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.
J Dairy Sci. 2023 Oct;106(10):6688-6700. doi: 10.3168/jds.2023-23450. Epub 2023 Aug 7.
Milk-clotting enzyme (MCE) is the essential active agents in dairy processing. The traditional MCE is mainly obtained from animal sources, in which calf rennet is the most widely used in cheese industry. Traditional MCE substitute is becoming necessary due to its limited production and increased cheese consumption. A novel traditional MCE substitute was produced from Bacillus velezensis DB219 in this study. The DB219 MCE exhibited a notable specific activity of 6,110 Soxhlet units/mg and 3.16-fold purification yield with 28.87% recovery through ammonium sulfate fractionation and DEAE-Sepharose Fast Flow. The purified DB219 MCE was a metalloprotease with a molecular weight of 36 kDa. The DB219 MCE was weak acid resistance and stable at pH 6.0 to 10.0 and temperature <45°C. The highest milk-clotting activity was observed in substrate at pH 5.5 added with 20 to 30 mM CaCl. The Michaelis constant and maximal velocity for casein were 0.31 g/L and 14.22 μmol/min. The DB219 MCE preferred to hydrolyze β-casein instead of α-casein. The DB219 MCE hydrolyzed α-casein, β-casein, and κ-casein to generate significantly different peptides in comparison with calf rennet and ES6023 MCE (fungal MCE) through SDS-PAGE and reversed-phase HPLC analysis. The DB219 MCE mainly cleaved Thr124-Ile125 and Ser104-Phe105 bonds in κ-casein and had unique casein cleavage sites and peptide composition through LC-MS/MS analysis. The DB219 MCE was potential to be a new milk coagulant and enriched kinds of traditional MCE substitute.
凝乳酶（MCE）是乳制品加工中的重要活性物质。传统的 MCE 主要来源于动物源，其中小牛皱胃酶在奶酪工业中应用最广泛。由于其产量有限且奶酪消耗量增加，传统 MCE 的替代品变得很有必要。本研究从地衣芽孢杆菌 DB219 中生产出一种新型传统 MCE 替代品。DB219 MCE 的比活为 6110Soxhlet 单位/mg，经硫酸铵分级沉淀和 DEAE-琼脂糖快速流纯化后，纯化收率为 3.16 倍，回收率为 28.87%。纯化的 DB219 MCE 是一种金属蛋白酶，分子量为 36 kDa。DB219 MCE 具有弱酸性抗性，在 pH6.0 至 10.0 和温度<45°C 的条件下稳定。在添加 20 至 30 mM CaCl 的底物中，DB219 MCE 的凝乳活性最高。DB219 MCE 对酪蛋白的米氏常数和最大速度分别为 0.31 g/L 和 14.22 μmol/min。与小牛皱胃酶和 ES6023 MCE（真菌 MCE）相比，DB219 MCE 更喜欢水解β-酪蛋白而不是α-酪蛋白。通过 SDS-PAGE 和反相 HPLC 分析，DB219 MCE 水解α-酪蛋白、β-酪蛋白和κ-酪蛋白生成的肽明显不同。通过 LC-MS/MS 分析，DB219 MCE 主要在κ-酪蛋白中裂解 Thr124-Ile125 和 Ser104-Phe105 键，并具有独特的酪蛋白裂解位点和肽组成。DB219 MCE 具有成为新型牛奶凝固剂和丰富传统 MCE 替代品的潜力。
