AprMH1 subtilisin from Bacillus zhangzhouensis MH1: Molecular cloning, characterization, and homology modeling

The growing demand for robust proteases in industrial applications, particularly those based on the widely used subtilisin family, necessitates the development of novel and improved enzymes. This study reports exploration and characterization of a subtilisin, AprMH1, isolated from a recently identified Bacillus zhangzhouensis MH1 (NCBI Acct. No. MZ569437). The isolated aprMH1 gene, consisting of 1146 bp and encoding a 381 amino acid protein, was successfully cloned and overexpressed in Bacillus subtilis DB104. The recombinant AprMH1 protease, belonging to the S8 subtilase family and sharing 99.27 % similarity with keratinase Ker1 (ANQ68333.1) of B. pumilus CCUG 66887, displayed remarkable biochemical properties that offer significant industrial potential. It was produced in a one-liter fermenter, yielding 400,000 U after 48 h, and purified to homogeneity by ion-exchange chromatography with a 3.4-fold purification. AprMH1, a monomer of 27.8 kDa, exhibited optimum activity at pH 10 and 55 °C. It remained stable between pH 7-11 and retained 64 % of activity at 50 °C for 1 h. It also remained stable with non-ionic surfactants and its stability with SDS was enhanced by propylene glycol. Structural insights were gained through 3D modeling and comparative analysis with subtilisin Carlsberg (P00780.2), Apr1 (P07518.1), and Savinase (P29600.1). AprMH1 was found to belong to a distinct alkaline subgroup within the true subtilisin subfamily, exhibiting superior activity and stability at higher temperatures, making it a promising candidate for applications in harsh industrial environments. These novel properties highlight the potential of AprMH1 for future industrial processes, especially in the detergent sector.