Motomitsu Kitaoka

National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan

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Publications (158)378.77 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The glycoside hydrolase family (GH) 130 is composed of inverting phosphorylases that catalyze reversible phosphorolysis of β-d-mannosides. Here we report a glycoside hydrolase as a new member of GH130. Dfer_3176 from Dyadobacter fermentans showed no synthetic activity using α-d-mannose 1-phosphate but it released α-d-mannose from β-1,2-mannooligosaccharides with an inversion of the anomeric configuration, indicating that Dfer_3176 is a β-1,2-mannosidase. Mutational analysis indicated that two glutamic acid residues are critical for the hydrolysis of β-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 β-d-mannosidic bond.
    FEBS letters 10/2015; DOI:10.1016/j.febslet.2015.10.008 · 3.17 Impact Factor
  • Motomitsu Kitaoka ·
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    ABSTRACT: Phosphorylases are useful catalysts for the practical preparation of various sugars. The number of known specificities was 13 in 2002 and is now 30. The drastic increase in available genome sequences has facilitated the discovery of novel activities. Most of these novel phosphorylase activities have been identified through the investigations of glycoside hydrolase families containing known phosphorylases. Here, the diversity of phosphorylases in each family is described in detail.
    Applied Microbiology and Biotechnology 08/2015; 99(20). DOI:10.1007/s00253-015-6927-0 · 3.34 Impact Factor
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    ABSTRACT: 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 (GH) 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 (GlcUA) were determined at resolutions of up to 1.6 Å. 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 GlcUA. Structural and sequence comparisons with other GH94 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. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 06/2015; 290(30). DOI:10.1074/jbc.M115.664664 · 4.57 Impact Factor
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    ABSTRACT: Infant gut-associated bifidobacteria possess a metabolic pathway to utilize lacto-N-biose (Gal-β1,3-GlcNAc) and galacto-N-biose (Gal-β1,3-GalNAc) from human milk and glycoconjugates specifically. In this pathway, N-acetylhexosamine 1-kinase (NahK) catalyzes the phosphorylation of GlcNAc or GalNAc at the anomeric C1 position with ATP. Crystal structures of NahK have only been determined in the closed state. In this study, we determined open state structures of NahK in three different forms (apo, ADP complex, and ATP complex). A comparison of the open and closed state structures revealed an induced fit structural change defined by two rigid domains. ATP binds to the small N-terminal domain, and binding of the N-acetylhexosamine substrate to the large C-terminal domain induces a closing conformational change with a rotation angle of 16°. In the nucleotide binding site, two magnesium ions bridging the α-γ and β-γ phosphates were identified. A mutational analysis indicated that a residue coordinating both of the two magnesium ions (Asp228) is essential for catalysis. The involvement of two magnesium ions in the catalytic machinery is structurally similar to the catalytic structures of protein kinases and aminoglycoside phosphotransferases, but distinct from the structures of other anomeric kinases or sugar 6-kinases. These findings help to elucidate the possible evolutionary adaptation of substrate specificities and induced fit mechanism.
    Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics 05/2015; 1854(5). DOI:10.1016/j.bbapap.2015.01.011 · 2.75 Impact Factor
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    ABSTRACT: We describe the novel substrate specificities of two independently evolved lacto-N-biosidases (LnbX and LnbB) towards the sugar chains of globo- and ganglio-series glycosphingolipids. LnbX, a non-classified member of the glycoside hydrolase family, isolated from Bifidobacterium longum subsp. longum, was shown to liberate galacto-N-biose (GNB: Galβ1-3GalNAc) and 2'-fucosyl GNB (a type-4 trisaccharide) from Gb5 pentasaccharide and globo H hexasaccharide, respectively. LnbB, a member of the glycoside hydrolase family 20 isolated from Bifidobacterium bifidum, was shown to release GNB from Gb5 and GA1 oligosaccharides. This is the first report describing enzymatic release of β-linked GNB from natural substrates. These unique activities may play a role in modulating the microbial composition in the gut ecosystem, and may serve as new tools for elucidating the functions of sugar chains of glycosphingolipids. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Carbohydrate research 03/2015; 408:18-24. DOI:10.1016/j.carres.2015.03.005 · 1.93 Impact Factor
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    ABSTRACT: The aerobic soil bacterium Cellvibrio vulgaris has a β-mannan-degradation gene cluster, including unkA, epiA, man5A, and aga27A. Among these genes, epiA has been assigned to encode an epimerase for converting d-mannose to d-glucose, even though the amino acid sequence of EpiA is similar to that of cellobiose 2-epimerases (CEs). UnkA, whose function currently remains unknown, shows a high sequence identity to 4-O-β-d-mannosyl-d-glucose phosphorylase. In this study, we have investigated CE activity of EpiA and the general characteristics of UnkA using recombinant proteins from Escherichia coli. Recombinant EpiA catalyzed the epimerization of the 2-OH group of sugar residue at the reducing end of cellobiose, lactose, and β-(1→4)-mannobiose in a similar manner to other CEs. Furthermore, the reaction efficiency of EpiA for β-(1→4)-mannobiose was 5.5 × 10(4)-fold higher than it was for d-mannose. Recombinant UnkA phosphorolyzed β-d-mannosyl-(1→4)-d-glucose and specifically utilized d-glucose as an acceptor in the reverse reaction, which indicated that UnkA is a typical 4-O-β-d-mannosyl-d-glucose phosphorylase.
    Bioscience Biotechnology and Biochemistry 02/2015; 79(6):1-9. DOI:10.1080/09168451.2015.1012146 · 1.06 Impact Factor
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    ABSTRACT: 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.
    01/2015; 62(2). DOI:10.5458/jag.jag.JAG-2014_011
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    ABSTRACT: 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-β-oligomannan using β-1,2-mannobiose and d-mannose as the optimal acceptors, respectively, in the presence of the donor α-d-mannose 1-phosphate. Kinetic analysis of the phosphorolytic reaction toward 1,2-β-oligomannan revealed that these enzymes followed a typical sequential Bi Bi mechanism. The kinetic parameters of the phosphorolysis of 1,2-β-oligomannan indicate that Teth514_1788 and Teth514_1789 prefer 1,2-β-oligomannans containing a DP ≥3 and β-1,2-Man2, respectively. These results indicate that the two enzymes are novel inverting phosphorylases that exhibit distinct chain-length specificities toward 1,2-β-oligomannan. Here, we propose 1,2-β-oligomannan:phosphate α-d-mannosyltransferase as the systematic name and 1,2-β-oligomannan phosphorylase as the short name for Teth514_1788 and β-1,2-mannobiose:phosphate α-d-mannosyltransferase as the systematic name and β-1,2-mannobiose phosphorylase as the short name for Teth514_1789.
    PLoS ONE 12/2014; 9(12):e114882. DOI:10.1371/journal.pone.0114882 · 3.23 Impact Factor
  • Yuan Liu · Mamoru Nishimoto · Motomitsu Kitaoka ·
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    ABSTRACT: Three sugar 1-phosphates that are donor substrates for phosphorylases were produced at the gram scale from phosphoenolpyruvic acid and the corresponding sugars by the combined action of pyruvate kinase and the corresponding anomeric kinases in good yields. These sugar 1-phosphates were purified through two electrodialysis steps. α-d-Galactose 1-phosphate was finally isolated as crystals of dipotassium salts. α-d-Mannose 1-phosphate and 2-acetamido-2-deoxy-α-d-glucose 1-phosphate were isolated as crystals of bis(cyclohexylammonium) salts.
    Carbohydrate Research 10/2014; 401. DOI:10.1016/j.carres.2014.10.014 · 1.93 Impact Factor
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    ABSTRACT: The Bifidobacterium genus harbours several health promoting members of the gut microbiota. Bifidobacteria display metabolic specialization by preferentially utilizing dietary or host derived β-galactosides. This study investigates the biochemistry and structure of a glycoside hydrolase family 42 (GH42) β-galactosidase from the probiotic Bifidobacterium animalis subsp. lactis Bl-04 (BlGal42A). BlGal42A displays a preference for undecorated β1-6 and β1-3 linked galactosides and populates a phylogenetic cluster with close bifidobacterial homologues implicated in the utilization of N-acetyl substituted β1-3 galactosides from human milk and mucin. A long loop containing an invariant tryptophan in GH42, proposed to bind substrate at subsite +1, is identified here as specificity signature within this clade of bifidobacterial enzymes. Galactose binding at the subsite −1 of the active site induced conformational changes resulting in an extra polar interaction and the ordering of a flexible loop that narrows the active site. The amino-acid sequence of this loop provides an additional specificity signature within this GH42 clade. The phylogenetic relatedness of enzymes targeting β1-6 and β1-3 galactosides likely reflects structural differences between these substrates and β1-4 galactosides, containing an axial galactosidic bond. These data advance our molecular understanding of the evolution of sub-specificities that support metabolic specialization in the gut niche.
    Molecular Microbiology 10/2014; 94(5). DOI:10.1111/mmi.12815 · 4.42 Impact Factor
  • Ryota Fujii · Motomitsu Kitaoka · Kiyoshi Hayashi ·
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    ABSTRACT: We describe a simple and easy protocol to introduce random mutations into plasmid DNA: error-prone rolling circle amplification. A template plasmid is amplified via rolling circle amplification with decreased fidelity in the presence of MnCl2 and is used to transform a host strain resulting in a mutant library with several random point mutations per kilobase through the entire plasmid. The primary advantage of this method is its simplicity. This protocol does not require the design of specific primers or thermal cycling. The reaction mixture can be used for direct transformation of a host strain. This method allows rapid preparation of randomly mutated plasmid libraries, enabling wider application of random mutagenesis.
    Methods in Molecular Biology 07/2014; 1179:23-9. DOI:10.1007/978-1-4939-1053-3_2 · 1.29 Impact Factor
  • Ryota Fujii · Motomitsu Kitaoka · Kiyoshi Hayashi ·
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    ABSTRACT: Although proteins can be artificially improved by random insertion and deletion mutagenesis methods, these procedures are technically difficult. Here we describe a simple method called random insertional-deletional strand exchange mutagenesis (RAISE). This method is based on gene shuffling and can be used to introduce a wide variety of insertions, deletions, and substitutions. RAISE involves three steps: DNA fragmentation, attachment of a random short sequence, and reconstruction. This yields unique mutants and can be a powerful technique for protein engineering.
    Methods in Molecular Biology 07/2014; 1179:151-8. DOI:10.1007/978-1-4939-1053-3_10 · 1.29 Impact Factor
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    ABSTRACT: 2-O-α-Glucosylglycerol phosphorylase (GGP) from Bacillus selenitireducens catalyzes both the reversible phosphorolysis of 2-O-α-glucosylglycerol (GG) and the hydrolysis of β-D-glucose 1-phosphate (βGlc1P). 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 with the glucose analogue 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, βGlc1P 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 βGlc1P hydrolysis. Y572F also showed significantly reduced activities for both of these reactions. In contrast, W381F showed significantly reduced reverse phosphorolytic activity but retained βGlc1P 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 βGlc1P 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.
    Journal of Biological Chemistry 05/2014; 289(26). DOI:10.1074/jbc.M114.573212 · 4.57 Impact Factor
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    ABSTRACT: 4-O-β-d-Mannosyl-d-glucose phosphorylase (MGP), found in anaerobes, converts 4-O-β-d-mannosyl-d-glucose (Man-Glc) to α-d-mannosyl phosphate and d-glucose. It participates in mannan metabolism with cellobiose 2-epimerase (CE), which converts β-1,4-mannobiose to Man-Glc. A putative MGP gene is present in the genome of the thermophilic aerobe Rhodothermus marinus (Rm) upstream of the gene encoding CE. Konjac glucomannan enhanced production by R. marinus of MGP, CE, and extracellular mannan endo-1,4-β-mannosidase. Recombinant RmMGP catalyzed the phosphorolysis of Man-Glc through a sequential bi–bi mechanism involving ternary complex formation. Its molecular masses were 45 and 222 kDa under denaturing and nondenaturing conditions, respectively. Its pH and temperature optima were 6.5 and 75 °C, and it was stable between pH 5.5–8.3 and below 80 °C. In the reverse reaction, RmMGP had higher acceptor preferences for 6-deoxy-d-glucose and d-xylose than R. albus NE1 MGP. In contrast to R. albus NE1 MGP, RmMGP utilized methyl β-d-glucoside and 1,5-anhydro-d-glucitol as acceptor substrates.
    Bioscience Biotechnology and Biochemistry 04/2014; 78(2):263-270. DOI:10.1080/09168451.2014.882760 · 1.06 Impact Factor
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    ABSTRACT: 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-β-oligoglucans (Sopn: n denotes degree of polymerization) using sophorose (Sop2) as the acceptor and α-d-glucose 1-phosphate (Glc1P) as the donor. Lin1839r recognized glucose as a very weak acceptor substrate to form polymeric 1,2-β-glucan. The degree of polymerization of the 1,2-β-glucan gradually decreased with long-term incubation to generate a series of Sopns. 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 Sop2. These results indicate that the Lin1839 protein is a novel inverting phosphorylase that catalyzes reversible phosphorolysis of 1,2-β-glucan with a degree of polymerization of ≥3. We propose 1,2-β-oligoglucan: phosphate α-glucosyltransferase as the systematic name and 1,2-β-oligoglucan phosphorylase as the short name for this Lin1839 protein.
    PLoS ONE 03/2014; 9(3):e92353. DOI:10.1371/journal.pone.0092353 · 3.23 Impact Factor
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    Takanori Nihira · Yuka Saito · Ken'ichi Ohtsubo · Hiroyuki Nakai · Motomitsu Kitaoka ·
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    ABSTRACT: The glycoside hydrolase family (GH) 65 is a family of inverting phosphorylases that act on α-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 β-d-glucose 1-phosphate (βGlc1P) as donor. During the search for a suitable acceptor, we found that Bsel_2816 possessed hydrolytic activity on βGlc1P 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 H2 (18)O incorporation experiment and the anomeric analysis during the hydrolysis of βGlc1P 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-α-d-glucosylglycerol (GG) at the rate of 180 s(-1). Bsel_2816 phosphorolyzed GG through sequential Bi-Bi mechanism with a k cat of 95 s(-1). We propose 2-O-α-d-glucopyranosylglycerol: phosphate β-d-glucosyltransferase as the systematic name and 2-O-α-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.
    PLoS ONE 01/2014; 9(1):e86548. DOI:10.1371/journal.pone.0086548 · 3.23 Impact Factor

  • 01/2014; 61(2):59-66. DOI:10.5458/jag.jag.JAG-2013_013
  • Yu Ogawa · Kazuhiro Noda · Satoshi Kimura · Motomitsu Kitaoka · Masahisa Wada ·
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    ABSTRACT: In-vitro synthesis of (1→3)-β-D-glucan was performed using laminaribiose phosphorylase obtained by an extraction of Euglena gracilis with sucrose phosphorylase. The synthetic product was a linear (1→3)-β-D-glucan with a narrow distribution of degree of polymerization (DP) centered on DP=30. X-ray diffraction and electron microscopy revealed that the glucan molecules obtained were self-organized as highly crystalline hexagonal lamellae. This synthetic product has quite high structural homogeneity at every level from primary to higher-order structure, which is a great advantage for the detailed analyses of physiological functions of (1→3)-β-D-glucan.
    International journal of biological macromolecules 12/2013; 64. DOI:10.1016/j.ijbiomac.2013.12.027 · 2.86 Impact Factor
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    ABSTRACT: Glycoside hydrolase family 42 (GH42) includes β-galactosidases catalyzing the release of galactose from the non-reducing end of different β-D-galactosides. Health-promoting probiotic bifidobacteria, which are important members of the human gastrointestinal tract microbiota, produce GH42 enzymes enabling utilization of β-galactosides exerting prebiotic effects. However, insight into the specificity of individual GH42 enzymes with respect to substrate monosaccharide composition, glycosidic linkage and degree of polymerization is lagging. Kinetic analysis of natural and synthetic substrates resembling various milk and plant galactooligosaccharides, distinguishes the three GH42 members, Bga42A, Bga42B, and Bga42C, encoded by the probiotic Bifidobacterium longum subsp. infantis ATCC 15697, and revealed the glycosyl residue at subsite +1 and its linkage to the terminal galactose at subsite-1 to be key specificity determinants. Bga42A thus prefers the β1-3-galactosidic linkage from human milk and other β1-3- and β1-6-galactosides with glucose or galactose situated at subsite +1. By contrast Bga42B very efficiently hydrolyses 4-galactosyllactose (Galβ1-4Galβ1-4Glc) as well as 4-galactobiose (Galβ1-4Gal) and 4-galactotriose (Galβ1-4Galβ1-4Gal). The specificity of Bga42C resembles that of Bga42B, but the activity was one order of magnitude lower. Based on enzyme kinetics, gene organization and phylogenetic analyses Bga42C is proposed to act in the metabolism of arabinogalactan-derived oligosaccharides. The distinct kinetic signatures of the three GH42 enzymes correlate to unique sequence motifs denoting specific clades in a GH42 phylogenetic tree providing novel insight into GH42 subspecificities. Overall the data illustrate the metabolic adaptation of bifidobacteria to the β-galactoside rich gut niche and emphasize the importance and diversity of β-galactoside metabolism in probiotic bifidobacteria.
    Glycobiology 11/2013; 24(2). DOI:10.1093/glycob/cwt104 · 3.15 Impact Factor
  • Yoshiyuki Koyama · Masafumi Hidaka · Mamoru Nishimoto · Motomitsu Kitaoka ·
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    ABSTRACT: Galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) is the key enzyme in the enzymatic production of lacto-N-biose I. For the purpose of industrial use, we improved the thermostability of GLNBP by evolutionary engineering in which five substitutions in the amino acid sequence were selected from a random mutagenesis GLNBP library constructed using error-prone polymerase chain reaction. Among them, C236Y and D576V mutants showed considerably improved thermostability. Structural analysis of C236Y revealed that the hydroxyl group of Tyr236 forms a hydrogen bond with the carboxyl group of E319. The C236Y and D576V mutations together contributed to the thermostability. The C236Y/D576V mutant exhibited 20°C higher thermostability than the wild type.
    Protein Engineering Design and Selection 09/2013; 26(11). DOI:10.1093/protein/gzt049 · 2.54 Impact Factor

Publication Stats

2k Citations
378.77 Total Impact Points


  • 2010-2015
    • National Agriculture and Food Research Organization
      Tsukuba, Ibaraki, Japan
  • 2001-2015
    • National Food Research Institute
      Ibaragi, Ōsaka, Japan
  • 2013
    • Niigata University
      • Faculty of Agriculture
      Niahi-niigata, Niigata, Japan
  • 2009
    • The Ohio State University
      • Department of Chemistry and Biochemistry
      Columbus, Ohio, United States
  • 2008
    • Ishikawa Prefectural University
      Ноноичи, Ishikawa, Japan
  • 2004
    • The University of Tokyo
      • Department of Biotechnology
      Tōkyō, Japan
  • 2000
    • Yamagata University
      • Department of Material and Biological Chemistry
      Ямагата, Yamagata, Japan