Kojibiose

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Tomoyuki Nishimoto - One of the best experts on this subject based on the ideXlab platform.

Shigeharu Fukuda - One of the best experts on this subject based on the ideXlab platform.

  • structural and mutational analysis of substrate recognition in Kojibiose phosphorylase
    FEBS Journal, 2014
    Co-Authors: Satoshi Okada, Shigeharu Fukuda, Takuo Yamamoto, Tomoyuki Nishimoto, Hiroto Chaen, Hikaru Watanabe, Takayoshi Wakagi, Shinya Fushinobu
    Abstract:

    Glycoside hydrolase (GH) family 65 contains phosphorylases acting on maltose (Glc-α1,4-Glc), Kojibiose (Glc-α1,2-Glc), trehalose (Glc-α1,α1,-Glc), and nigerose (Glc-α1,3-Glc). These phosphorylases can efficiently catalyze the reverse reactions with high specificities, and thus can be applied to the practical synthesis of α-glucosyl oligosaccharides. Here, we determined the crystal structures of Kojibiose phosphorylase from Caldicellulosiruptor saccharolyticus in complex with glucose and phosphate and in complex with Kojibiose and sulfate, providing the first structural insights into the substrate recognition of a glycoside hydrolase family 65 enzyme. The loop 3 region comprising the active site of Kojibiose phosphorylase is significantly longer than the active sites of other enzymes, and three residues around this loop, Trp391, Glu392, and Thr417, recognize Kojibiose. Various mutants mimicking the residue conservation patterns of other phosphorylases were constructed by mutation at these three residues. Activity measurements of the mutants against four substrates indicated that Trp391 and Glu392, especially the latter, are required for the Kojibiose activity. Database Structural data are available in the Protein Data Bank database under accession numbers 3WIQ and 3WIR. Structured digital abstract CsKP and CsKP bind by x-ray crystallography (View interaction) .

  • enzymatic properties of recombinant Kojibiose phosphorylase from caldicellulosiruptor saccharolyticus atcc43494
    Bioscience Biotechnology and Biochemistry, 2011
    Co-Authors: Takuo Yamamoto, Tomoyuki Nishimoto, Mio Nishiokosaka, Seisuke Izawa, Hajime Aga, Hiroto Chaen, Shigeharu Fukuda
    Abstract:

    One Kojibiose phoshorylase (KP) homolog gene was cloned from Caldicellulosiruptor saccharolyticus ATCC43494. Recombinant KP from C. saccharolyticus (Cs-KP) expressed in Escherichia coli showed highest activity at pH 6.0 at 85 °C, and was stable from pH 3.5 to 10.0 and up to 85 °C for phosphorolysis. Cs-KP showed higher productivity of kojioligosaccharides of DP≧4 than KP from Thermoanaerobacter brockii ATCC35047.

  • construction and characterization of chimeric enzymes of Kojibiose phosphorylase and trehalose phosphorylase from thermoanaerobacter brockii
    Carbohydrate Research, 2006
    Co-Authors: Takuo Yamamoto, Kazuhisa Mukai, Hiroshi Yamashita, Michio Kubota, Hiroto Chaen, Hikaru Watanabe, Shigeharu Fukuda
    Abstract:

    Chimeric phosphorylases were constructed of the Kojibiose phosphorylase (KP) gene and the trehalose phosphorylase (TP) gene from Thermoanaerobacter brockii. Four chimeric enzymes had KP activity, and another had TP activity. Chimera V-III showed not TP, but KP activity, although only 125 amino acid residues in 785 residues of chimera V-III were from that of KP. Chimera V-III had 1% of the specific activity of the wild-type KP. Furthermore, the temperature profile and kinetic parameters of chimera V-III were remarkably changed as compared to those of the wild-type KP. The results of the molecular mass of chimera V-III using GPC (76,000 Da) strongly suggested that the chimera V-III protein exists as a monomer in solution, whereas wild-type KP and TP are hexamer and dimer structures, respectively. The result of the substrate specificity for phosphorolysis was that the chimera acted on nigerose, sophorose and laminaribiose, in addition to Kojibiose. Furthermore, chimera V-III was also able to act on sophorose and laminaribiose in the absence of inorganic phosphate, and produced two trisaccharides, beta-D-glucosyl-(1-->6)-laminaribiose and laminaritriose, from laminaribiose.

  • acceptor recognition of Kojibiose phosphorylase from thermoanaerobacter brockii syntheses of glycosyl glycerol and myo inositol
    Journal of Bioscience and Bioengineering, 2006
    Co-Authors: Takuo Yamamoto, Tomoyuki Nishimoto, Michio Kubota, Hajime Aga, Hiroto Chaen, Hikaru Watanabe, Shigeharu Fukuda
    Abstract:

    The glucosyl transfer reaction of Kojibiose phosphorylase (KP; EC 2.4.1.230) was examined using glycerol or myo-inositol as an acceptor. In the case of glycerol, KP produced two main transfer products: saccharides A and B. The structure of saccharide A was O-alpha-D-glucopyranosyl-(1-->1)-glycerol and that of saccharide B was O-alpha-D-glucopyranosyl-(1-->2)-O-alpha-D-glucopyranosyl-(1-->1)-glycerol. These results show that KP transferred a glucose residue to the hydroxyl group at position 1 of glycerol. On the other hand, when myo-inositol was used as an acceptor, KP produced four transfer products: saccharides 1-4. The structures of saccharides 1 and 2 were O-alpha-D-glucopyranosyl-(1-->1)- and O-alpha-D-glucopyranosyl-(1-->5)-myo-inositol, respectively; those of saccharides 3 and 4 were O-alpha-D-glucopyranosyl-(1-->2)-O-alpha-D-glucopyranosyl-(1-->1)- and O-alpha-D-glucopyranosyl-(1-->2)-O-alpha-D-glucopyranosyl-(1-->5)-myo-inositol, respectively. KP transferred a glucose residue to the hydroxyl group at position 1 or 5 of myo-inositol. On the basis of the structures of their glucosyl transfer products, glycerol and myo-inositol were found to have a common structure with three hydroxyl groups corresponding to the hydroxyl group of the glucose molecule at positions 2, 3 and 4. The conformation of these three hydroxyl groups in the structure is equatorial. This structure is the substrate recognition site of KP. It has been suggested that KP strictly recognizes the structures of glycerol and myo-inositol, and catalyzes the transfer reaction of a glucose residue to the hydroxyl group at position 1 in glycerol, and at position 1 or 5 in myo-inositol, corresponding to position 2 in glucose.

  • hyper expression of Kojibiose phosphorylase gene and trehalose phosphorylase gene from thermoanaerobacter brockii atcc35047 in bacillus subtilis and selaginose synthesis utilizing two phosphorylases
    Journal of Bioscience and Bioengineering, 2005
    Co-Authors: Takuo Yamamoto, Kazuhiko Maruta, Kazuhisa Mukai, Hiroshi Yamashita, Tomoyuki Nishimoto, Michio Kubota, Hiroto Chaen, Hikaru Watanabe, Shigeharu Fukuda
    Abstract:

    The Kojibiose phosphorylase (KP) gene and trehalose phosphorylase (TP) gene from Thermoanaerobacter brockii ATCC35047 were intracellularly hyper-expressed under the control of the Bacillus amyloliquefaciens α-amylase promoter in Bacillus subtilis. The production yields were estimated to be 2.1 g of KP and 4.9 g of TP per liter of medium. Selaginose, non-reducing trisaccharide, was synthesized from trehalose utilizing the recombinant KP and TP from B. subtilis. Selaginose was not hydrolyzed by salivary amylase, artificial gastric juice, pancreatic amylase, or small intestinal enzymes.

Yoshio Tsujisaka - One of the best experts on this subject based on the ideXlab platform.

  • enzymatic synthesis of a 2 o α d glucopyranosyl cyclic tetrasaccharide by Kojibiose phosphorylase
    Carbohydrate Research, 2005
    Co-Authors: Hikaru Watanabe, Shigeharu Fukuda, Masashi Kurimoto, Tomoyuki Nishimoto, Michio Kubota, Hajime Aga, Takanobu Higashiyama, Yoshio Tsujisaka
    Abstract:

    The glucosyl transfer reaction of Kojibiose phosphorylase (KPase) from Thermoanaerobacter brockii ATCC35047 was examined using cyclo-{-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->} (CTS) as an acceptor. KPase produced four transfer products, saccharides 1-4. The structure of a major product, saccharide 4, was 2-O-alpha-d-glucopyranosyl-CTS, cyclo-{-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->6)-[alpha-d-Glcp-(1-->2)]-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->}. The other transfer products, saccharides 1-3, were 2-O-alpha-kojibiosyl-, 2-O-alpha-kojitriosyl-, and 2-O-alpha-kojitetraosyl-CTS, respectively. These results showed that KPase transferred a glucose residue to the C-2 position at the ring glucose residue of CTS. This enzyme also catalyzed the chain-extending reaction of the side chain of 2-O-alpha-d-glycopyranosyl-CTS.

  • Enhancement of thermostability of Kojibiose phosphorylase from Thermoanaerobacter brockii ATCC35047 by random mutagenesis
    Journal of bioscience and bioengineering, 2005
    Co-Authors: Takuo Yamamoto, Kazuhisa Mukai, Hiroshi Yamashita, Shigeharu Fukuda, Masashi Kurimoto, Michio Kubota, Yoshio Tsujisaka
    Abstract:

    Random mutation by error-prone PCR was introduced into Kojibiose phosphorylase from Thermoanaerobacter brockii ATCC35047. One thermostable mutant enzyme, D513N, was isolated. The D513N mutant enzyme showed an optimum temperature of 67.5–70°C (the wild type, 65°C), and thermostability up to 67.5°C (the wild type, up to 60°C). The half-lives of D513N were estimated to be 135 h at 60°C, 110 min at 70°C and 6 min at 75°C, respectively. They were about 1.6-fold, 7-fold and 6-fold longer than those of the wild-type enzyme, respectively.

  • Cloning and sequencing of Kojibiose phosphorylase gene from Thermoanaerobacter brockii ATCC35047.
    Journal of bioscience and bioengineering, 2004
    Co-Authors: Takuo Yamamoto, Kazuhiko Maruta, Kazuhisa Mukai, Hiroshi Yamashita, Shigeharu Fukuda, Masashi Kurimoto, Tomoyuki Nishimoto, Michio Kubota, Yoshio Tsujisaka
    Abstract:

    Abstract A gene encoding Kojibiose phosphorylase was cloned from Thermoanaerobacter brockii ATCC35047. The kojP gene encodes a polypeptide of 775 amino acid residues. The deduced amino acid sequence was homologous to those of trehalose phosphorylase from T. brockii and maltose phosphorylases from Bacillus sp. and Lactobacillus brevis with 35%, 29% and 28% identities, respectively. Kojibiose phosphorylase was efficiently overexpressed in Escherichia coli JM109. The DNA sequence of 3956 bp analyzed in this study contains three open reading frames (ORFs) downstream of kojP . The four ORFs, kojP, kojE, kojF , and kojG , form a gene cluster. The amino acid sequences deduced from kojE and kojF are similar to those of the N-terminal and C-terminal regions of a sugar-binding periplasmic protein from Thermoanaerobacter tengcongensis MB4. Furthermore, the amino acid sequence deduced from kojG is similar to that of a permease of the ABC-type sugar transport systems from T. tengcongensis MB4. Each of three amino acid substitutions, D362N, K614Q and E642Q, caused a complete loss of Kojibiose phosphorylase activity. These results suggest that D362, K614 and E642 play an important role in catalysis. Another mutation, D459N, increased K m values for Kojibiose (7-fold that for the wild type), β-G1P (11-fold) and glucose (7-fold), whereas K m for inorganic phosphate was minimally affected by this mutation, suggesting that D459 may be involved in the binding to saccharides.

  • enzymatic synthesis of kojioligosaccharides using Kojibiose phosphorylase
    Journal of Bioscience and Bioengineering, 2001
    Co-Authors: Hiroto Chaen, Shigeharu Fukuda, Masashi Kurimoto, Tomoyuki Nishimoto, Tetsuya Nakada, Yoshio Tsujisaka
    Abstract:

    Abstract We have attempted to synthesize kojioligosaccharides (oligosaccharides having the α-1,2 glycosidic linkage at the nonreducing end) using two methods. In the first, mixtures of various proportions of glucose and β- d -glucose-1-phosphate (β-G1P) were allowed to react in the presence of Kojibiose phosphorylase (KPase). In the second, maltose was allowed to react with KPase and maltose phosphorylase (MPase) simultaneously. In the former method, kojioligosaccharides having only the α-1,2 glucosidic linkage were synthesized and the average degree of polymerization (D.P.) of oligosaccharides increased with decreasing proportions of glucose. In the second method, kojioligosaccharides were obtained at approximately 70% yields under optimum conditions. 4-α- d -Kojibiosyl-glucose, kojitriose and kojitetraose, the principal kojioligosaccharides synthesized, were not hydrolyzed by salivary amylase, artificial gastric juice, pancreatic amylase, or small intestinal enzymes.

  • enzymatic synthesis of novel oligosaccharides from l sorbose maltose and sucrose using Kojibiose phosphorylase
    Journal of Bioscience and Bioengineering, 2001
    Co-Authors: Hiroto Chaen, Shigeharu Fukuda, Masashi Kurimoto, Tomoyuki Nishimoto, Tetsuya Nakada, Yoshio Tsujisaka
    Abstract:

    Abstract Glucosyl- l -sorbose, -maltose, and -sucrose were synthesized using Kojibiose phosphorylase (KPase) from Thermoanaerobacter brockii ATCC35047 with β- d -glucose-1-phosphate (β-G1P) as a glucosyl donor. One disaccharide and two trisaccharides thus synthesized were isolated by Toyopearl HW-40S column chromatography. The results of KPase digestion, methylation analysis, and 13C-NMR studies indicated that these oligosaccharides were α- d -glucopyranosyl-(1→5)-α- l -sorbopyranose, α- d -glucopyranosyl-(1→2)-α- d -glucopyranosyl-(1→4)- d -glucopyranose (4-α- d -kojibiosyl-glucose), and α- d -glucopyranosyl-(1→2)-α- d -glucopyranosyl-(1→2)-β- d -fructofuranoside, which are all novel oligosaccharides. Glucosyl- l -sorbose was partially hydrolyzed to glucose and l -sorbose by α-glucosidases, while glucosyl-sucrose and glucosyl-maltose were not hydrolyzed by glucoamylase, α-glucosidases, or CGTase.

Norio Shiomi - One of the best experts on this subject based on the ideXlab platform.

  • Chemistry Central Journal Three novel oligosaccharides synthesized using Thermoanaerobacter brockii Kojibiose phosphorylase
    2020
    Co-Authors: Natsuko Takahashi, Tomoyuki Nishimoto, Eri Fukushi, Shuichi Onodera, Jun Kawabata, Noureddine Benkeblia, Norio Shiomi
    Abstract:

    Abstract Background: Recently synthesized novel oligosaccharides have been produced primarily by hydrolases and glycosyltransferases, while phosphorylases have also been subject of few studies. Indeed, phosphorylases are expected to give good results via their reversible reaction. The purpose of this study was to synthesis other novel oligosaccharides using Kojibiose phosphorylase

  • Structures of xylosylfructoside, and saccharides , and synthesized by Kojibiose phosphorylase
    2011
    Co-Authors: Natsuko Takahashi, Tomoyuki Nishimoto, Eri Fukushi, Shuichi Onodera, Jun Kawabata, Noureddine Benkeblia, Norio Shiomi
    Abstract:

    Copyright information:Taken from "Three novel oligosaccharides synthesized using Kojibiose phosphorylase"http://journal.chemistrycentral.com/content/1/1/18Chemistry Central Journal 2007;1():18-18.Published online 28 Jun 2007PMCID:PMC1994063.

  • HPAEC of saccharides produced from xylosylfructoside and β-D-G1P using Kojibiose phosphorylase
    2011
    Co-Authors: Natsuko Takahashi, Tomoyuki Nishimoto, Eri Fukushi, Shuichi Onodera, Jun Kawabata, Noureddine Benkeblia, Norio Shiomi
    Abstract:

    Copyright information:Taken from "Three novel oligosaccharides synthesized using Kojibiose phosphorylase"http://journal.chemistrycentral.com/content/1/1/18Chemistry Central Journal 2007;1():18-18.Published online 28 Jun 2007PMCID:PMC1994063. The enzyme reaction was carried out with 0.12 M xylosylfructoside and 0.10 M β-D-G1P at 50°C for 54 h

  • isolation and identification of novel tri and tetra saccharides synthesized by thermoanaerobacter brockii Kojibiose phosphorylase
    Journal of applied glycoscience, 2007
    Co-Authors: Natsuko Takahashi, Tomoyuki Nishimoto, Eri Fukushi, Shuichi Onodera, Jun Kawabata, Norio Shiomi
    Abstract:

    Novel tri- and tetra-saccharides were synthesized by glucosyltransfer from β-D-glucose 1-phosphate (β-D-G1P) to palatinose using Thermoanaerobacter brockii Kojibiose phosphorylase. There saccharides were isolated using carbon-Celite column chromatography and preparative high performance liquid chromatography. Gas liquid chromatography analysis of methyl derivatives, MALDI-TOF MS and NMR measurements were used for structural confirmation of the saccharides. The 1H and 13C NMR signals of the saccharides were assigned using 2D-NMR including COSY, HSQC, HSQC-TOCSY and HMBC. These oligosaccharides were identified as 2G-α-D-glucopyranosyl-palatinose; O-α-D-glucopyranosyl-(1→2)-O-α-D-glucopyranosyl-(1→6)-D-fructofuranose and 2G(2-α-D-glucopyranosyl)2-palatinose; O-α-D-glucopyranosyl-(1→2)-O-α-D-glucopyranosyl-(1→2)-O-α-D-glucopyranosyl-(1→6)-D-fructofuranose.

  • Three novel oligosaccharides synthesized using Thermoanaerobacter brockii Kojibiose phosphorylase
    Chemistry Central Journal, 2007
    Co-Authors: Natsuko Takahashi, Tomoyuki Nishimoto, Eri Fukushi, Shuichi Onodera, Jun Kawabata, Noureddine Benkeblia, Norio Shiomi
    Abstract:

    Background Recently synthesized novel oligosaccharides have been produced primarily by hydrolases and glycosyltransferases, while phosphorylases have also been subject of few studies. Indeed, phosphorylases are expected to give good results via their reversible reaction. The purpose of this study was to synthesis other novel oligosaccharides using Kojibiose phosphorylase. Results Three novel oligosaccharides were synthesized by glucosyltransfer from β-D-glucose 1-phosphate (β-D-G1P) to xylosylfructoside [ O -α-D-xylopyranosyl-(1→2)-β-D-fructofuranoside] using Thermoanaerobacter brockii Kojibiose phosphorylase. These oligosaccharides were isolated using carbon-Celite column chromatography and preparative high performance liquid chromatography. Gas liquid chromatography analysis of methyl derivatives, MALDI-TOF MS and NMR measurements were used for structural characterisation. The ^1H and ^13C NMR signals of each saccharide were assigned using 2D-NMR including COSY (correlated spectroscopy), HSQC (herteronuclear single quantum coherence), CH_2-selected E-HSQC (CH_2-selected Editing-HSQC), HSQC-TOCSY (HSQC-total correlation spectroscopy) and HMBC (heteronuclear multiple bond correlation). Conclusion The structure of three synthesized saccharides were determined, and these oligosaccharides have been identified as O -α-D-glucopyranosyl-(1→2)- O -α-D-xylopyranosyl-(1→2)-β-D-fructofuranoside (saccharide 1 ), O -α-D-glucopyranosyl-(1→2)- O -α-D-glucopyranosyl-(1→2)- O -α-D-xylopyranosyl-(1→2)-β-D-fructofuranoside (saccharide 2 ) and O -α-D-glucopyranosyl-(1→[2- O -α-D-glucopyranosyl-1]_2→2)- O -α-D-xylopyranosyl-(1→2)-β-D-fructofuranoside (saccharide 3 ).

Hiroyuki Nakai - One of the best experts on this subject based on the ideXlab platform.

  • The kinetic parameters for the synthetic reactions catalyzed by Teth514_1788 and Teth514_1789.
    2014
    Co-Authors: Kazuhiro Chiku, Takanori Nihira, Motomitsu Kitaoka, Erika Suzuki, Mamoru Nishimoto, Ken'ichi Ohtsubo, Hiroyuki Nakai
    Abstract:

    aThe kinetic parameters were calculated by fitting the initial velocities to various concentrations of acceptor substrates in the presence of 10 mm α-Man1P using the Michaelis-Menten equation.b,cThe kinetic parameters were calculated by fitting the initial velocities toward various concentrations of donor substrates in the presence of 10 mm β-1,2-Man2 (b) or 10 mmd-mannose (c) using the Michaelis-Menten equation.dNot detectable. To investigate the acceptor specificities, the synthetic reactions were examined using the following putative carbohydrate acceptors: d-allose, d-altrose, d-arabinose, d-fructose, d-galactosamine, d-galactose, d-galacturonic acid, d-glucosamine, d-glucose, d-glucuronic acid, d-lyxose, d-talose, d-ribose, d-xylose, l-arabinose, l-rhamnose, 1,5-anhydro-d-glucitol, 2-deoxy-d-glucose, methyl-α-d-glucoside, methyl-β-d-glucoside, 3-O-methyl-d-glucose, N-acetyl-d-galactosamine, N-acetyl-d-glucosamine, N-acetyl-d-mannosamine, cellobiose, gentiobiose, isomaltose, Kojibiose, lactose, lactulose, laminaribiose, maltose, melibiose, nigerose, N,N′-diacetylchitobiose, sophorose, sucrose, trehalose, and xylobiose.The kinetic parameters for the synthetic reactions catalyzed by Teth514_1788 and Teth514_1789.

  • 2-O-a-D-Glucosylglycerol Phosphorylase from Bacillus selenitireducens MLS10 Possessing Hydrolytic Activity on b-D-Glucose 1-Phosphate
    2013
    Co-Authors: Takanori Nihira, Yuka Saito, Hiroyuki Nakai, Motomitsu Kitaoka
    Abstract:

    The glycoside hydrolase family (GH) 65 is a family of inverting phosphorylases that act on a-glucosides. A GH65 protein (Bsel_2816) from Bacillus selenitireducens MLS10 exhibited inorganic phosphate (Pi)-dependent hydrolysis of Kojibiose at the rate of 0.43 s21. No carbohydrate acted as acceptor for the reverse phosphorolysis using b-D-glucose 1-phosphate (bGlc1P) as donor. During the search for a suitable acceptor, we found that Bsel_2816 possessed hydrolytic activity on bGlc1P with a kcat of 2.8 s 21; moreover, such significant hydrolytic activity on sugar 1-phosphate had not been reported for any inverting phosphorylase. The H2 18O 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-a-D-glucosylglycerol (GG) at the rate of 180 s21. Bsel_2816 phosphorolyzed GG through sequential Bi-Bi mechanism with a kcat of 95 s 21. We propose 2-O-a-D-glucopyranosylglycerol: phosphate b-D-glucosyltransferase as the systematic name and 2-O-a-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

  • identification of bacillus selenitireducens mls10 maltose phosphorylase possessing synthetic ability for branched α d glucosyl trisaccharides
    Carbohydrate Research, 2012
    Co-Authors: Takanori Nihira, Yuka Saito, Motomitsu Kitaoka, Kenichi Otsubo, Hiroyuki Nakai
    Abstract:

    Abstract 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 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-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. These results suggested that Bsel2056 possessed a binding space to accommodate the bulky C2 substituent of d -glucose.

  • rational engineering of lactobacillus acidophilus ncfm maltose phosphorylase into either trehalose or Kojibiose dual specificity phosphorylase
    Protein Engineering Design & Selection, 2010
    Co-Authors: Hiroyuki Nakai, Bent O Petersen, Yvonne Westphal, Adiphol Dilokpimol, Maher Abou Hachem, Jens O Duus, H A Schols, Birte Svensson
    Abstract:

    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 (alpha1, alpha1)- 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.

  • 2-O-α-D-glucosylglycerol phosphorylase from Bacillus selenitireducens MLS10 possessing hydrolytic activity on β-D-glucose 1-phosphate.
    Public Library of Science (PLoS), 2024
    Co-Authors: Takanori Nihira, Yuka Saito, Hiroyuki Nakai, Ken'ichi Ohtsubo, Motomitsu Kitaoka
    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

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