Recombinant Expression and Fermentation Optimization of B.stearothermophilu Maltogenic Amylases in Bacillus subtilis

Abstract:

To obtain the recombinant expression of gene amyM for maltogenic amylases from Bacillus stearothermophilus in Bacillus subtilis，the target gene was PCR-amplified using plasmid opt-amyM/T as template，and ligated with the expression vector pHY300PLK，then transformed into the expression host Bacillus subtilis CCTCC M 2016536. The activity of maltogenic amylases reached 250.7 U/mL after cultivated in TB culture for 48 h. By optimizing the types of nitrogen source and the complex of it，the concentration of nitrogen source and carbon source etc. The optimal fermentation condition was determined as：25.0 g/L yeast extract and 5.0 g/L soy peptone of complex nitrogen source，5.0 g/L of glycerin，initial pH of medium 6.5 and the optimal temperature was 41℃. Under optimal conditions，the production of maltogenic amylases reached 396.7 U/mL，which was 1.6 times of that before optimization. Further experiments were determined the enzymatic properties of maltogenic amylase: the optimum temperature of maltogenic amylase was 60 ℃，the optimum pH was 5.5，the half-life was 325 h，respectively. The K_m was 0.95 g/L and the specific activity is 2 646 U/mg.