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A novel bacterial β-N-acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation and reverse hydrolysis activities.
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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 toward 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 4878.6 U/mg of protein. CmNAGase had a molecular mass of 92 kDa, and its optimum activity was at pH 5.4 and 40 °C. The V max, K m, K cat, and K cat/K m 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, suggest a possible application in the production of GlcNAc or higher N-Acetyl COSs.
SUBMITTER: Zhang A
PROVIDER: S-EPMC7324980 | biostudies-literature | 2020
REPOSITORIES: biostudies-literature
Publications
A novel bacterial β-<i>N</i>-acetyl glucosaminidase from <i>Chitinolyticbacter meiyuanensis</i> possessing transglycosylation and reverse hydrolysis activities.
Biotechnology for biofuels 20200629
<h4>Background</h4><i>N</i>-Acetyl glucosamine (GlcNAc) and <i>N</i>-Acetyl chitooligosaccharides (<i>N</i>-Acetyl COSs) exhibit many biological activities, and have been widely used in the pharmaceutical, agriculture, food, and chemical industries. Particularly, higher <i>N</i>-Acetyl COSs with degree of polymerization from 4 to 7 ((GlcNAc)<sub>4</sub>-(GlcNAc)<sub>7</sub>) show good antitumor and antimicrobial activity, as well as possessing strong stimulating activity toward natural killer ce ...[more]