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

Alei Zhang; Xiaofang Mo; Ning Zhou; Yingying Wang; Guoguang Wei; Jie Chen; Kequan Chen; Pingkai Ouyang

doi:10.1186/s13068-020-01754-4

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

Biotechnol Biofuels. 2020 Jun 29:13:115. doi: 10.1186/s13068-020-01754-4. eCollection 2020.

Authors

Alei Zhang¹, Xiaofang Mo¹, Ning Zhou¹, Yingying Wang¹, Guoguang Wei¹, Jie Chen¹, Kequan Chen¹, Pingkai Ouyang¹

Affiliation

¹ State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211800 People's Republic of China.

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.

Keywords: Exo-acting activity; N-Acetyl chitooligosaccharides; N-Acetyl glucosamine; Reverse hydrolysis; Transglycosylation; β-N-acetyl glucosaminidase.