The Experts below are selected from a list of 2916 Experts worldwide ranked by ideXlab platform
Laixi Wang - One of the best experts on this subject based on the ideXlab platform.
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Glycan Remodeling of Human Erythropoietin (EPO) Through Combined Mammalian Cell Engineering and Chemoenzymatic Transglycosylation
2017Co-Authors: Qiang Yang, Shilei Zhu, Roushu Zhang, Chun Mun Loke, John F. Cipollo, Laixi WangAbstract:The tremendous structural heterogeneity of N-glycosylation of glycoproteins poses a great challenge for deciphering the biological functions of specific glycoforms and for developing protein-based therapeutics. We have previously reported a chemoenzymatic glycan remodeling method for producing homogeneous glycoforms of N-glycoproteins including intact antibodies, which consist of endoglycosidase-catalyzed deglycosylation and novel glycosynthase-catalyzed Transglycosylation, but its application to complex glycoproteins carrying multiple N-glycans remains to be examined. We report here site-selective chemoenzymatic glycosylation remodeling of recombinant human erythropoietin (EPO) that contains three N-glycans. We found that the generation of a HEK293S GnT I knockout FUT8 overexpressing cell line enabled the production of an unusual Man5GlcNAc2Fuc glycoform, which could be converted to the core-fucosylated GlcNAc-EPO intermediate acceptor for enzymatic Transglycosylation. With this acceptor, homogeneous sialylated glycoform or azide-tagged glycoform were produced using the glycosynthase (EndoF3-D165A) catalyzed Transglycosylation. Interestingly, a remarkable site-selectivity was observed in the Transglycosylation reactions, leading to the introduction of two N-glycans selectively at the Asn-38 and Asn-83 sites, which was confirmed by a detailed MS/MS analysis of the Transglycosylation product. Finally, a different N-glycan was attached at the third (Asn-24) site by pushing the enzymatic Transglycosylation with a distinct glycan oxazoline, achieving the site-selective glycosylation modification of the protein. This study represents the first example of site-selective chemoenzymatic glycan engineering of complex glycoproteins carrying multiple N-glycans
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Glycosynthase Mutants of Endoglycosidase S2 Show Potent Transglycosylation Activity and Remarkably Relaxed Substrate Specificity for Antibody Glycosylation Remodeling.
The Journal of biological chemistry, 2016Co-Authors: Xin Tong, Qiang Yang, John P. Giddens, Laixi WangAbstract:Glycosylation can exert a profound impact on the structures and biological functions of antibodies. Glycosylation remodeling using the endoglycosidase-catalyzed deglycosylation and Transglycosylation approach is emerging as a promising platform to produce homogeneous glycoforms of antibodies, but the broad application of this method will require the availability of highly efficient glycosynthase mutants. We describe in this paper a systematic site-directed mutagenesis of an endoglycosidase from Streptococcus pyogenes of serotype M49 (Endo-S2) and the evaluation of the resulting mutants for their hydrolysis and Transglycosylation activities. We found that mutations at the Asp-184 residue gave mutants that demonstrated significantly different properties, some possessed potent Transglycosylation activity with diminished hydrolysis activity but others did not, which would be otherwise difficult to predict without the comparative study. In contrast to the previously reported Endo-S mutants that are limited to action on complex type N-glycans, the Endo-S2 glycosynthases described here, including D184M and D184Q, were found to have remarkably relaxed substrate specificity and were capable of transferring three major types (complex, high-mannose, and hybrid type) of N-glycans for antibody glycosylation remodeling. In addition, the Endo-S2 glycosynthase mutants were found to be much more active in general than the Endo-S mutants for Transglycosylation. The usefulness of these Endo-S2 glycosynthase mutants was exemplified by an efficient glycosylation remodeling of two therapeutic monoclonal antibodies, rituximab and trastuzumab (Herceptin).
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efficient transfer of sialo oligosaccharide onto proteins by combined use of a glycosynthase like mutant of mucor hiemalis endoglycosidase and synthetic sialo complex type sugar oxazoline
Biochimica et Biophysica Acta, 2010Co-Authors: Midori Umekawa, Takayuki Higashiyama, Wei Huang, Laixi Wang, Shinichiro Shoda, Yurie Koga, Tomonari Tanaka, Masato Noguchi, Atsushi Kobayashi, Hisashi AshidaAbstract:Abstract Background An efficient method for synthesizing homogenous glycoproteins is essential for elucidating the structural and functional roles of glycans of glycoproteins. We have focused on the Transglycosylation activity of endo-β- N -acetylglucosaminidase from Mucor hiemalis (Endo-M) as a tool for glycoconjugate syntheses, since it can transfer en bloc the oligosaccharide of not only high-mannose type but also complex-type N-glycan onto various acceptors having an N -acetylglucosamine residue. However, there are two major bottlenecks for its practical application: the low yield of the Transglycosylation product and the difficulty to obtain the activated sugar oxazoline substrate, especially the sialo-complex type one. Methods We carried out the Transglycosylation using a glycosynthase-like N175Q mutant of Endo-M, which was found to possess enhanced Transglycosylation activity with sugar oxazoline as a donor substrate, in combination with an easy preparation of the sialo-complex-type sugar oxazoline from natural sialoglycopeptide in egg yolk. Results Endo-M-N175Q showed efficient Transglycosylation toward sialo-complex-type sugar oxazoline onto bioactive peptides and bovine ribonuclease B, and each sialylated compound was obtained in significantly high yield. Conclusions Highly efficient and simple chemo-enzymatic syntheses of various sialylated compounds were enabled, by a combination of a simple synthesis of sialo-complex-type sugar oxazoline and the Endo-M-N175Q catalyzed Transglycosylation. General significance Our method would be very useful for a practical synthesis of biologically important glycopeptides and glycoproteins.
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enzymatic Transglycosylation for glycoconjugate synthesis
Current Opinion in Chemical Biology, 2009Co-Authors: Laixi Wang, Wei HuangAbstract:Remarkable advances have been made in recent years in exploiting the Transglycosylation activity of glycosidases and glycosynthase mutants for oligosaccharide and glycoconjugate synthesis. New glycosynthases were generated from retaining glycosidases, inverting glycosidases, and those that proceed in a mechanism of substrate-assisted catalysis. Directed evolution coupled with elegant screening methods has led to the discovery of an expanding number of glycosynthase mutants that show improved catalytic activity and/or altered substrate specificity. In particular, enzymatic Transglycosylation strategy has been recently extended to the synthesis of complex glycoconjugates, including glycosphingolipids, N-glycoproteins, and other glycosylated natural products.
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endo β n acetylglucosaminidase catalyzed polymerization of β glcp 1 4 glcpnac oxazoline a revisit to enzymatic Transglycosylation
Carbohydrate Research, 2009Co-Authors: Hirofumi Ochiai, Wei Huang, Laixi WangAbstract:An alternative synthesis of beta-Glcp-(1-->4)-GlcpNAc oxazoline is described, and its enzymatic reaction with the endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) was re-investigated. Under normal Transglycosylation conditions with a catalytic amount of enzyme, Endo-A showed only marginal activity for Transglycosylation with the disaccharide oxazoline, consistent with our previous observations. However, when used in a relatively large quantity, Endo-A could promote the Transglycosylation of the disaccharide oxazoline to a GlcpNAc-Asn acceptor. In addition to the initial Transglycosylation product, a series of large oligosaccharides were also formed due to the tandem Transglycosylation to the terminal glucose residues in the intermediate products. In the absence of an external acceptor, Endo-A could polymerize the disaccharide oxazoline to form oligo- and polysaccharides having the -4-beta-(Glcp-(1-->4)-beta -GlcpNAc)-1-repeating units. This is the first example of an endo-beta-N-acetylglucosaminidase-promoted polymerization of activated oligosaccharide substrates. This enzymatic polymerization may find useful applications for the synthesis of novel artificial polysaccharides.
Kiyotaka Fujita - One of the best experts on this subject based on the ideXlab platform.
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mutants of mucor hiemalis endo β n acetylglucosaminidase show enhanced Transglycosylation and glycosynthase like activities
Journal of Biological Chemistry, 2008Co-Authors: Midori Umekawa, Wei Huang, Hisashi Ashida, Laixi Wang, Kiyotaka Fujita, Kenji YamamotoAbstract:Abstract Endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M), a family 85 glycoside hydrolase, acts on the β1,4 linkage of N,N′-diacetylchitobiose moiety in the N-linked glycans of glycoproteins and catalyzes not only the hydrolysis reaction but also the Transglycosylation reaction that transfers the releasing sugar chain to an acceptor other than water to form a new glycosidic linkage. The Transglycosylation activity of Endo-M holds a great promise for the chemo-enzymatic synthesis and glyco-engineering of glycoproteins, but the inherent hydrolytic activity for product hydrolysis and low Transglycosylation have hampered its broad applications. This paper describes the site-directed mutagenesis on residues in the putative catalytic region of Endo-M to generate mutants with superior Transglycosylation activity. Two interesting mutants were discovered. The Y217F mutant was found to possess much enhanced Transglycosylation activity and yet much diminished hydrolytic activity in comparison with the wild-type Endo-M. Kinetic analyses revealed that the Km value of Y217F for an acceptor substrate 4-methylumbelliferyl-β-l-N-acetylglucosaminide was only one-tenth of that of the wild-type, implicating a much higher affinity of Y217F for the acceptor substrate than the wild-type. The other mutant, N175A, acts like a glycosynthase. It was found that mutation at Asn175“knocked out” the hydrolytic activity, but the mutant was able to take the highly active sugar oxazolines (the transition state mimics) as donor substrates for Transglycosylation. This is the first glycosynthase derived from endo-β-N-acetylglucosaminidases that proceed via a substrate-assisted mechanism. Our findings provide further insights on the substrate-assisted mechanism of GH85. The usefulness of the novel glycosynthase was exemplified by the efficient synthesis of a human immunodeficiency virus, type 1 (HIV-1) glycopeptide with potent anti-HIV activity.
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molecular cloning of mucor hiemalis endo β n acetylglucosaminidase and some properties of the recombinant enzyme
Archives of Biochemistry and Biophysics, 2004Co-Authors: Kiyotaka Fujita, Hidehiko Kumagai, Kazuo Kobayashi, Akihiro Iwamatsu, Makoto Takeuchi, Kenji YamamotoAbstract:Endo-M, endo-beta-N-acetylglucosaminidase from Mucor hiemalis, is known as a useful enzyme for the synthesis of neoglycopeptides due to its Transglycosylation activity. We cloned the Endo-M gene encoding a putative 744 amino acids, which shows high identity to glycoside hydrolase family 85 endo-beta-N-acetylglucosaminidases. The gene encoding Endo-M was expressed in protease-deficient Candida boidinii with a molecular mass of 85 kDa as a monomeric form. Recombinant Endo-M could liberate both high-mannose type and biantennary complex type oligosaccharides from glycopeptides, which was same as the native enzyme. The Km and Kcat values for DNS-Man6GlcNAc2Asn were 0.51 mM and 8.25 s(-1), respectively. Recombinant Endo-M also exhibited Transglycosylation activity toward high-mannose type and biantennary complex type oligosaccharides, which were transferred to alcohols, monosaccharides, oligosaccharides, and glycosides. To investigate about the catalytically essential amino acids of Endo-M, site-directed mutagenesis was performed, and it was found that mutants E177G and E177Q completely abolished the hydrolytic activity and W228R partially abolished the Transglycosylation activity.
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Transglycosylation reaction of mucor hiemalis endo β n acetylglucosaminidase using sugar derivatives modified at c 1 or c 2 as oligosaccharide acceptors
Carbohydrate Research, 2004Co-Authors: Takashi Yamanoi, Kenji Yamamoto, Kenji Osumi, Eri Akaike, Yoshiki Oda, Maki Tsutsumida, Kiyotaka FujitaAbstract:Abstract We investigated the Transglycosylation reaction of the recombinant endo-β- N -acetylglucosaminidase from Mucor hiemalis (Endo-M) expressed in Candida boidinii using such sugar derivatives as N-acylated d -glucosamines, C -glucosyl derivatives, and a 2-O-glycosylated disaccharide as acceptors. We found that a variety of sugar derivatives modified at C-1 or C-2 could be used as acceptors for Transglycosylation by Endo-M to create novel oligosaccharides.
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high efficiency of transferring a native sugar chain from a glycopeptide by a microbial endoglycosidase in organic solvents
Carbohydrate Research, 2004Co-Authors: Eri Akaike, Kenji Yamamoto, Masaya Fujita, Kenji Osumi, Takashi Yamanoi, Maki Tsutsumida, Kiyotaka FujitaAbstract:We examined the Transglycosylation reaction by the recombinant endo-beta-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) expressed in Candida boidinii in media containing organic solvents. The recombinant Endo-M could transglycosylate a disialo biantennary complex-type oligosaccharide from hen egg yolk glycopeptide to p-nitrophenyl N-acetyl-beta-D-glucosaminide even in the presence of 30% acetone, dimethyl sulfoxide, or methanol. The yield of the Transglycosylation product reached 21-34% of the total amount of acceptor, while the yield was only about 14% in aqueous solution.
Kenji Yamamoto - One of the best experts on this subject based on the ideXlab platform.
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mutants of mucor hiemalis endo β n acetylglucosaminidase show enhanced Transglycosylation and glycosynthase like activities
Journal of Biological Chemistry, 2008Co-Authors: Midori Umekawa, Wei Huang, Hisashi Ashida, Laixi Wang, Kiyotaka Fujita, Kenji YamamotoAbstract:Abstract Endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M), a family 85 glycoside hydrolase, acts on the β1,4 linkage of N,N′-diacetylchitobiose moiety in the N-linked glycans of glycoproteins and catalyzes not only the hydrolysis reaction but also the Transglycosylation reaction that transfers the releasing sugar chain to an acceptor other than water to form a new glycosidic linkage. The Transglycosylation activity of Endo-M holds a great promise for the chemo-enzymatic synthesis and glyco-engineering of glycoproteins, but the inherent hydrolytic activity for product hydrolysis and low Transglycosylation have hampered its broad applications. This paper describes the site-directed mutagenesis on residues in the putative catalytic region of Endo-M to generate mutants with superior Transglycosylation activity. Two interesting mutants were discovered. The Y217F mutant was found to possess much enhanced Transglycosylation activity and yet much diminished hydrolytic activity in comparison with the wild-type Endo-M. Kinetic analyses revealed that the Km value of Y217F for an acceptor substrate 4-methylumbelliferyl-β-l-N-acetylglucosaminide was only one-tenth of that of the wild-type, implicating a much higher affinity of Y217F for the acceptor substrate than the wild-type. The other mutant, N175A, acts like a glycosynthase. It was found that mutation at Asn175“knocked out” the hydrolytic activity, but the mutant was able to take the highly active sugar oxazolines (the transition state mimics) as donor substrates for Transglycosylation. This is the first glycosynthase derived from endo-β-N-acetylglucosaminidases that proceed via a substrate-assisted mechanism. Our findings provide further insights on the substrate-assisted mechanism of GH85. The usefulness of the novel glycosynthase was exemplified by the efficient synthesis of a human immunodeficiency virus, type 1 (HIV-1) glycopeptide with potent anti-HIV activity.
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molecular cloning of mucor hiemalis endo β n acetylglucosaminidase and some properties of the recombinant enzyme
Archives of Biochemistry and Biophysics, 2004Co-Authors: Kiyotaka Fujita, Hidehiko Kumagai, Kazuo Kobayashi, Akihiro Iwamatsu, Makoto Takeuchi, Kenji YamamotoAbstract:Endo-M, endo-beta-N-acetylglucosaminidase from Mucor hiemalis, is known as a useful enzyme for the synthesis of neoglycopeptides due to its Transglycosylation activity. We cloned the Endo-M gene encoding a putative 744 amino acids, which shows high identity to glycoside hydrolase family 85 endo-beta-N-acetylglucosaminidases. The gene encoding Endo-M was expressed in protease-deficient Candida boidinii with a molecular mass of 85 kDa as a monomeric form. Recombinant Endo-M could liberate both high-mannose type and biantennary complex type oligosaccharides from glycopeptides, which was same as the native enzyme. The Km and Kcat values for DNS-Man6GlcNAc2Asn were 0.51 mM and 8.25 s(-1), respectively. Recombinant Endo-M also exhibited Transglycosylation activity toward high-mannose type and biantennary complex type oligosaccharides, which were transferred to alcohols, monosaccharides, oligosaccharides, and glycosides. To investigate about the catalytically essential amino acids of Endo-M, site-directed mutagenesis was performed, and it was found that mutants E177G and E177Q completely abolished the hydrolytic activity and W228R partially abolished the Transglycosylation activity.
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chemoenzymatic synthesis of neoglycopeptides using endo β n acetylglucosaminidase from mucor hiemalis
ChemInform, 2004Co-Authors: Katsuji Haneda, Toshiyuki Inazu, Mamoru Mizuno, Kenji YamamotoAbstract:Publisher Summary Endo- β -N-acetylglucosaminidase is an enzyme that hydrolytically cleaves the N,N´ -diacetylchitobiose moiety of the asparagines N-linked oligosaccharide of various glycoproteins and releases intact oligosaccharides. This enzyme is a unique endoglycosidase that leaves one N-acetyl-d-glucosamine (GlcNAc) residue on the protein moiety. Several microbial endo- β -N-acetylglucosaminidases show the Transglycosylation activity. Endo- β -N-acetylglucosaminidases of Arthrobacter protophormiae (Endo-A) and of mucor hiemalis (Endo-M) also show the Transglycosylation activity. Endo-A acts on a high mannose-type oligosaccharide, whereas Endo-M acts on complex-type, high mannose-type, and hybrid-type oligosaccharides and can transfer the oligosaccharides from glycopeptides to suitable acceptors with a GlcNAc residue during hydrolysis of the glycopeptides. This chapter focuses on a chemoenzymatic synthetic method for a glycopeptides combined with the chemical synthesis of a peptide containing GlcNAc as a glycosylation tag and the Transglycosylation catalyzed by Endo-M.
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Transglycosylation reaction of mucor hiemalis endo β n acetylglucosaminidase using sugar derivatives modified at c 1 or c 2 as oligosaccharide acceptors
Carbohydrate Research, 2004Co-Authors: Takashi Yamanoi, Kenji Yamamoto, Kenji Osumi, Eri Akaike, Yoshiki Oda, Maki Tsutsumida, Kiyotaka FujitaAbstract:Abstract We investigated the Transglycosylation reaction of the recombinant endo-β- N -acetylglucosaminidase from Mucor hiemalis (Endo-M) expressed in Candida boidinii using such sugar derivatives as N-acylated d -glucosamines, C -glucosyl derivatives, and a 2-O-glycosylated disaccharide as acceptors. We found that a variety of sugar derivatives modified at C-1 or C-2 could be used as acceptors for Transglycosylation by Endo-M to create novel oligosaccharides.
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high efficiency of transferring a native sugar chain from a glycopeptide by a microbial endoglycosidase in organic solvents
Carbohydrate Research, 2004Co-Authors: Eri Akaike, Kenji Yamamoto, Masaya Fujita, Kenji Osumi, Takashi Yamanoi, Maki Tsutsumida, Kiyotaka FujitaAbstract:We examined the Transglycosylation reaction by the recombinant endo-beta-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) expressed in Candida boidinii in media containing organic solvents. The recombinant Endo-M could transglycosylate a disialo biantennary complex-type oligosaccharide from hen egg yolk glycopeptide to p-nitrophenyl N-acetyl-beta-D-glucosaminide even in the presence of 30% acetone, dimethyl sulfoxide, or methanol. The yield of the Transglycosylation product reached 21-34% of the total amount of acceptor, while the yield was only about 14% in aqueous solution.
Wei Huang - One of the best experts on this subject based on the ideXlab platform.
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efficient transfer of sialo oligosaccharide onto proteins by combined use of a glycosynthase like mutant of mucor hiemalis endoglycosidase and synthetic sialo complex type sugar oxazoline
Biochimica et Biophysica Acta, 2010Co-Authors: Midori Umekawa, Takayuki Higashiyama, Wei Huang, Laixi Wang, Shinichiro Shoda, Yurie Koga, Tomonari Tanaka, Masato Noguchi, Atsushi Kobayashi, Hisashi AshidaAbstract:Abstract Background An efficient method for synthesizing homogenous glycoproteins is essential for elucidating the structural and functional roles of glycans of glycoproteins. We have focused on the Transglycosylation activity of endo-β- N -acetylglucosaminidase from Mucor hiemalis (Endo-M) as a tool for glycoconjugate syntheses, since it can transfer en bloc the oligosaccharide of not only high-mannose type but also complex-type N-glycan onto various acceptors having an N -acetylglucosamine residue. However, there are two major bottlenecks for its practical application: the low yield of the Transglycosylation product and the difficulty to obtain the activated sugar oxazoline substrate, especially the sialo-complex type one. Methods We carried out the Transglycosylation using a glycosynthase-like N175Q mutant of Endo-M, which was found to possess enhanced Transglycosylation activity with sugar oxazoline as a donor substrate, in combination with an easy preparation of the sialo-complex-type sugar oxazoline from natural sialoglycopeptide in egg yolk. Results Endo-M-N175Q showed efficient Transglycosylation toward sialo-complex-type sugar oxazoline onto bioactive peptides and bovine ribonuclease B, and each sialylated compound was obtained in significantly high yield. Conclusions Highly efficient and simple chemo-enzymatic syntheses of various sialylated compounds were enabled, by a combination of a simple synthesis of sialo-complex-type sugar oxazoline and the Endo-M-N175Q catalyzed Transglycosylation. General significance Our method would be very useful for a practical synthesis of biologically important glycopeptides and glycoproteins.
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enzymatic Transglycosylation for glycoconjugate synthesis
Current Opinion in Chemical Biology, 2009Co-Authors: Laixi Wang, Wei HuangAbstract:Remarkable advances have been made in recent years in exploiting the Transglycosylation activity of glycosidases and glycosynthase mutants for oligosaccharide and glycoconjugate synthesis. New glycosynthases were generated from retaining glycosidases, inverting glycosidases, and those that proceed in a mechanism of substrate-assisted catalysis. Directed evolution coupled with elegant screening methods has led to the discovery of an expanding number of glycosynthase mutants that show improved catalytic activity and/or altered substrate specificity. In particular, enzymatic Transglycosylation strategy has been recently extended to the synthesis of complex glycoconjugates, including glycosphingolipids, N-glycoproteins, and other glycosylated natural products.
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endo β n acetylglucosaminidase catalyzed polymerization of β glcp 1 4 glcpnac oxazoline a revisit to enzymatic Transglycosylation
Carbohydrate Research, 2009Co-Authors: Hirofumi Ochiai, Wei Huang, Laixi WangAbstract:An alternative synthesis of beta-Glcp-(1-->4)-GlcpNAc oxazoline is described, and its enzymatic reaction with the endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) was re-investigated. Under normal Transglycosylation conditions with a catalytic amount of enzyme, Endo-A showed only marginal activity for Transglycosylation with the disaccharide oxazoline, consistent with our previous observations. However, when used in a relatively large quantity, Endo-A could promote the Transglycosylation of the disaccharide oxazoline to a GlcpNAc-Asn acceptor. In addition to the initial Transglycosylation product, a series of large oligosaccharides were also formed due to the tandem Transglycosylation to the terminal glucose residues in the intermediate products. In the absence of an external acceptor, Endo-A could polymerize the disaccharide oxazoline to form oligo- and polysaccharides having the -4-beta-(Glcp-(1-->4)-beta -GlcpNAc)-1-repeating units. This is the first example of an endo-beta-N-acetylglucosaminidase-promoted polymerization of activated oligosaccharide substrates. This enzymatic polymerization may find useful applications for the synthesis of novel artificial polysaccharides.
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expeditious chemoenzymatic synthesis of homogeneous n glycoproteins carrying defined oligosaccharide ligands
Journal of the American Chemical Society, 2008Co-Authors: Hirofumi Ochiai, Wei Huang, Laixi WangAbstract:An efficient chemoenzymatic method for the construction of homogeneous N-glycoproteins was described that explores the Transglycosylation activity of the endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) with synthetic sugar oxazolines as the donor substrates. First, an array of large oligosaccharide oxazolines were synthesized and evaluated as substrates for the Endo-A-catalyzed Transglycosylation by use of ribonuclease B as a model system. The experimental results showed that Endo-A could tolerate modifications at the outer mannose residues of the Man3GlcNAc-oxazoline core, thus allowing introduction of large oligosaccharide ligands into a protein and meanwhile preserving the natural, core N-pentasaccharide (Man3GlcNAc2) structure in the resulting glycoprotein upon Transglycosylation. In addition to ligands for galectins and mannose-binding lectins, azido functionality could be readily introduced at the N-pentasaccharide (Man3GlcNAc2) core by use of azido-containing Man3GlcNAc oxaz...
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mutants of mucor hiemalis endo β n acetylglucosaminidase show enhanced Transglycosylation and glycosynthase like activities
Journal of Biological Chemistry, 2008Co-Authors: Midori Umekawa, Wei Huang, Hisashi Ashida, Laixi Wang, Kiyotaka Fujita, Kenji YamamotoAbstract:Abstract Endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M), a family 85 glycoside hydrolase, acts on the β1,4 linkage of N,N′-diacetylchitobiose moiety in the N-linked glycans of glycoproteins and catalyzes not only the hydrolysis reaction but also the Transglycosylation reaction that transfers the releasing sugar chain to an acceptor other than water to form a new glycosidic linkage. The Transglycosylation activity of Endo-M holds a great promise for the chemo-enzymatic synthesis and glyco-engineering of glycoproteins, but the inherent hydrolytic activity for product hydrolysis and low Transglycosylation have hampered its broad applications. This paper describes the site-directed mutagenesis on residues in the putative catalytic region of Endo-M to generate mutants with superior Transglycosylation activity. Two interesting mutants were discovered. The Y217F mutant was found to possess much enhanced Transglycosylation activity and yet much diminished hydrolytic activity in comparison with the wild-type Endo-M. Kinetic analyses revealed that the Km value of Y217F for an acceptor substrate 4-methylumbelliferyl-β-l-N-acetylglucosaminide was only one-tenth of that of the wild-type, implicating a much higher affinity of Y217F for the acceptor substrate than the wild-type. The other mutant, N175A, acts like a glycosynthase. It was found that mutation at Asn175“knocked out” the hydrolytic activity, but the mutant was able to take the highly active sugar oxazolines (the transition state mimics) as donor substrates for Transglycosylation. This is the first glycosynthase derived from endo-β-N-acetylglucosaminidases that proceed via a substrate-assisted mechanism. Our findings provide further insights on the substrate-assisted mechanism of GH85. The usefulness of the novel glycosynthase was exemplified by the efficient synthesis of a human immunodeficiency virus, type 1 (HIV-1) glycopeptide with potent anti-HIV activity.
Sha Li - One of the best experts on this subject based on the ideXlab platform.
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Transglycosylation of stevioside to improve the edulcorant quality by lower substitution using cornstarch hydrolyzate and cgtase
Food Chemistry, 2013Co-Authors: Wei Li, Sha Li, Qiuyan XiaoAbstract:Stevioside is an abundant sweetener extracted from Stevia rebaudiana leaf with a bitter aftertaste. Enzymatic Transglycosylation of stevioside is a solution to improve the edulcorant quality of stevioside, but highly derivatised stevioside coming with high conversion of stevioside is undesired. In this experiment, the Transglycosylation of stevioside was investigated by using a commercial cyclodextrin glucanotransferase and cornstarch hydrolyzate. With controlled parameters, the product was mainly composed of mono- and di-glucosylated stevioside while the highest stevioside conversion reached 77.11%. Neither kinetic nor thermodynamic factor stimulated the formation of high substituted steviosides. The simultaneous hydrolysis in the reaction might inhibit the yield of highly substituted steviosides.
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Transglycosylation of stevioside to improve the edulcorant quality by lower substitution using cornstarch hydrolyzate and cgtase
Food Chemistry, 2013Co-Authors: Wei Li, Sha Li, Qiuyan XiaoAbstract:Stevioside is an abundant sweetener extracted from Stevia rebaudiana leaf with a bitter aftertaste. Enzymatic Transglycosylation of stevioside is a solution to improve the edulcorant quality of stevioside, but highly derivatised stevioside coming with high conversion of stevioside is undesired. In this experiment, the Transglycosylation of stevioside was investigated by using a commercial cyclodextrin glucanotransferase and cornstarch hydrolyzate. With controlled parameters, the product was mainly composed of mono- and di-glucosylated stevioside while the highest stevioside conversion reached 77.11%. Neither kinetic nor thermodynamic factor stimulated the formation of high substituted steviosides. The simultaneous hydrolysis in the reaction might inhibit the yield of highly substituted steviosides.
