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

Abstract

<jats:title>Abstract</jats:title> <jats:p><jats:bold>Background:</jats:bold> <jats:italic>N</jats:italic>-acetyl glucosamine (GlcNAc) and <jats:italic>N</jats:italic>-acetyl chitooligosaccharides (<jats:italic>N</jats:italic>-acetyl COSs) exhibit many biological activities, and have been widely used in the pharmaceutical, agriculture, food, and chemical industries. Particularly, higher <jats:italic>N</jats:italic>-acetyl COSs with degree of polymerization from 4 to 7 ((GlcNAc)<jats:sub>4</jats:sub>−(GlcNAc)<jats:sub>7</jats:sub>) show good antitumor and antimicrobial activity, as well as possessing strong stimulating activity towards natural killer cells. Thus, it is of great significance to discover a β-<jats:italic>N</jats:italic>-acetyl glucosaminidase (NAGase) that can not only produce GlcNAc, but also synthesize <jats:italic>N</jats:italic>-acetyl COSs. <jats:bold>Results: </jats:bold>The gene encoding the novel β-<jats:italic>N</jats:italic>-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 (12−35%) with the corresponding domain of most well-characterized NAGases. The <jats:italic>Cm</jats:italic>NAGase gene was highly expressed with an active form in<jats:italic> Escherichia coli </jats:italic>BL21 (DE3) cells. The specific activity of purified <jats:italic>Cm</jats:italic>NAGase toward <jats:italic>p</jats:italic>-nitrophenyl-<jats:italic>N</jats:italic>-acetyl glucosaminide (<jats:italic>p</jats:italic>NP-GlcNAc) was 4,878.6 U/mg of protein. <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>max</jats:sub>,<jats:italic> K</jats:italic><jats:sub>m</jats:sub>, <jats:italic>K</jats:italic><jats:sub>cat</jats:sub>, and <jats:italic>K</jats:italic><jats:sub>cat</jats:sub>/<jats:italic>K</jats:italic><jats:sub>m</jats:sub> of <jats:italic>Cm</jats:italic>NAGase for <jats:italic>p</jats:italic>NP-GlcNAc were 16,666.67 μmol min<jats:sup>-1 </jats:sup>mg<jats:sup>-1</jats:sup>, 0.50 μmol mL<jats:sup>-1</jats:sup>, 25,555.56 s<jats:sup>-1</jats:sup>, and 51,111.12 mL μmol<jats:sup>-1</jats:sup> s<jats:sup>-1</jats:sup>, respectively. Analysis of the hydrolysis products of <jats:italic>N</jats:italic>-acetyl COSs and colloidal chitin revealed that <jats:italic>Cm</jats:italic>NAGase is a typical exo-acting NAGase. Particularly, <jats:italic>Cm</jats:italic>NAGase can synthesize higher <jats:italic>N</jats:italic>-acetyl COSs ((GlcNAc)<jats:sub>3</jats:sub>−(GlcNAc)<jats:sub>7</jats:sub>) from (GlcNAc)<jats:sub>2</jats:sub>−(GlcNAc)<jats:sub>6</jats:sub>, respectively, showed that it possesses transglycosylation activity. In addition, <jats:italic>Cm</jats:italic>NAGase also has reverse hydrolysis activity toward GlcNAc, synthesizing various linked GlcNAc dimers. <jats:bold>Conclusions:</jats:bold> The observations recorded in this study that <jats:italic>Cm</jats:italic>NAGase is a novel NAGase with exo-acting, transglycosylation, and reverse hydrolysis activities, suggests a possible application in the production of GlcNAc or higher <jats:italic>N</jats:italic>-acetyl COSs.</jats:p>

Discovering and Designing a Chimeric Hyperthermophilic Chitinase for Crystalline Chitin Degradation

“…Continuous hydrolysis with high concentration of HCl can degrade it into N-acetyl-Dglucosamine. 4 These physical and chemical methods are energy-extensive and can generate serious environmental pollution. 5 In contrast, directly hydrolyzing chitin with chitinase is mild and environmentally friendly.…”