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

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

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 Cm NAGase, was cloned from Chitinolyticbacter meiyuanensis SYBC-H1. The deduced amino acid sequence of Cm NAGase contains a glycoside hydrolase family 20 catalytic module that shows low identity (12–35%) with the corresponding domain of most well-characterized NAGases. The Cm NAGase gene was highly expressed with an active form in Escherichia coli BL21 (DE3) cells. The specific activity of purified Cm NAGase toward p -nitrophenyl- N -acetyl glucosaminide ( p NP-GlcNAc) was 4878.6 U/mg of protein. Cm NAGase 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 Cm NAGase for p NP-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 Cm NAGase is a typical exo-acting NAGase. Particularly, Cm NAGase can synthesize higher N -Acetyl COSs ((GlcNAc) 3 –(GlcNAc) 7 ) from (GlcNAc) 2 –(GlcNAc) 6 , respectively, showed that it possesses transglycosylation activity. In addition, Cm NAGase also has reverse hydrolysis activity toward GlcNAc, synthesizing various linked GlcNAc dimers. Conclusions The observations recorded in this study that Cm NAGase 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.