Inducible heterogenous expression of bacterial maltogenic amylase in Bacillus subtilis

Abstract:

[Objective] To achieve the efficient heterogenous expression of a bacterial maltogenic amylase in Bacillus subtilis and investigate the characterization of the recombinant enzyme. [Methods] We cloned the genes of xylose isomerase promoter region and its regulatory protein from Bacillus megatherium into an Escherichia coli / Bacillus sp. shuttle expression vector, and constructed an recombinant expression plasmid which harbored an encoding gene of maltogenic amylase from Bacillus licheniformis. Then we transformed the expression plasmid into Bacillus subtilis and optimized the induction conditions of the transformant to increase the production of the recombinant enzyme. [Results] The recombinant Bacillus subtilis which inductively expressed the maltogenic amylase was obtained. The optimal induction condition was as follow, that 1% inducer was added into fermentation medium after 9 h-incubation at 45 °C. The recombinant maltogenic amylase had a molecular size of 67 kD. From the study of the enzymatic properties of the enzyme, it was found that, with soluble starch as substrate, the reaction products were 60.42% maltose and few glucose. Furthermore, the recombinant enzyme showed an optimal activity at 45 °C with pH 6.5. Ca2+、Co2+ and EDTA could improve the efficiency of enzyme reaction. [Conclusion] With induction of xylose, the bacterial maltogenic amylase was efficiently expressed in the recombinant Bacillus subtilis, and the maximal yield reached 296.64 U/mL, which showed good application prospect in industry.

Key words:Bacillus subtilis, Maltogenic amylase, Xylose promoter, Induction conditions, Enzymatic properties