Production, purification and characterization of a milk clotting enzyme from Bacillus methanolicus LB-1

Bacillus methanolicus LB-1 isolated from traditional rice wine was found to produce a milk clotting enzyme (MCE), and its fermentation conditions were optimized using response surface methodology. Then the MCE was produced by ethanol precipitation, and further chromatography separation resulted in a 10.46-fold purification with a 59.28% recovery. The MCA (milk clotting activity) of the purified MCE reached 597,310 +/- 0.13 SU/g. The optimal temperature of the MCE was determined to be 50 degrees C and it was stable in the low temperature range of 40-45 degrees C. The MCE had an optimum pH of 6.5, and it was stable under neutral conditions. Calcium chloride at the concentration of 25mM was found to be the most effective stimulus. The MCE was identified by LC-MS to be a putative protein (ID I3EB99) containing 759 amino acids with a molecular weight of 80.37kDa and a pI of 9.23.