Using glycerol-inducible expression system to overexpressed maltooligosaccharide-forming α-amylase in Bacillus subtilis

Abstract

In order to meet the desire of maltopentaose (G5) in industrial application, we developed a glycerol-inducible expression system in Bacillus subtilis to overexpress maltooligosaccharide-forming α-amylase from Bacillus cereus ATCC 14579 (BcMFAse). Verifying the glycerol-inducible promoter, optimizing fermentation conditions, comparing homologous promoter and constructing double translation initiation sites were studied. Results shown that the optimal induced time for glycerol-inducible promoter is at 8 h, the optimal induced concentration of glycerol is 1% and the optimized fermentation medium was consisted of 2% tryptone, 0.6% yeast exact, 1% NaCl and 0.6% casein hydrolysate with highest BcMFAse activity (~1549.9 U/mL) promoted by PGlpD in 500 mL triangular flask. Comparing to the homologous promoter, PGlpDL from Bacillus paralicheniformis A4-3 exhibited stronger ability to promoted the expression of BcMFAse and the maximum BcMFAse activity was ~2364.6 U/mL. The BcMFAse activity achieved ~3137.5 U/mL by constructing double translation initiation sites (TISs) at 5´-untranslated region(5´-UTR) of promoter PGlpDL. This study provided a high-efficiency way for overexpressing the BcMFAse in B. subtilis, which would economically producing G5 on industry.