Purification and characteristics of a novel milk-clotting metalloprotease from Bacillus velezensis DB219

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 weight of 36 kDa. The DB219 MCE was weak acid <45 degrees C. The highest milk-clotting activity was observed in substrate at pH 5.5 added with 20 to 30 mM CaCl2. The Michaelis constant and maximal velocity for casein were 0.31 g/L and 14.22 mu mol/min. The DB219 MCE preferred to hydrolyze beta-casein instead of alpha-casein. The DB219 MCE hydrolyzed alpha-casein, beta-casein, and comparison with calf rennet and ES6023 MCE (fungal MCE) through SDS-PAGE and reversed-phase HPLC kinds of traditional MCE substitute.

Automated summary

A novel traditional MCE substitute was produced from Bacillus velezensis DB219 with high activity and purification yield. It showed the highest milk-clotting activity at weak acid condition, particularly at pH 5.5 with the addition of CaCl2. DB219 MCE preferred hydrolyzing β-casein and had weaker hydrolytic activity towards α-casein.