Alkaline-adapted beta-mannanase of Bacillus pumilus:gene heterologous expression and enzyme characterization

Abstract:

Abstract:［Objective］We expressed a novel alkaline-adapted beta-mannanase gene and characterized the enzyme for potential industrial applications.［Methods］We obtained a mannanase gene (named manB) from Bacillus pumilus Nsic-2 and expressed the gene manB in Escherichia coli and Bacillus subtilis． Furthermore，we characterized the enzyme.［Results］The gene manB had an open reading frame of 1104 bp that encoded a polypeptide of 367-amino-acid betamannanase (ManB)．The protein sequence showed the highest identity with the beta-mannanase from B．pumilus CCAM080065． We expressed the gene manB in E.coli BL21 (DE3) with the enzyme activity of 11021. 3 U/mL．Compared with other mannanases，ManB showed higher stability under alkaline conditions and was stable at pH6.0－9.0．The specific activity of purified ManB was 4191±107 U/mg．The Km and Vmax values of purified ManBwere 35.7 mg/mL and 14.9 μmol/(mL·min)，respectively．Meanwhile，we achieved recombinant protein secretion expression in B．subtilis WB800N.［Conclusion］ We achieved heterologous expression of the gene manB and characterized its enzyme．The alkaline-adapted ManBshowed potential value in industrial applications due to its pH stability.

Key words:Keywords: Bacillus pumilus，β-mannanase，gene cloning，enzymatic properties，Bacillus subtilis expression