Characteristics and application in cheese making of newly isolated milk-clotting enzyme from Bacillus megaterium LY114

Milk-clotting enzyme (MCE) is a crucial active agent in cheese making. It is necessary to find traditional MCE substitutes due to the limited production of traditional MCE (e.g., calf rennet) and increased cheese consumption. Bacillus megaterium strain LY114 with good milk-clotting activity (MCA) (448 SU/mL) and a high MCA/proteolytic activity (PA) ratio (6.0) was isolated and identified from agricultural soil in Laiyang (Shandong, China) through 16S rRNA sequencing of 45 strains. The Bacillus megaterium LY114 MCE had a remarkable specific activity (7532 SU/mg) and displayed a 4.83-fold purification yield with 34.17% recovery through ammonium sulfate fractionation and DEAE-Sepharose Fast Flow. The purified LY114 MCE was a metalloprotease with a molecular weight of 30 kDa. LY114 MCE was stable at pH 5.0-7.0 and temperature <40 degrees C. The highest MCA appeared at a substrate pH of 5.5 with 30 mM CaCl2. The Michaelis constant (Km) and maximal velocity (Vm) for casein were 0.31 g/L and 14.16 mu mol/min, respectively. LY114 MCE preferred to hydrolyze alpha-casein (alpha-CN) rather than beta-casein (beta-CN) and had unique alpha-CN, beta-CN and kappa-casein (kappa-CN) cleavage sites. LY114 MCE hydrolyzed casein to generate significantly different peptides compared with calf rennet and fungal MCE as determined by SDS-PAGE analysis. Chemical index analysis and sensory evaluation confirmed the usefulness of LY114 MCE in cheese making. LY114 MCE had the potential to be used in dairy processing and enriched traditional MCE substitutes.