Purification and characterization of a novel milk-clotting enzyme produced by Bacillus amyloliquefaciens GSBa-1

A novel milk-clotting enzyme (MCE) produced by Bacillus amyloliquefaciens GSBa-1 was purified and identified to belong to the peptidase M4 family, and the mature peptide with milk-clotting activity (MCA) was a neutral metalloproteinase with molecular mass of about 38 kDa. The optimal pH and temperature were determined to be at 5.5 and 57 degrees C for MCA, and at 7.0 and 57 degrees C for the proteolytic activity, respectively. The MCE exhibited slight autolysis that could be inhibited by Ca2+ and Na+. Hydrolysis of caseins revealed that kappa-casein exhibited higher sensitivity to the MCE action than alpha- and beta-casein. By in-gel tryptic digestion and LC-MS/MS analysis of the major peptide (about 13 kDa) generated from hydrolysis of kappa-casein by the MCE, the cleavage site was identified to be at Lys 111-Lys 112, which was different from those of other MCEs reported earlier. The MCE from B. amyloliquefaciens GSBa-1 could serve as a novel milk coagulant for potential application in making cheese with desired proteolysis.