A novel β-N-acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation activity and its use in generating long-chain N-acetyl chitooligosaccharides

Background: N-acetyl glucosamine (GlcNAc) and N-acetyl chitooligosaccharides (N-acetyl COSs) exhibit antitumor and antimicrobial activities, and have been widely used in the pharmaceutical, agriculture, food, and chemical industries. Thus, it is crucial to discover a NAGase that can both synthesize GlcNAc and 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 with the corresponding domain of the well-characterized NAGases. CmNAGase gene was highly expressed with soluble form in Escherichia coli BL21 (DE3) cells, whereupon it had a specific activity of 4,878.6 U/mg of protein toward p-nitrophenyl-N-acetyl glucosaminide. CmNAGase had a molecular mass of 92 kDa, and its optimum activity was at pH 5.4 and 40ºC. The Vmax, Km, and Kcat of CmNAGase were 833.33 μmol·L-1 ·min-1, 10.9 mmol, and 6.37 ´ 108 mM·mg-1, respectively. Analysis of the hydrolysis products of N-acetyl chitooligosaccharides and colloidal chitin revealed that CmNAGase exhibits exo-acting activities. Particularly, it possesses transglycosylation activity, which can synthesize (GlcNAc)n+1 from (GlcNAc)n (n=1−6), respectively. In addition, CmNAGase also can catalyze GlcNAc to its dimers with various linked forms. Conclusions: The observations recorded in this study that CmNAGase is an exo NAGase with unique transglycosylation activity, suggests a possible application in the production of long-chain N-acetyl CHOs. This is first report of a bacterial NAGase, which can produce long-chain N-acetyl COSs via transglycosylation activity.

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