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Purpose: This study was conducted to find a microbial milk-clotting enzyme (MCE) with a high and stable milk-clotting activity (MCA) to proteolytic activity (PA) ratio suitable for the cheese industry. Methods: Microbial strains were isolated from soil suspensions cultured in solid casein medium. 16S rDNA of representative isolates were sequenced to identify the microbial species. Nutrition and fermentation conditions were systematically examined to optimize MCA of the selected MCE. Protease inhibitors were used to identify the type of MCE. The casein hydrolysis was analyzed through reversed-phase HPLC (RP-HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results: The Bacillus licheniformis BL312 was identified from 50 bacterial strains. BL312 MCE achieved a maximal MCA (460 ± 15 SU/mL) at 48 h that was 2.7-fold higher than the control, and the MCA/PA ratio (9.0) and pH (6.6) remained stable throughout the fermentation process. Medium containing 30 g/L wheat bran shorts, 5 g/L glucose, and 3 g/L corn steep liquor was sufficient for optimal BL312 MCE production. Fermentation conditions of an inoculum size of 7.0% (v/v), fermentation temperature of 37 °C, agitation speed of 210 rpm, and initial pH 6.6 were required to achieve maximal MCA. BL312 MCE was inhibited by phenylmethanesulfonyl fluoride (PMSF) and high concentrations of ethylenediaminetetraacetic acid (EDTA) (5–25 mM). The αs-casein (αs-CN) and β-casein (β-CN) hydrolysates generated by BL312 MCE and calf rennet were different. Conclusions: BL312 MCE is a serine/alkaline protease that exhibits high MCA and various hydrolysis for caseins in comparison with calf rennet. [ABSTRACT FROM AUTHOR]

A bacterium producing an extracellular milk-clotting enzyme was isolated from yak grazing soil in the Tianzhu Tibetan autonomous county on the Tibetan Plateau and identified as Bacillus amyloliquefaciens. We used single-factor testing to study the optimum physical conditions and nutritional parameters for production of the milk-clotting enzyme, while the temperature and pH stability of the enzyme were also studied. The optimum conditions for production of the milk-clotting enzyme were: temperature, 37°C; inoculum size, 4% (1.8 × 10 CFU/mL); agitation speed, 170 rpm; and initial pH of the medium, 6.2. The maximum milk-clotting activity and milk-clotting activity per proteolysis activity were found using wheat bran juice at a concentration 18 g/100 mL, 4% (w/v) sucrose and 2% (w/v) skim milk powder. These optimized conditions resulted in a 2.68-fold increase in production of the milk-clotting enzyme. The crude enzyme exhibited good temperature and pH stability; 80% of the milk-clotting activity was retained after incubation at 40°C for 30 min and more than 70% of the activity was retained between pH 5.5 and 7.5. This B. amyloliquefaciens milk-clotting enzyme has potential as a calf rennet substitute. [ABSTRACT FROM AUTHOR]