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Atsuo Kimura - One of the best experts on this subject based on the ideXlab platform.
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A novel intracellular dextranase derived from Paenibacillus sp. 598K with an ability to degrade cycloIsomaltooligosaccharides
Applied Microbiology and Biotechnology, 2019Co-Authors: Daiki Mizushima, Atsuo Kimura, Takatsugu Miyazaki, Yuh Shiwa, Keitarou Kimura, Shiho Suzuki, Nobuyuki Fujita, Hirofumi Yoshikawa, Shinichi Kitamura, Hiroshi HaraAbstract:Paenibacillus sp. 598K produces cycloIsomaltooligosaccharides (CIs) in culture from dextran and starch. CIs are cyclic oligosaccharides consisting of seven or more α-(1 → 6)-linked- d -glucose residues. The extracellular enzyme CI glucanotransferase (PsCITase), which is the member of glycoside hydrolase family 66, catalyzes the final stage of CI production and produces mainly cycloisomaltoheptaose. We have discovered a novel intracellular CI-degrading dextranase (PsDEX598) from Paenibacillus sp. 598K. The 69.7-kDa recombinant PsDEX598 does not digest isomaltotetraose or shorter Isomaltooligosaccharides, but digests longer ones of at least up to isomaltoheptaose. It also digests oligoCIs of cycloisomaltoheptaose, cycloisomaltooctaose, and cycloisomaltononaose better than it does with megaloCIs of cycloisomaltodecaose, cycloisomaltoundecaose, and cycloisomaltododecaose, as well as an α-(1 → 6)- d -glucan of dextran 40. PsDEX598 is produced intracellularly when culture medium is supplemented with cycloisomaltoheptaose or dextran, but not with Isomaltooligosaccharides (a mixture of isomaltose, isomaltotriose, and panose), starch, or glucose. The whole genomic DNA sequence of the strain 598K implies that it harbors two genes for enzymes belonging to glycoside hydrolase family 66 (PsCITase and PsDEX598), and PsDEX598 is the only dextranase in the strain. PsDEX598 does not have any carbohydrate-binding modules (CBMs) and has a low similarity (< 30%) with other family 66 dextranases, and the catalytic amino acids of this enzyme are predicted to be Asp191, Asp303, and Glu368. The strain Paenibacillus sp. 598K appears to take up CI-7, so these findings indicate that this bacterium can degrade CIs using a dextranase within the cells and so utilize them as a carbon source for growth.
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Engineered dextranase from Streptococcus mutans enhances the production of longer Isomaltooligosaccharides.
Bioscience Biotechnology and Biochemistry, 2018Co-Authors: Patcharapa Klahan, Masayuki Okuyama, Kohei Jinnai, Min Ma, Asako Kikuchi, Yuya Kumagai, Takayoshi Tagami, Atsuo KimuraAbstract:ABSTRACTHerein, we investigated enzymatic properties and reaction specificities of Streptococcus mutans dextranase, which hydrolyzes α-(1→6)-glucosidic linkages in dextran to produce isomaltooligos...
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structural elucidation of dextran degradation mechanism by streptococcus mutans dextranase belonging to glycoside hydrolase family 66
Journal of Biological Chemistry, 2012Co-Authors: Nobuhiro Suzuki, Haruhide Mori, Kazumi Funane, Mitsuru Momma, Zui Fujimoto, Masayuki Okuyama, Atsuo KimuraAbstract:Abstract Dextranase is an enzyme that hydrolyzes dextran α-1,6 linkages. Streptococcus mutans dextranase (SmDex) belongs to glycoside hydrolase family 66, producing Isomaltooligosaccharides of various sizes, and consisting of at least five amino acid sequence regions. The crystal structure of the conserved fragment from Gln-100 to Ile-732 of SmDex, devoid of its N and C-terminal variable regions, was determined at 1.6 A resolution and found to contain three structural domains. Domain N possessed an immunoglobulin-like β-sandwich fold, domain A the enzyme's catalytic module, comprising a (β/α)8-barrel, and domain C formed a β-sandwich structure containing two Greek key motifs. Two ligand complex structures were also determined and, in the enzyme/isomaltotriose complex structure, the bound Isomaltooligosaccharide with four glucose moieties was observed in the catalytic glycone cleft and considered to be the transglycosylation product of the enzyme, indicating the presence of four subsites -4 to -1 in the catalytic cleft. The complexed structure with 4′,5′-epoxypentyl-α-D-glucopyranoside, a suicide substrate of the enzyme, revealed that the epoxide ring reacted to form a covalent bond with the Asp-385 sidechain. These structures collectively indicated that Asp-385 was the catalytic nucleophile and Glu-453 the acid/base of the double displacement mechanism, in which the enzyme showed a retaining catalytic character. This is the first structural report for the enzyme belonging to glycoside hydrolase family 66, elucidating the enzyme's catalytic machinery.
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Isolation of Bacillus and Paenibacillus Bacterial Strains That Produce Large Molecules of Cyclic Isomaltooligosaccharides
Bioscience Biotechnology and Biochemistry, 2008Co-Authors: Kazumi Funane, Atsuo Kimura, Kazue Terasawa, Yasuko Mizuno, Shigehachi Gibu, Tadaaki Tokashiki, Yasuyuki Kawabata, Mikihiko KobayashiAbstract:Cyclic Isomaltooligosaccharides (CIs) usually consist of 7 to 12 glucose units, although only CI-10 has strong inclusion complex-forming ability. Four Bacillus strains and two Paenibacillus strains were isolated as novel CI-producing bacteria. Among these, five strains produced small amounts of CI-7 to CI-9, but mainly produced CI-10 to CI-12. Larger CIs, up to CI-17, were also identified.
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a novel cyclic Isomaltooligosaccharide cycloisomaltodecaose ci 10 produced by bacillus circulans t 3040 displays remarkable inclusion ability compared with cyclodextrins
Journal of Biotechnology, 2007Co-Authors: Kazumi Funane, Atsuo Kimura, Kazue Terasawa, Yasuko Mizuno, Shigehachi Gibu, Tadaaki Tokashiki, Yasuyuki Kawabata, Takeshi Miyagi, Mikihiko KobayashiAbstract:Abstract Cyclodextrans (CIs) are cyclic Isomaltooligosaccharides and only CI-7, CI-8, and CI-9 were known. CI-7, CI-8, and CI-9, consisting of seven, eight, and nine glucoses, respectively, bound by α-(1 → 6) linkages, are known to be produced by T-3040 strain of Bacillus circulans. However, we have found, using 13 C NMR and mass spectrometry, that this strain also produces CI-10, CI-11 and CI-12. These large CIs are very soluble in water and inhibit the glucan synthesis of glucansucrases to the same degree as do the smaller CIs. The CIs were thought to be poor at forming inclusion complexes with chemical compounds, due to their flexible α-(1 → 6)-glucosidic structure. Among these six CIs, CI-10 was much better at forming an inclusion complex, and it ability to do so was as good as cyclodextrins, as determined by its ability to stabilize the color of Victoria blue B. Therefore, CI-10 may be the most commercially useful CI.
Lei Zhang - One of the best experts on this subject based on the ideXlab platform.
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Bioinspired preparation of polydopamine microcapsule for multienzyme system construction
Green Chemistry, 2020Co-Authors: Lei Zhang, Ruijie Meng, Shizhang Qiao, Yanjun Jiang, Zhongyi Jiang, Rui Wang, Jian Li, Yang ZhengAbstract:Inspired by the structural organization of mitochondria and the bioadhesive principle, a simple and versatile approach to construct a multienzyme system is developed. More specifically, the multienzyme system is composed of a polydopamine (PDA) microcapsule scaffold and three spatially separated enzymes. The PDA microcapsules are prepared through the rapid, spontaneous self-polymerization of dopamine on the surface of CaCO3 microparticle template, followed by dissolution of the template using EDTA. The wall thickness of the microcapsules can be tuned by the dopamine concentration in an aqueous solution. The three enzymes are respectively immobilized through physical encapsulation in the lumen, in situ entrapment within the wall and chemical attachment on the out surface under extremely mild conditions. As an example, a multienzyme system, containing α-amylase, β-amylase and glucosidase, was constructed to convert starch into Isomaltooligosaccharide, and the multienzyme system displays higher catalytic activity and enhanced operational stability. The method developed in this study will establish a powerful platform for the facile construction of multienzyme cascade systems.
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Bioinspired stability improvement of layer-by-layer microcapsules using a bioadhesive for enzyme encapsulation
Reactive and Functional Polymers, 2016Co-Authors: Lei Zhang, Penggao Cheng, Yingqiao Wang, Na Tang, Wei Du, Jun Xiang, Xuekui WangAbstract:Capsules prepared by the layer-by-layer (LbL) technique are gaining interest. This work proposed a novel approach to improving the mechanical stability of LbL microcapsules under mild conditions by the self-polymerisation of dopamine among different polymers in aqueous solution. More specifically, common poly(sodium styrenesulfonate)/poly(sodium 4-styrenesulfonate) (PSS/PAH) microcapsules were prepared using layer-by-layer method. The as-prepared microcapsule was then treated with dopamine and compared with PSS/PAH microcapsules fabricated by a similar protocol without dopamine treatment. The morphology, structure, and chemical composition of the microcapsules were characterised, and the underlying mechanism was tentatively discussed. To explore the application potential of the microcapsules, glucosidase was encapsulated for the conversion of maltose to Isomaltooligosaccharides.
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Bioinspired preparation of polydopamine microcapsule for multienzyme system construction
Green Chemistry, 2011Co-Authors: Lei Zhang, Ruijie Meng, Jiafu Shi, Yuanyuan Zhu, Shizhang Qiao, Yanjun Jiang, Zhongyi Jiang, Rui Wang, Jian Li, Yang ZhengAbstract:Inspired by the structural organization of mitochondria and the bioadhesive principle, a simple and versatile approach to construct a multienzyme system is developed. More specifically, the multienzyme system is composed of a polydopamine (PDA) microcapsule scaffold and three spatially separated enzymes. The PDA microcapsules are prepared through the rapid, spontaneous self-polymerization of dopamine on the surface of CaCO(3) microparticle template, followed by dissolution of the template using EDTA. The wall thickness of the microcapsules can be tuned by the dopamine concentration in an aqueous solution. The three enzymes are respectively immobilized through physical encapsulation in the lumen, in situ entrapment within the wall and chemical attachment on the out surface under extremely mild conditions. As an example, a multienzyme system, containing alpha-amylase, beta-amylase and glucosidase, was constructed to convert starch into Isomaltooligosaccharide, and the multienzyme system displays higher catalytic activity and enhanced operational stability. The method developed in this study will establish a powerful platform for the facile construction of multienzyme cascade systems.
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Sandwich-structured enzyme membrane reactor for efficient conversion of maltose into Isomaltooligosaccharides
Bioresource Technology, 2010Co-Authors: Lei Zhang, Zhongyi Jiang, Yang Zheng, Yanlei Su, Yanjun JiangAbstract:A novel enzyme membrane reactor with sandwich structure has been developed by confining glucosidase between two sheets of ultrafiltration membranes to effectively convert maltose to Isomaltooligosaccharides (IMOs). The hydrophilic ultrafiltration membranes, which were prepared by phase inversion method using PES as bulk polymer and Pluronic F127 as both surface modification and pore formation agent, exhibited the desirable enzyme adsorption-resistant property. The scanning electron microscopy (SEM) photographs showed that two sheets of PES/Pluronic F127 membranes were packed tightly and glucosidase was kept in a free state within a nanoscale space. When the weight ratio of Pluronic F127 to PES was 30%, glucosidase could be completely rejected by the membranes. Due to the sandwich structuring of the membrane reactor and the high hydrophilicity of the PES/Pluronic F127 membrane surface, maltose conversion and yield reached 100% and 58% under the optimum experimental conditions (pH 6.0, 50 degrees C), respectively. (C) 2010 Elsevier Ltd. All rights reserved.
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Immobilized transglucosidase in biomimetic polymer–inorganic hybrid capsules for efficient conversion of maltose to Isomaltooligosaccharides
Biochemical Engineering Journal, 2009Co-Authors: Lei Zhang, Yanjun Jiang, Zhongyi Jiang, Wei ChengAbstract:Abstract Isomaltooligosaccharides (IMOs) are relatively new functional food ingredients which have great potential to improve the quality of many foods due to their low calories, no cariogenicity and safety for diabetics. To convert maltose to IMOs efficiently, α-transglucosidase was immobilized in a kind of alginate–chitosan–calcium phosphate hybrid capsules (Alg–Chi–CaP), which were prepared through a facile bio-inspired mineralization process. The surface morphology of Alg–Chi–CaP capsule and alginate–chitosan capsule (Alg–Chi) was characterized by scanning electron microscopy (SEM). Due to the presence of inorganic shell, immobilization efficiency of transglucosidase in Alg–Chi–CaP capsules was higher than that in Alg–Chi capsules. The optimal temperature (60 °C) and pH (6.0) value for enzymatic conversion catalyzed by transglucosidase immobilized Alg–Chi–CaP capsules were identical to those catalyzed by free transglucosidase. As compared to the free enzyme, transglucosidase in Alg–Chi–CaP capsules exhibited significantly higher recycling stability and storage stability in a broader temperature and pH range.
Kazumi Funane - One of the best experts on this subject based on the ideXlab platform.
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Isomaltooligosaccharide binding structure of paenibacillus sp 598k cycloIsomaltooligosaccharide glucanotransferase
Bioscience Reports, 2017Co-Authors: Zui Fujimoto, Mitsuru Momma, Nobuhiro Suzuki, Daiki Mizushima, Keitarou Kimura, Naomi Kishine, Ryuichiro Suzuki, Kazumi FunaneAbstract:Paenibacillus sp. 598K cycloIsomaltooligosaccharide glucanotransferase (CITase), a member of glycoside hydrolase family 66 (GH66), catalyses the intramolecular transglucosylation of dextran to produce CIs with seven or more degrees of polymerization. To clarify the cyclization reaction and product specificity of the enzyme, we determined the crystal structure of PsCITase. The core structure of PsCITase consists of four structural domains: a catalytic (β/α) 8 -domain and three β-domains. A family 35 carbohydrate-binding module (first CBM35 region of Paenibacillus sp. 598K CITase, (PsCBM35-1)) is inserted into and protrudes from the catalytic domain. The ligand complex structure of PsCITase prepared by soaking the crystal with cycloisomaltoheptaose yielded bound sugars at three sites: in the catalytic cleft, at the joint of the PsCBM35-1 domain and at the loop region of PsCBM35-1. In the catalytic site, soaked cycloisomaltoheptaose was observed as a linear isomaltoheptaose, presumably a hydrolysed product from cycloisomaltoheptaose by the enzyme and occupied subsites –7 to –1. Beyond subsite –7, three glucose moieties of another isomaltooiligosaccharide were observed, and these positions are considered to be distal subsites –13 to –11. The third binding site is the canonical sugar-binding site at the loop region of PsCBM35-1, where the soaked cycloisomaltoheptaose is bound. The structure indicated that the concave surface between the catalytic domain and PsCBM35-1 plays a guiding route for the long-chained substrate at the cyclization reaction.
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Bacteroides thetaiotaomicron VPI‐5482 glycoside hydrolase family 66 homolog catalyzes dextranolytic and cyclization reactions
FEBS Journal, 2012Co-Authors: Eiji Yamamoto, Min Sun Kang, Wataru Saburi, Haruhide Mori, Kazumi Funane, Mitsuru Momma, Hiroyuki Nakai, Masayuki Okuyama, Zui FujimotoAbstract: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 > 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.
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structural elucidation of dextran degradation mechanism by streptococcus mutans dextranase belonging to glycoside hydrolase family 66
Journal of Biological Chemistry, 2012Co-Authors: Nobuhiro Suzuki, Haruhide Mori, Kazumi Funane, Mitsuru Momma, Zui Fujimoto, Masayuki Okuyama, Atsuo KimuraAbstract:Abstract Dextranase is an enzyme that hydrolyzes dextran α-1,6 linkages. Streptococcus mutans dextranase (SmDex) belongs to glycoside hydrolase family 66, producing Isomaltooligosaccharides of various sizes, and consisting of at least five amino acid sequence regions. The crystal structure of the conserved fragment from Gln-100 to Ile-732 of SmDex, devoid of its N and C-terminal variable regions, was determined at 1.6 A resolution and found to contain three structural domains. Domain N possessed an immunoglobulin-like β-sandwich fold, domain A the enzyme's catalytic module, comprising a (β/α)8-barrel, and domain C formed a β-sandwich structure containing two Greek key motifs. Two ligand complex structures were also determined and, in the enzyme/isomaltotriose complex structure, the bound Isomaltooligosaccharide with four glucose moieties was observed in the catalytic glycone cleft and considered to be the transglycosylation product of the enzyme, indicating the presence of four subsites -4 to -1 in the catalytic cleft. The complexed structure with 4′,5′-epoxypentyl-α-D-glucopyranoside, a suicide substrate of the enzyme, revealed that the epoxide ring reacted to form a covalent bond with the Asp-385 sidechain. These structures collectively indicated that Asp-385 was the catalytic nucleophile and Glu-453 the acid/base of the double displacement mechanism, in which the enzyme showed a retaining catalytic character. This is the first structural report for the enzyme belonging to glycoside hydrolase family 66, elucidating the enzyme's catalytic machinery.
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Isolation of Bacillus and Paenibacillus Bacterial Strains That Produce Large Molecules of Cyclic Isomaltooligosaccharides
Bioscience Biotechnology and Biochemistry, 2008Co-Authors: Kazumi Funane, Atsuo Kimura, Kazue Terasawa, Yasuko Mizuno, Shigehachi Gibu, Tadaaki Tokashiki, Yasuyuki Kawabata, Mikihiko KobayashiAbstract:Cyclic Isomaltooligosaccharides (CIs) usually consist of 7 to 12 glucose units, although only CI-10 has strong inclusion complex-forming ability. Four Bacillus strains and two Paenibacillus strains were isolated as novel CI-producing bacteria. Among these, five strains produced small amounts of CI-7 to CI-9, but mainly produced CI-10 to CI-12. Larger CIs, up to CI-17, were also identified.
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a novel cyclic Isomaltooligosaccharide cycloisomaltodecaose ci 10 produced by bacillus circulans t 3040 displays remarkable inclusion ability compared with cyclodextrins
Journal of Biotechnology, 2007Co-Authors: Kazumi Funane, Atsuo Kimura, Kazue Terasawa, Yasuko Mizuno, Shigehachi Gibu, Tadaaki Tokashiki, Yasuyuki Kawabata, Takeshi Miyagi, Mikihiko KobayashiAbstract:Abstract Cyclodextrans (CIs) are cyclic Isomaltooligosaccharides and only CI-7, CI-8, and CI-9 were known. CI-7, CI-8, and CI-9, consisting of seven, eight, and nine glucoses, respectively, bound by α-(1 → 6) linkages, are known to be produced by T-3040 strain of Bacillus circulans. However, we have found, using 13 C NMR and mass spectrometry, that this strain also produces CI-10, CI-11 and CI-12. These large CIs are very soluble in water and inhibit the glucan synthesis of glucansucrases to the same degree as do the smaller CIs. The CIs were thought to be poor at forming inclusion complexes with chemical compounds, due to their flexible α-(1 → 6)-glucosidic structure. Among these six CIs, CI-10 was much better at forming an inclusion complex, and it ability to do so was as good as cyclodextrins, as determined by its ability to stabilize the color of Victoria blue B. Therefore, CI-10 may be the most commercially useful CI.
Yang Zheng - One of the best experts on this subject based on the ideXlab platform.
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Bioinspired preparation of polydopamine microcapsule for multienzyme system construction
Green Chemistry, 2020Co-Authors: Lei Zhang, Ruijie Meng, Shizhang Qiao, Yanjun Jiang, Zhongyi Jiang, Rui Wang, Jian Li, Yang ZhengAbstract:Inspired by the structural organization of mitochondria and the bioadhesive principle, a simple and versatile approach to construct a multienzyme system is developed. More specifically, the multienzyme system is composed of a polydopamine (PDA) microcapsule scaffold and three spatially separated enzymes. The PDA microcapsules are prepared through the rapid, spontaneous self-polymerization of dopamine on the surface of CaCO3 microparticle template, followed by dissolution of the template using EDTA. The wall thickness of the microcapsules can be tuned by the dopamine concentration in an aqueous solution. The three enzymes are respectively immobilized through physical encapsulation in the lumen, in situ entrapment within the wall and chemical attachment on the out surface under extremely mild conditions. As an example, a multienzyme system, containing α-amylase, β-amylase and glucosidase, was constructed to convert starch into Isomaltooligosaccharide, and the multienzyme system displays higher catalytic activity and enhanced operational stability. The method developed in this study will establish a powerful platform for the facile construction of multienzyme cascade systems.
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Bioinspired preparation of polydopamine microcapsule for multienzyme system construction
Green Chemistry, 2011Co-Authors: Lei Zhang, Ruijie Meng, Jiafu Shi, Yuanyuan Zhu, Shizhang Qiao, Yanjun Jiang, Zhongyi Jiang, Rui Wang, Jian Li, Yang ZhengAbstract:Inspired by the structural organization of mitochondria and the bioadhesive principle, a simple and versatile approach to construct a multienzyme system is developed. More specifically, the multienzyme system is composed of a polydopamine (PDA) microcapsule scaffold and three spatially separated enzymes. The PDA microcapsules are prepared through the rapid, spontaneous self-polymerization of dopamine on the surface of CaCO(3) microparticle template, followed by dissolution of the template using EDTA. The wall thickness of the microcapsules can be tuned by the dopamine concentration in an aqueous solution. The three enzymes are respectively immobilized through physical encapsulation in the lumen, in situ entrapment within the wall and chemical attachment on the out surface under extremely mild conditions. As an example, a multienzyme system, containing alpha-amylase, beta-amylase and glucosidase, was constructed to convert starch into Isomaltooligosaccharide, and the multienzyme system displays higher catalytic activity and enhanced operational stability. The method developed in this study will establish a powerful platform for the facile construction of multienzyme cascade systems.
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Sandwich-structured enzyme membrane reactor for efficient conversion of maltose into Isomaltooligosaccharides
Bioresource Technology, 2010Co-Authors: Lei Zhang, Zhongyi Jiang, Yang Zheng, Yanlei Su, Yanjun JiangAbstract:A novel enzyme membrane reactor with sandwich structure has been developed by confining glucosidase between two sheets of ultrafiltration membranes to effectively convert maltose to Isomaltooligosaccharides (IMOs). The hydrophilic ultrafiltration membranes, which were prepared by phase inversion method using PES as bulk polymer and Pluronic F127 as both surface modification and pore formation agent, exhibited the desirable enzyme adsorption-resistant property. The scanning electron microscopy (SEM) photographs showed that two sheets of PES/Pluronic F127 membranes were packed tightly and glucosidase was kept in a free state within a nanoscale space. When the weight ratio of Pluronic F127 to PES was 30%, glucosidase could be completely rejected by the membranes. Due to the sandwich structuring of the membrane reactor and the high hydrophilicity of the PES/Pluronic F127 membrane surface, maltose conversion and yield reached 100% and 58% under the optimum experimental conditions (pH 6.0, 50 degrees C), respectively. (C) 2010 Elsevier Ltd. All rights reserved.
Zui Fujimoto - One of the best experts on this subject based on the ideXlab platform.
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Isomaltooligosaccharide binding structure of paenibacillus sp 598k cycloIsomaltooligosaccharide glucanotransferase
Bioscience Reports, 2017Co-Authors: Zui Fujimoto, Mitsuru Momma, Nobuhiro Suzuki, Daiki Mizushima, Keitarou Kimura, Naomi Kishine, Ryuichiro Suzuki, Kazumi FunaneAbstract:Paenibacillus sp. 598K cycloIsomaltooligosaccharide glucanotransferase (CITase), a member of glycoside hydrolase family 66 (GH66), catalyses the intramolecular transglucosylation of dextran to produce CIs with seven or more degrees of polymerization. To clarify the cyclization reaction and product specificity of the enzyme, we determined the crystal structure of PsCITase. The core structure of PsCITase consists of four structural domains: a catalytic (β/α) 8 -domain and three β-domains. A family 35 carbohydrate-binding module (first CBM35 region of Paenibacillus sp. 598K CITase, (PsCBM35-1)) is inserted into and protrudes from the catalytic domain. The ligand complex structure of PsCITase prepared by soaking the crystal with cycloisomaltoheptaose yielded bound sugars at three sites: in the catalytic cleft, at the joint of the PsCBM35-1 domain and at the loop region of PsCBM35-1. In the catalytic site, soaked cycloisomaltoheptaose was observed as a linear isomaltoheptaose, presumably a hydrolysed product from cycloisomaltoheptaose by the enzyme and occupied subsites –7 to –1. Beyond subsite –7, three glucose moieties of another isomaltooiligosaccharide were observed, and these positions are considered to be distal subsites –13 to –11. The third binding site is the canonical sugar-binding site at the loop region of PsCBM35-1, where the soaked cycloisomaltoheptaose is bound. The structure indicated that the concave surface between the catalytic domain and PsCBM35-1 plays a guiding route for the long-chained substrate at the cyclization reaction.
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Bacteroides thetaiotaomicron VPI‐5482 glycoside hydrolase family 66 homolog catalyzes dextranolytic and cyclization reactions
FEBS Journal, 2012Co-Authors: Eiji Yamamoto, Min Sun Kang, Wataru Saburi, Haruhide Mori, Kazumi Funane, Mitsuru Momma, Hiroyuki Nakai, Masayuki Okuyama, Zui FujimotoAbstract: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 > 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.
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structural elucidation of dextran degradation mechanism by streptococcus mutans dextranase belonging to glycoside hydrolase family 66
Journal of Biological Chemistry, 2012Co-Authors: Nobuhiro Suzuki, Haruhide Mori, Kazumi Funane, Mitsuru Momma, Zui Fujimoto, Masayuki Okuyama, Atsuo KimuraAbstract:Abstract Dextranase is an enzyme that hydrolyzes dextran α-1,6 linkages. Streptococcus mutans dextranase (SmDex) belongs to glycoside hydrolase family 66, producing Isomaltooligosaccharides of various sizes, and consisting of at least five amino acid sequence regions. The crystal structure of the conserved fragment from Gln-100 to Ile-732 of SmDex, devoid of its N and C-terminal variable regions, was determined at 1.6 A resolution and found to contain three structural domains. Domain N possessed an immunoglobulin-like β-sandwich fold, domain A the enzyme's catalytic module, comprising a (β/α)8-barrel, and domain C formed a β-sandwich structure containing two Greek key motifs. Two ligand complex structures were also determined and, in the enzyme/isomaltotriose complex structure, the bound Isomaltooligosaccharide with four glucose moieties was observed in the catalytic glycone cleft and considered to be the transglycosylation product of the enzyme, indicating the presence of four subsites -4 to -1 in the catalytic cleft. The complexed structure with 4′,5′-epoxypentyl-α-D-glucopyranoside, a suicide substrate of the enzyme, revealed that the epoxide ring reacted to form a covalent bond with the Asp-385 sidechain. These structures collectively indicated that Asp-385 was the catalytic nucleophile and Glu-453 the acid/base of the double displacement mechanism, in which the enzyme showed a retaining catalytic character. This is the first structural report for the enzyme belonging to glycoside hydrolase family 66, elucidating the enzyme's catalytic machinery.
