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

  • Liu Li
  • Zhe Zheng
  • Xiao Zhao
  • Fengyu Wu
  • Jian Zhang
  • Zhennai YangEmail author


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 °C and it was stable in the low temperature range of 40–45 °C. The MCE had an optimum pH of 6.5, and it was stable under neutral conditions. Calcium chloride at the concentration of 25 mM 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 80.37 kDa and a pI of 9.23.


Bacillus methanolicus Milk clotting enzyme Production characterization identification 



This work is supported by National Natural Science Foundation of China (Grant No. 31601488, No. 31571857).


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Copyright information

© The Korean Society of Food Science and Technology 2019

Authors and Affiliations

  • Liu Li
    • 1
  • Zhe Zheng
    • 1
  • Xiao Zhao
    • 1
  • Fengyu Wu
    • 1
  • Jian Zhang
    • 1
  • Zhennai Yang
    • 1
    • 2
    Email author
  1. 1.Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Laboratory of Food Quality and SafetyBeijing Technology and Business UniversityBeijingChina
  2. 2.School of Food and Chemical EngineeringBeijing Technology and Business UniversityBeijingChina

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