα-グルコシダーゼが作用し，直接グルコースを遊離する経路) の存在を示した．さらに，植物α-グルコシダーゼには，触媒ドメインとは独立して機能する澱粉粒吸着ドメインがC末端に存在することを解明した．本ドメインに保存された芳香族アミノ酸に対する部位特異的変異導入により，澱粉粒吸着に関与する残基を推定した．植物種子中には，複数のα-グルコシダーゼが存在する．我々は，イネ種子中に可溶性および不溶性 (界面活性剤により可溶化が可能) の性質を示す2種のアイソザイムを見出した．両酵素の発現様式から，14品種のイネ種子を二つのグループ (グループ1，2) に大別した．グループ1は，不溶性酵素のみが乾燥種子に検出されるイネ品種である．不溶性α-グルコシダーゼが登熟期で合成され，完熟に伴い種子中に保存されるが，発芽の進行につれ消失する．発芽後に可溶性酵素が新たに合成される．グループ2の品種では，可溶性および不溶性の酵素が乾燥種子に検出された．両酵素は登熟期で合成され，発芽期間中の活性は変化せず，一定のレベルで推移する．グループ1からは赤米を，グループ2では日本晴を実験対象に選び，α-グルコシダーゼの機能解析を行った．翻訳後修飾やゲノム遺伝子発現調節によるアイソフォームやアイソザイムの形成機構ならびに精製酵素を用いた性質の解明を行った．多様なα-グルコシダーゼが関与する澱粉代謝の一端が明らかにされた．N末領域に生じる翻訳後限定分解は，植物酵素に対し一般的に観察される現象であった．

DOI： 10.5458/jag.53.137

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：TAYLOR & FRANCIS LTD  

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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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.tetasy.2004.11.046

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

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記述言語：日本語   出版者・発行元：(一社)日本応用糖質科学会  

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記述言語：日本語   出版者・発行元：セルロース学会  

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記述言語：日本語   出版者・発行元：日本応用糖質科学会  

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記述言語：日本語   出版者・発行元：日本応用糖質科学会  

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記述言語：日本語   出版者・発行元：日本応用糖質科学会  

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記述言語：日本語   出版者・発行元：日本応用糖質科学会  

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記述言語：日本語   出版者・発行元：日本応用糖質科学会  

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記述言語：日本語   出版者・発行元：日本応用糖質科学会  

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記述言語：日本語   出版者・発行元：日本応用糖質科学会  

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記述言語：日本語   出版者・発行元：日本応用糖質科学会  

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記述言語：日本語   出版者・発行元：日本応用糖質科学会  

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記述言語：日本語   出版者・発行元：日本応用糖質科学会  

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記述言語：英語   出版者・発行元：日本応用糖質科学会  

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DOI： 10.5458/bag.4.2_147

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：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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記述言語：英語   出版者・発行元：新潟大学農学部  

We discovered an inverting maltose phosphorylase (Bsel2056) belonging to glycoside hydrolase family 65 from Bacillus selenitireducens MLS10, which possesses synthetic ability for α-D-glucosyl disaccharides and trisaccharides through the reverse phosphorolysis with β-D-glucose 1-phosphate as the donor. Bsel2056 showed the preference 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-α-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-α-D-glucopyranosyl-[1,2-α-D-glucopyranosyl]-D-glucose and 1,4-α-D-glucopyranosyl-[1,2-β-D-glucopyranosyl]-D-glucose, from 1,2-α-D-glucopyranosyl-D-glucose (kojibiose) and 1,2-β-D-glucopyranosyl-D-glucose (sophorose), respectively, as the acceptors. Modeling structure analysis using Lactobacillus brevis GH65 maltose phosphorylase as the template and superimposition of an inhibitory pseudomaltotetrasacchaide acarbose from a complex with Aspergillus awamori GH15 glucoamylase suggested that Bsel2056 possessed a binding space to accommodate the bulky C2 substituent of D-glucose.糖質加リン酸分解酵素(ホスホリラーゼ)は、その反応の可逆性と厳密な反応位置特異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16種類のみ報告されており、新たな特異性を有するホスホリラーゼの発見が望まれている。そこで今回Bacillus selenitireducens MLS10が有するGlycoside Hydrolase Family 65に属するマルトースホスホリラーゼ遺伝子(bsel2056)をクローニングし、組換え酵素の糖受容体特異性を調査した。結果、グルコースの２位にアミノ基を有するグルコサミンなど８種の単糖類を糖受容体とし、α-1,4- グルコ２糖を生産することが明らかになった。さらに既知のマルトースホスホリラーゼとは異なり、本酵素は１、２結合を有するグルコ２糖すなわちコウジビオースおよびソフォロースを糖受容体として認識し、分岐グルコ３糖を生産した。また3D モデリング解析の結果から、糖受容体として結合するグルコースの２位の水酸基周辺に本酵素特有の嵩高い置換基(アミノ基やグルコース残基)を収納するための結合スペースが認められた。

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その他リンク： http://hdl.handle.net/10191/22583

記述言語：日本語   出版者・発行元：日本応用糖質科学会  

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記述言語：英語   出版者・発行元：日本応用糖質科学会  

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DOI： 10.5458/bag.2.2_117

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記述言語：英語   出版者・発行元：新潟大学農学部  

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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記述言語：英語   出版者・発行元：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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記述言語：日本語   出版者・発行元：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

DOI： 10.1271/kagakutoseibutsu.49.669

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記述言語：英語   出版者・発行元：新潟大学農学部  

α-D-ガラクトース1-リン酸（Gal 1-P）の比色定量として、ガラクトース定量法にホスファターゼを組み合わせる方法が知られるが、ガラクトースおよびガラクトース含有オリゴ糖が混在する試料中でのGal 1-Pの定量法としては不都合があった。そこで我々はガラクトース代謝系酵素群に着目し、Gal 1-Pのみを検出およびGal 1-Pとガラクトースを同時に検出する2つの比色定量法を開発した。各種濃度のGal 1-Pに発色試薬（UDP-グルコース-ヘキソース-1-リン酸ウリジリルトランスフェラーゼ、ホスホグルコムターゼ、グルコース-6-リン酸デヒドロゲナーゼ、NAD＋またはThio-NAD＋、UDP-グルコース、グルコース1、6-ビスリン酸、MgCl2含有トリス緩衝液）を混合し、経時的に340nm（NAD＋）または400nm（Thio-NAD＋）の吸収を測定した結果、20分程度で平衡に達し吸光度はGal 1-P濃度に比例した。また上記試薬にムタロターゼ、ガラクトキナーゼおよびATPを追加することにより、各種濃度のガラクトースに対し同様の結果が得られた。また加リン酸分解酵素ラクト-N-ビオースホスホリラーゼの反応によって生じるGal 1-Pを、発色試薬中にて反応停止操作を伴なわず連続的にモニタリングした結果、算出された活性値は各酵素濃度に比例しており、本定量法を用いた活性測定法の妥当性・有用性が示された。

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その他リンク： http://hdl.handle.net/10191/18620

記述言語：英語   出版者・発行元：新潟大学農学部  

A gene cluster involved in maltose/maltodextrin metabolism was identified in the genome of Lactobacillus acidophilusNCFM. Enzymatic properties was described for MalP belonging to glycoside hydrolase family 65 (GH65), which is encodedby malP (GenBank: AAV43670.1) located in the gene cluster. MalP catalyses phosphorolysis of maltose with inversion ofthe anomeric configuration releasing β-glucose 1-phosphate and glucose. Broad acceptor specificity was demonstrated byreverse phosphorolysis using various carbohydrate acceptors and β-glucose 1-phosphate as donor. MalP showed strongpreference for monosaccharide acceptors with equatorial 3-OH and 4-OH, and reacted also with the 2-deoxy amino sugars.By contrast none of the tested di- and trisaccharides served as acceptor. Disaccharide yields obtained from 50 mM β-glucose 1-phosphate 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 α-D-glucopyranosyl-(1→4)-Dglucopyranose,α -D-glucopyranosyl-(1→4)-D-glucosaminopyranose,α -D-glucopyranosyl-(1→4)-N-acetyl-D-glucosaminopyranose,α -D-glucopyranosyl-(1→4)-D-mannopyranose, α-D-glucopyranosyl-(1→4)-D-xylopyranose, and α-D-glucopyranosyl-(1→4)-L-fucopyranose. Additionally MalP catalysed synthesis of the α-(1 → 4)-glucosidic disaccharides from maltose in a coupledphosphorolysis/reverse phosphorolysis one-pot reaction. Thus phosphorolysis of maltose to β-glucose 1-phosphatecircumvented addition of costly β-glucose 1-phosphate for reverse phosphorolysis with different monosacchaide acceptors.This strategy can be applied to large-scale production of valuable oligosaccharides from low-cost carbohydrates as catalysedby phosphorylases with different substrate specificity.Lactobacillus acidophilus NCFM のゲノム内に、マルトース/マルトデキストリン代謝に関与する遺伝子クラスターを見出した。本クラスター中には、グルコシドハイドロレースファミリー65に属する機能未知な糖質関連酵素（MalP）をコードする遺伝子が含まれている。そこで大腸菌を宿主として組み換え酵素を生産し、その酵素化学的諸性質を調査した結果、MalP はマルトースに特異的に作用する糖質加リン酸分解酵素であることが明らかになった。加えてMalP が触媒する糖質合成反応を用いて、６種のα（- 1→4）- グルコ二糖を高収率生産に成功した。

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その他リンク： http://agriknowledge.affrc.go.jp/RN/2010814991

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