Purification and structural characterization of a novel milk-clotting enzyme from Bacillus velezensis

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. Physicochemical property prediction indicated that the enzyme is an acid protease with an isoelectric point of 4.98 and an instability index of 36.93, suggesting good stability. Secondary structure prediction showed that the α-helix content was 31.75%, which is higher than that of milk-clotting enzymes from Bos taurus, Camel, and other sources. Sequence alignment revealed that the proportions of identical amino acid residues between this enzyme and those from Bos taurus, Camel, Cryphonectria parasitica, Rhizomucor pusillus, and Bacillus subtilis were all below 15%, indicating significant differences. This study provides a theoretical basis for the development and application of novel microbial milk-clotting enzymes.Structural prediction revealed a 422-amino-acid enzyme with more α-helices than β-sheets and a pocket-shaped active site.