Enzymatic synthesis of N-acetylglucosaminobioses by reverse hydrolysis: characterisation and application of the library of fungal β-N-acetylhexosaminidases

Rauvolfová, Jana; Weignerová, Lenka; Kuzma, Marek; Přikrylová, Věra; Macková, Martina; Pišvejcová, Andrea; Křen, Vladimı́r

doi:10.1016/j.molcatb.2004.02.007

Cited by 14 publications

(21 citation statements)

References 26 publications

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Section: Reverse Hydrolysis Activity Of Cmnagase Toward Glcnacmentioning

confidence: 99%

“…The reverse hydrolysis reaction of GH20 NAGases toward GlcNAc can form many connection con gurations of the GlcNAc dimer, for example by β(1→3), β(1→4), and β(1→6) glycosidic bonds, which is mainly determined by the regioselectivity of glycosidase [30]. To date, studies on the synthesis of GlcNAc dimer from GlcNAc were mainly reported by Rauvolfová et al, which produce β-GlcNAc-(1→3)-GlcNAc, β-GlcNAc-(1→4)-GlcNAc, and β-GlcNAc-(1→6)-GlcNAc using the library of fungal GH20 NAGases [30]. For example, the GH20 NAGases from Acremonium persicinum CCF 1850, A. oryzae CCF 1066, Aspergillus avipes CCF 2026, A. avus CCF 1129, P. oxalicum CCF 2315, and A. terreus CCF 2539, which only produced β-GlcNAc-(1→6)-GlcNAc from GlcNAc [30,31].…”

Section: Reverse Hydrolysis Activity Of Cmnagase Toward Glcnacmentioning

confidence: 99%

“…To date, studies on the synthesis of GlcNAc dimer from GlcNAc were mainly reported by Rauvolfová et al, which produce β-GlcNAc-(1→3)-GlcNAc, β-GlcNAc-(1→4)-GlcNAc, and β-GlcNAc-(1→6)-GlcNAc using the library of fungal GH20 NAGases [30]. For example, the GH20 NAGases from Acremonium persicinum CCF 1850, A. oryzae CCF 1066, Aspergillus avipes CCF 2026, A. avus CCF 1129, P. oxalicum CCF 2315, and A. terreus CCF 2539, which only produced β-GlcNAc-(1→6)-GlcNAc from GlcNAc [30,31]. The GH20 NAGases from P. funiculosum CCF 1994, P. funiculosum CCF 2325, P. chrysogenum CCF 1269, and Trichoderma harzianum CCF 2687 synthesized β-GlcNAc-(1→3)-GlcNAc, β-GlcNAc-(1→4)-GlcNAc, and β-GlcNAc-(1→6)-GlcNAc from GlcNAc [30].…”

Section: Reverse Hydrolysis Activity Of Cmnagase Toward Glcnacmentioning

confidence: 99%

“…NAGases can be found in a wide variety of organisms including bacteria, fungal, insects, plants, and animals [24][25][26][27]. However, studies about the GH20 NAGases with transglycosylation and reverse hydrolysis activities are mainly derived from fungal sources [28][29][30][31][32][33][34]. There are few reports about the bacterial NAGases possessing transglycosylation and reverse hydrolysis activities [21,[35][36][37].…”

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A novel bacterial β-N-acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation and reverse hydrolysis activities

Zhang

Zhou

et al. 2020

Preprint

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Abstract Background: N-acetyl glucosamine (GlcNAc) and N-acetyl chitooligosaccharides (N-acetyl COSs) exhibit many biological activities, and have been widely used in the pharmaceutical, agriculture, food, and chemical industries. Particularly, higher N-acetyl COSs with degree of polymerization from 4 to 7 ((GlcNAc)4−(GlcNAc)7) show good antitumor and antimicrobial activity, as well as possessing strong stimulating activity towards natural killer cells. Thus, it is of great significance to discover a β-N-acetyl glucosaminidase (NAGase) that can not only produce GlcNAc, but also synthesize N-acetyl COSs. Results: The gene encoding the novel β-N-acetyl glucosaminidase, designated CmNAGase, was cloned from Chitinolyticbacter meiyuanensis SYBC-H1. The deduced amino acid sequence of CmNAGase contains a glycoside hydrolase family 20 catalytic module that shows low identity (12−35%) with the corresponding domain of most well-characterized NAGases. The CmNAGase gene was highly expressed with an active form in Escherichia coli BL21 (DE3) cells. The specific activity of purified CmNAGase toward p-nitrophenyl-N-acetyl glucosaminide (pNP-GlcNAc) was 4,878.6 U/mg of protein. CmNAGase had a molecular mass of 92 kDa, and its optimum activity was at pH 5.4 and 40ºC. The Vmax, Km, Kcat, and Kcat/Km of CmNAGase for pNP-GlcNAc were 16,666.67 μmol min-1 mg-1, 0.50 μmol mL-1, 25,555.56 s-1, and 51,111.12 mL μmol-1 s-1, respectively. Analysis of the hydrolysis products of N-acetyl COSs and colloidal chitin revealed that CmNAGase is a typical exo-acting NAGase. Particularly, CmNAGase can synthesize higher N-acetyl COSs ((GlcNAc)3−(GlcNAc)7) from (GlcNAc)2−(GlcNAc)6, respectively, showed that it possesses transglycosylation activity. In addition, CmNAGase also has reverse hydrolysis activity toward GlcNAc, synthesizing various linked GlcNAc dimers. Conclusions: The observations recorded in this study that CmNAGase is a novel NAGase with exo-acting, transglycosylation, and reverse hydrolysis activities, suggests a possible application in the production of GlcNAc or higher N-acetyl COSs.

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Section: Reverse Hydrolysis Activity Of Cmnagase Toward Glcnacmentioning

confidence: 99%

Section: Reverse Hydrolysis Activity Of Cmnagase Toward Glcnacmentioning

confidence: 99%

Section: Reverse Hydrolysis Activity Of Cmnagase Toward Glcnacmentioning

confidence: 99%

mentioning

confidence: 99%

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A novel bacterial β-N-acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation and reverse hydrolysis activities

Zhang

Zhou

et al. 2020

Preprint

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“…The regioselectivity of glycosidases mainly depends on two main factors: the sources of the enzymes and the kinds of substrates (5,54,55). The ␤-Nacetylhexosaminidase from A. oryzae synthesizes GlcNAc␤1-3/1-6Gal␤1-4Glc via reverse hydrolysis by using GlcNAc and lactose (44).…”

Section: Figmentioning

confidence: 99%

Efficient and Regioselective Synthesis of β-GalNAc/GlcNAc-Lactose by a Bifunctional Transglycosylating β- N -Acetylhexosaminidase from Bifidobacterium bifidum

Chen

Jin

et al. 2016

Appl Environ Microbiol

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ABSTRACT␤-N-Acetylhexosaminidases have attracted interest particularly for oligosaccharide synthesis, but their use remains limited by the rarity of enzyme sources, low efficiency, and relaxed regioselectivity of transglycosylation. In this work, genes of 13 ␤-Nacetylhexosaminidases, including 5 from Bacteroides fragilis ATCC 25285, 5 from Clostridium perfringens ATCC 13124, and 3 from Bifidobacterium bifidum JCM 1254, were cloned and heterogeneously expressed in Escherichia coli. The resulting recombinant enzymes were purified and screened for transglycosylation activity. A ␤-N-acetylhexosaminidase named BbhI, which belongs to glycoside hydrolase family 20 and was obtained from B. bifidum JCM 1254, possesses the bifunctional property of efficiently transferring both GalNAc and GlcNAc residues through ␤1-3 linkage to the Gal residue of lactose. The effects of initial substrate concentration, pH, temperature, and reaction time on transglycosylation activities of BbhI were studied in detail. With the use of 10 mM pNP-␤-GalNAc or 20 mM pNP-␤-GlcNAc as the donor and 400 mM lactose as the acceptor in phosphate buffer (pH 5.8), BbhI synthesized GalNAc␤1-3Gal␤1-4Glc and GlcNAc␤1-3Gal␤1-4Glc at maximal yields of 55.4% at 45°C and 4 h and 44.9% at 55°C and 1.5 h, respectively. The model docking of BbhI with lactose showed the possible molecular basis of strict regioselectivity of ␤1-3 linkage in ␤-N-acetylhexosaminyl lactose synthesis. IMPORTANCEOligosaccharides play a crucial role in many biological events and therefore are promising potential therapeutic agents. However, their use is limited because large-scale production of oligosaccharides is difficult. The chemical synthesis requires multiple protecting group manipulations to control the regio-and stereoselectivity of glycosidic bonds. In comparison, enzymatic synthesis can produce oligosaccharides in one step by using glycosyltransferases and glycosidases. Given the lower price of their glycosyl donor and their broader acceptor specificity, glycosidases are more advantageous than glycosyltransferases for large-scale synthesis. ␤-N-Acetylhexosaminidases have attracted interest particularly for ␤-N-acetylhexosaminyl oligosaccharide synthesis, but their application is affected by having few enzyme sources, low efficiency, and relaxed regioselectivity of transglycosylation. In this work, we describe a microbial ␤-N-acetylhexosaminidase that exhibited strong transglycosylation activity and strict regioselectivity for ␤-N-acetylhexosaminyl lactose synthesis and thus provides a powerful synthetic tool to obtain biologically important GalNAc␤1-3Lac and GlcNAc␤1-3Lac. Oligosaccharides are widely distributed in nature and play a crucial role in many biological events, such as cell structure modulation, cell-cell recognition and communication, and cellmicrobe/toxin interaction and adhesion; therefore, they are promising and important potential therapeutic agents (1-3). However, their use is limited because of the difficulty of large-scale production of oligosaccharides. Their...

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Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2003–2004

Harvey

2008

Mass Spectrometry Reviews

100

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This review is the third update of the original review, published in 1999, on the application of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings the topic to the end of 2004. Both fundamental studies and applications are covered. The main topics include methodological developments, matrices, fragmentation of carbohydrates and applications to large polymeric carbohydrates from plants, glycans from glycoproteins and those from various glycolipids. Other topics include the use of MALDI MS to study enzymes related to carbohydrate biosynthesis and degradation, its use in industrial processes, particularly biopharmaceuticals and its use to monitor products of chemical synthesis where glycodendrimers and carbohydrate-protein complexes are highlighted.

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Enzymatic synthesis of N-acetylglucosaminobioses by reverse hydrolysis: characterisation and application of the library of fungal β-N-acetylhexosaminidases

Cited by 14 publications

References 26 publications

A novel bacterial β-N-acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation and reverse hydrolysis activities

A novel bacterial β-N-acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation and reverse hydrolysis activities

Efficient and Regioselective Synthesis of β-GalNAc/GlcNAc-Lactose by a Bifunctional Transglycosylating β- N -Acetylhexosaminidase from Bifidobacterium bifidum

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2003–2004

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