AUTHOR=Chen Yan , Zhou Ning , Chen Xueman , Wei Guoguang , Zhang Alei , Chen Kequan , Ouyang Pingkai 

TITLE=Characterization of a New Multifunctional GH20 β-N-Acetylglucosaminidase From Chitinibacter sp. GC72 and Its Application in Converting Chitin Into N-Acetyl Glucosamine

JOURNAL=Frontiers in Microbiology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.874908

DOI=10.3389/fmicb.2022.874908

ISSN=1664-302X

ABSTRACT=<p>In this study, a gene encoding β-<italic>N</italic>-acetylglucosaminidase, designated NAGaseA, was cloned from <italic>Chitinibacter</italic> sp. GC72 and subsequently functional expressed in <italic>Escherichia coli</italic> BL21 (DE3). NAGaseA contains a glycoside hydrolase family 20 catalytic domain that shows low identity with the corresponding domain of the well-characterized NAGases. The recombinant NAGaseA had a molecular mass of 92 kDa. Biochemical characterization of the purified NAGaseA revealed that the optimal reaction condition was at 40°C and pH 6.5, and exhibited great pH stability in the range of pH 6.5–9.5. The <italic>V</italic><sub><italic>ma</italic></sub><italic><sub><italic>x</italic></sub></italic>, <italic>K</italic><sub>m</sub>, <italic>k</italic><sub>cat</sub>, and <italic>k</italic><sub>cat</sub><italic>/K</italic><sub>m</sub> of NAGaseA toward <italic>p</italic>-nitrophenyl-<italic>N</italic>-acetyl glucosaminide (<italic>p</italic>NP-GlcNAc) were 3333.33 μmol min<sup>–1</sup> l<sup>–1</sup>, 39.99 μmol l<sup>–1</sup>, 4667.07 s<sup>–1</sup>, and 116.71 ml μmol<sup>–1</sup> s<sup>–1</sup>, respectively. Analysis of the hydrolysis products of <italic>N</italic>-acetyl chitin oligosaccharides (<italic>N</italic>-Acetyl COSs) indicated that NAGaseA was capable of converting <italic>N</italic>-acetyl COSs ((GlcNAc)<sub>2</sub>–(GlcNAc)<sub>6</sub>) into GlcNAc with hydrolysis ability order: (GlcNAc)<sub>2</sub> &gt; (GlcNAc)<sub>3</sub> &gt; (GlcNAc)<sub>4</sub> &gt; (GlcNAc)<sub>5</sub> &gt; (GlcNAc)<sub>6</sub>. Moreover, NAGaseA could generate (GlcNAc)<sub>3</sub>–(GlcNAc)<sub>6</sub> from (GlcNAc)<sub>2</sub>–(GlcNAc)<sub>5</sub>, respectively. These results showed that NAGaseA is a multifunctional NAGase with transglycosylation activity. In addition, significantly synergistic action was observed between NAGaseA and other sources of chitinases during hydrolysis of colloid chitin. Finally, 0.759, 0.481, and 0.986 g/l of GlcNAc with a purity of 96% were obtained using three different chitinase combinations, which were 1.61-, 2.36-, and 2.69-fold that of the GlcNAc production using the single chitinase. This observation indicated that NAGaseA could be a potential candidate enzyme in commercial GlcNAc production.</p>