Bacillus methanolicus LB-1 isolated from traditionalrice wine was found to produce a milk clottingenzyme (MCE), and its fermentation conditions wereoptimized using response surface methodology. Then theMCE was produced by ethanol precipitation, and furtherchromatography separation resulted in a 10.46-fold purificationwith a 59.28% recovery. The MCA (milk clottingactivity) of the purified MCE reached 597,310 ± 0.13 SU/g. The optimal temperature of the MCE was determined tobe 50 C and it was stable in the low temperature range of40–45 C. The MCE had an optimum pH of 6.5, and it wasstable under neutral conditions. Calcium chloride at theconcentration of 25 mM was found to be the most effectivestimulus. The MCE was identified by LC–MS to be aputative protein (ID I3EB99) containing 759 amino acidswith a molecular weight of 80.37 kDa and a pI of 9.23.