In this study, a gene encoding β--acetylglucosaminidase, designated NAGaseA, was cloned from sp. GC72 and subsequently functional expressed in 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 , , , and of NAGaseA toward -nitrophenyl--acetyl glucosaminide (NP-GlcNAc) were 3333.33 μmol min l, 39.99 μmol l, 4667.07 s, and 116.71 ml μmol s, resp... More
In this study, a gene encoding β--acetylglucosaminidase, designated NAGaseA, was cloned from sp. GC72 and subsequently functional expressed in 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 , , , and of NAGaseA toward -nitrophenyl--acetyl glucosaminide (NP-GlcNAc) were 3333.33 μmol min l, 39.99 μmol l, 4667.07 s, and 116.71 ml μmol s, respectively. Analysis of the hydrolysis products of -acetyl chitin oligosaccharides (-Acetyl COSs) indicated that NAGaseA was capable of converting -acetyl COSs ((GlcNAc)-(GlcNAc)) into GlcNAc with hydrolysis ability order: (GlcNAc) > (GlcNAc) > (GlcNAc) > (GlcNAc) > (GlcNAc). Moreover, NAGaseA could generate (GlcNAc)-(GlcNAc) from (GlcNAc)-(GlcNAc), 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.
