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

In this study, a gene encoding beta-N-acetylglucosaminidase, designated NAGaseA, was cloned from Chitinibacter sp. GC72 and subsequently functional expressed in Escherichia coli BL21 (DE3). NAGaseA contains a glycoside hydrolase family 20 catalytic domain that shows low identity with the corresponding domain of the wellcharacterized 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 degrees C and pH 6.5, and exhibited great pH stability in the range of pH 6.5-9.5. The V-max, K-m, k(cat), and k(cat)/K-m of NAGaseA toward p-nitrophenyl-Nacetyl glucosaminide (pNP-GlcNAc) were 3333.33 mu mol min(-1) l(-1), 39.99 mu mol l(-1), 4667.07 s(-1), and 116.71 ml mu mol(-1) s(-1), respectively. Analysis of the hydrolysis products of N-acetyl chitin oligosaccharides (N-Acetyl COSs) indicated that NAGaseA was capable of converting N-acetyl COSs ((GlcNAc)(2)-(GlcNAc)(6)) into GlcNAc with hydrolysis ability order: (GlcNAc)(2) > (GlcNAc)(3) > (GlcNAc)(4) > (GlcNAc)(5) > (GlcNAc)(6). Moreover, NAGaseA could generate (GlcNAc)(3)-(GlcNAc)(6) from (GlcNAc)(2)-(GlcNAc)(5), 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.

Automated summary

In this study, a gene encoding beta-N-acetylglucosaminidase, designated NAGaseA, was cloned and functionally expressed in Escherichia coli. NAGaseA showed both transglycosylation and hydrolytic activities, making it a potential candidate for GlcNAc production.