A Bacillus pumilus originated β-N-acetylglucosaminidase for chitin combinatory hydrolysis and exploration of its thermostable mechanism.

β-N-acetylglucosaminase (NAGase) plays pivotal roles in industrial applications. Here, a GH3 family NAGase encoding gene from Bacillus pumilus was cloned and expressed in Escherichia coli. The optimal temperature and pH of the recombinant BpNagZ were 70 °C and 6.0, respectively, and kept more than 40% residual activity at 70 °C for 30 min. Metal ions such as Zn2 +, Cu2+, Cd2+, Mg2+, and Ca2+, even chelating agent, EDTA had slight effects on the activity of BpNagZ, indicating that BpNagZ was not a metal-dependent enzyme. Compared with the homology protein, BsNagZ from B. subtilis, the thermostability and activity of BpNagZ improved significantly. Structural simulation and sequence alignment showed that the increases in secondary structure, the content of proline and valine, the number of hydrogen bond were the main factors affecting the thermostability of BpNagZ. Mutation analysis also verified that four prolines in the BpNagZ had obvious effects on the thermostability. Combinatory hydrolysis of colloidal chitin with acidic mammalian exochitinase (AMcase) and BpNagZ showed the maximum combinatory efficiency of GlcNAc can reach 87% during 2.25 h. These biochemical characteristics indicated that BpNagZ was a thermostable enzyme with high activity in industrial application.