Heterologous expression, biochemical characterization, and overproduction of alkaline α-amylase from Bacillus alcalophilus in Bacillus subtilis

Background: Alkaline alpha-amylases have potential applications for hydrolyzing starch under high pH conditions in the starch and textile industries and as ingredients in detergents for automatic dishwashers and laundries. While the alkaline alpha-amylase gains increased industrial interest, the yield of alkaline alpha-amylases from wild-type microbes is low, and the combination of genetic engineering and process optimization is necessary to achieve the overproduction of alkaline alpha-amylase. Results: The alkaline alpha-amylase gene from Bacillus alcalophilus JN21 (CCTCC NO. M 2011229) was cloned and expressed in Bacillus subtilis strain WB600 with vector pMA5. The recombinant alkaline alpha-amylase was stable at pH from 7.0 to 11.0 and temperature below 40 degrees C. The optimum pH and temperature of alkaline alpha-amylase was 9.0 and 50 degrees C, respectively. Using soluble starch as the substrate, the K-m and V-max of alkaline alpha-amylase were 9.64 g/L and 0.80 g/(L.min), respectively. The effects of medium compositions (starch, peptone, and soybean meal) and temperature on the recombinant production of alkaline alpha-amylase in B. subtilis were investigated. Under the optimal conditions (starch concentration 0.6% (w/v), peptone concentration 1.45% (w/v), soybean meal concentration 1.3% (w/v), and temperature 37 degrees C), the highest yield of alkaline alpha-amylase reached 415 U/mL. The yield of alkaline alpha-amylase in a 3-L fermentor reached 441 U/mL, which was 79 times that of native alkaline alpha-amylase from B. alcalophilus JN21. Conclusions: This is the first report concerning the heterologous expression of alkaline alpha-amylase in B. subtilis, and the obtained results make it feasible to achieve the industrial production of alkaline alpha-amylase with the recombinant B. subtilis.