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

Abstract

<jats:title>Abstract</jats:title> <jats:p><jats:bold>Background:</jats:bold> 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. <jats:bold>Results: </jats:bold>The gene encoding the novel β-N-acetyl glucosaminidase, designated <jats:italic>Cm</jats:italic>NAGase, was cloned from <jats:italic>Chitinolyticbacter meiyuanensis </jats:italic>SYBC-H1. The deduced amino acid sequence of <jats:italic>Cm</jats:italic>NAGase contains a glycoside hydrolase family 20 catalytic module that shows low identity with the corresponding domain of the well-characterized NAGases. <jats:italic>Cm</jats:italic>NAGase gene was highly expressed with soluble form in<jats:italic> Escherichia coli </jats:italic>BL21 (DE3) cells, whereupon it had a specific activity of 4,878.6 U/mg of protein toward <jats:italic>p</jats:italic>-nitrophenyl-N-acetyl glucosaminide. <jats:italic>Cm</jats:italic>NAGase had a molecular mass of 92 kDa, and its optimum activity was at pH 5.4 and 40ºC. The <jats:italic>V</jats:italic><jats:sub><jats:italic>max</jats:italic></jats:sub>,<jats:italic> K</jats:italic><jats:sub><jats:italic>m</jats:italic></jats:sub>, and <jats:italic>K</jats:italic><jats:sub><jats:italic>cat</jats:italic></jats:sub> of <jats:italic>Cm</jats:italic>NAGase were 833.33 μmol·L<jats:sup>-1 </jats:sup>·min<jats:sup>-1</jats:sup>, 10.9 mmol, and 6.37 ´ 10<jats:sup>8</jats:sup> mM·mg<jats:sup>-1</jats:sup>, respectively. Analysis of the hydrolysis products of <jats:italic>N</jats:italic>-acetyl chitooligosaccharides and colloidal chitin revealed that <jats:italic>Cm</jats:italic>NAGase exhibits exo-acting activities. Particularly, it possesses transglycosylation activity, which can synthesize (GlcNAc)<jats:sub>n+1</jats:sub> from (GlcNAc)<jats:sub>n (n=1−6)</jats:sub>, respectively. In addition, <jats:italic>Cm</jats:italic>NAGase also can catalyze GlcNAc to its dimers with various linked forms. <jats:bold>Conclusions:</jats:bold> The observations recorded in this study that <jats:italic>Cm</jats:italic>NAGase 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.</jats:p>