School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, Zhejiang, China.
College of Food Science and Technology, Zhejiang University of Technology, Hangzhou 310014, Zhejiang, China.
Food Res Int. 2023 Oct;172:113202. doi: 10.1016/j.foodres.2023.113202. Epub 2023 Jun 28.
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 °C. The highest MCA appeared at a substrate pH of 5.5 with 30 mM CaCl. The Michaelis constant (K) and maximal velocity (V) for casein were 0.31 g/L and 14.16 μmol/min, respectively. LY114 MCE preferred to hydrolyze α-casein (α-CN) rather than β-casein (β-CN) and had unique α-CN, β-CN and κ-casein (κ-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.
凝乳酶（MCE）是奶酪制作中的关键活性物质。由于传统 MCE（例如小牛皱胃酶）的产量有限且奶酪消耗量增加，因此有必要寻找传统 MCE 的替代品。通过对来自中国莱阳的农业土壤中的 45 株菌进行 16S rRNA 测序，分离并鉴定出一株具有良好凝乳活性（MCA）（448 SU/mL）和高 MCA/蛋白水解活性（PA）比值（6.0）的巨大芽孢杆菌 LY114。巨大芽孢杆菌 LY114 MCE 的比活显著（7532 SU/mg），通过硫酸铵分级沉淀和 DEAE-琼脂糖 Fast Flow，其纯化产率达到 4.83 倍，回收率为 34.17%。纯化的 LY114 MCE 是一种金属蛋白酶，分子量为 30 kDa。LY114 MCE 在 pH 5.0-7.0 和温度<40°C 下稳定。在底物 pH 5.5 和 30 mM CaCl 下，MCA 最高。酪蛋白的米氏常数（K）和最大速度（V）分别为 0.31 g/L 和 14.16 μmol/min。LY114 MCE 优先水解α-酪蛋白（α-CN）而不是β-酪蛋白（β-CN），并且具有独特的α-CN、β-CN 和κ-酪蛋白（κ-CN）切割位点。与小牛皱胃酶和真菌 MCE 相比，LY114 MCE 通过 SDS-PAGE 分析水解酪蛋白生成的肽明显不同。化学指数分析和感官评价证实了 LY114 MCE 在奶酪制作中的实用性。LY114 MCE 有可能用于乳制品加工和丰富传统 MCE 的替代品。
