High-Level Expression and Substrate-Binding Region Modification of a Novel BL312 Milk-Clotting Enzyme To Enhance the Ratio of Milk-Clotting Activity to Proteolytic Activity

A novel BL312 milk-clotting enzyme (MCE) exhibited high-level expression and remarkable milk-clotting activity (MCA) (865 +/- 20 SU/mL) that was 3.3-fold higher than the control by optimizing induction conditions in recombinant Escherichia. coli harboring pET24a-proMCE. Through substrate-binding region analyses and modification, MCE-G165A was identified from nine mutants and showed a proteolytic activity of 49.4 +/- 2.4 U/mL and an MCA/PA ratio of 18.2, which were respectively 1.9-fold lower and 2.0-fold higher than those of the control. The purified MCE-G165A (28 kDa) exhibited weak alpha(s)-casein, beta-casein, and strong kappa-casein (kappa-CN) hydrolysis levels as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reversed-phase high-performance liquid chromatography. The milk-clotting mechanism for MCE-G165A was the primary hydrolysis of Met106-Ala107 and Asn123-Thr124 bonds in kappa-CN, as determined by mass spectrometry. MCE-G165A showed different hydrolysis sites in casein, leading to various functional peptides. Feasible methods for obtaining MCEs suitable as calf rennet substitutes are presented.