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 β-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 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 V_max, K_m, k_cat, and k_cat/K_m of NAGaseA toward p-nitrophenyl-N-acetyl glucosaminide (pNP-GlcNAc) were 3333.33 μmol min^–1 l^–1, 39.99 μmol l^–1, 4667.07 s^–1, and 116.71 ml μmol^–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.