Milk-Clotting Enzyme from Bacillus velezensis DS-1 (Longnan Douchi): Purification, Structure Prediction, and Cheese Making

Abstract This study isolated a high milk-clotting enzyme-producing strain DS-1 from Longnan Douchi, identified as Bacillus velezensis based on 16S rDNA. The enzyme was purified by ammonium sulfate precipitation and DEAE-Sephadex A-25 chromatography, yielding 56.1 kDa, 6.57-fold purification, and 68.57% recovery. It showed optimal activity at pH 5.5 and 60 °C, stability at pH 5.5–9.5 and 35–45 °C, and belonged to the aspartic protease family. Structural prediction revealed a 422-amino-acid enzyme with more α-helices than β-sheets and a pocket-shaped active site. When applied to Cheddar cheese, DS-1 cheese showed higher moisture content, enhanced proteolysis indicators (WSN, TCA-N, PTA-N), and softer texture than the control. After three months, 58 volatile flavor compounds (vs. 49 in control) were detected, with total relative content of 97.68% (vs. 70.84% in control), and superior sensory scores. Thus, B. velezensis DS-1 rennet exhibits favorable properties for producing fast-ripening, flavor-rich Cheddar cheese, representing a promising microbial rennet.