Inducible heterologous expression and carbon catabolite repression of β-amylase from Bacillus megaterium in Bacillus subtilis

Abstract:

[Background] β-Amylases have been widely used in food and medical fields. Most industrial β-amylases are extracted from plants, hampering the application of β-amylase due to high costs. Microbial production of β-amylase has been reported before but has not been industrialized because of the low yields. [Objective] To achieve an efficient inducible expression of a β-amylase from Bacillus megaterium in Bacillus subtilis, relieve carbon catabolite repression (CCR) exerted on the expression of recombinant β-amylase and characterize the recombinant enzyme. [Methods] A xylose-induced vector was constructed to mediate the expression of the amyM gene from Bacillus megaterium 1514 encoding a β-amylase in Bacillus subtilis. CCR of the recombinant β-amylase was studied by site-directed mutagenesis of the catabolite responsive element (CRE) located within the signal peptide-encoding region of amyM. [Results] The recombinant Bacillus subtilis that inductively expressed the β-amylase was obtained. The yield of the recombinant enzyme was significantly improved by silent mutagenesis of conserved nucleotide within amyM-CRE. The recombinant β-amylase had a molecular size of 57 kD and hydrolyzed soluble starch to yield 72% maltose and a little glucose. The enzyme was optimally active at pH 6.0 and 50 °C. Co2+ and Ca2+ increased the efficiency of enzymatic hydrolysis. [Conclusion] Highly efficient expression of β-amylase was achieved to provide experimental support for the industrial production of β-amylase from fermentation.