Cloning and efficient expression of a thermostable and weak acidic β-mannanase gene

Abstract:

[Objective] The β-mannanase gene (man) from Bacillus subtilis BE-91 which is an efficient strain for hemi-cellulose degradation was cloned and expressed in Escherichia coli BL21(DE3) , then the enzymatic properties of the β-mannanase (Man) were studied. [Methods] The man was amplified from B. subtilis BE-91 by PCR, respectively linked to pEASY-E1 and pET28a, and finally expressed in E. coli BL21(DE3). After comparing the extracellular and intracellular β-mannanase activities from the gene engineering strains, the β-mannanase with highest activity was selected to study the enzymatic properties fully. [Results] The manA (GenBank: KP277209) was 960 bp in length, including a termination codon and encoded 319 amino acids. The highest activity of the extracellular β-mannanase from pEASY-man/BL strain was 229.1 IU/mL. The optimal temperature and pH of the β-mannanase were 65 °C and 6.0. The catalytic activity of the enzyme was stable at no more than 65 °C and pH 4.5?7.0 after being incubated for 30 min. It was promoted by Cu2+, Mn2+, Zn2+and Ca2+, while inhibited seriously by Ba2+ and Pb2+ at concentration of 1 mmol/L. [Conclusion] A precious β-mannanase gene has been excavated from B. subtilis BE-91, and its expression product with properties of thermostability and weak acidity may be available for feed additive.

Key words:Bacillus subtilis，β-Mannanase，Gene cloning，Prokaryotic expression，Enzymatic property