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

Background N-Acetyl glucosamine (GlcNAc) andN-Acetyl chitooligosaccharides (N-Acetyl COSs) exhibit many biological activities, and have been widely used in the pharmaceutical, agriculture, food, and chemical industries. Particularly, higherN-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 beta-N-acetyl glucosaminidase (NAGase) that can not only produce GlcNAc, but also synthesizeN-Acetyl COSs. Results The gene encoding the novel beta-N-acetyl glucosaminidase, designatedCmNAGase, was cloned fromChitinolyticbacter meiyuanensisSYBC-H1. The deduced amino acid sequence ofCmNAGase contains a glycoside hydrolase family 20 catalytic module that shows low identity (12-35%) with the corresponding domain of most well-characterized NAGases. TheCmNAGase gene was highly expressed with an active form inEscherichia coliBL21 (DE3) cells. The specific activity of purifiedCmNAGase towardp-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 degrees C. TheV(max),K-m,K-cat, andK(cat)/K(m)ofCmNAGase forpNP-GlcNAc were 16,666.67 mu mol min(-1) mg(-1), 0.50 mu mol mL(-1), 25,555.56 s(-1), and 51,111.12 mL mu mol(-1) s(-1), respectively. Analysis of the hydrolysis products ofN-Acetyl COSs and colloidal chitin revealed thatCmNAGase is a typical exo-acting NAGase. Particularly,CmNAGase can synthesize higherN-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 thatCmNAGase is a novel NAGase with exo-acting, transglycosylation, and reverse hydrolysis activities, suggest a possible application in the production of GlcNAc or higherN-Acetyl COSs.