Recombinant serine protease from Bacillus subtilis BGN4: Biochemical and structural characterization with antibiofilm potential

Proteases are crucial biocatalysts used in biotechnology, medicine, and industry. The demand for new proteases with biotechnological applications necessitates their development. This report details the cloning, expression, and characterization of an alkaline serine protease with notable potential for industrial use. An alkaline protease gene from Bacillus subtilis BGN4 (MN093306) was cloned and expressed in Escherichia coli BL21 (DE3), and then the protein was purified by affinity chromatography. The ~48 kDa protease exhibited optimal activity at pH 10 and 50 °C. The purified protease showed a specific activity of 77.41 U/mg, with a K m of 0.0597 μM, a V max of 0.597 U/mL/min, and a k cat of 1.19 min −1 , indicating high substrate affinity and catalytic efficiency. The enzyme was completely inhibited by PMSF, confirming its serine protease nature. Protease activity was enhanced in the presence of SDS and was stimulated by metal ions such as Ca 2+ and Mg 2+ . The protease retained stability in organic solvents such as chloroform and n-hexane. Substrate specificity analysis revealed maximum activity toward azocasein and casein, indicating selective substrate preference. MALDI-TOF confirmed the protease's identity with 54.3% sequence coverage. Circular dichroism showed 42% α-helix, 28% β-sheet, and 30% random coils, while AlphaFold predicted an active-site binding pocket with catalytic residues Ser351, His183, and Asp354. The protease demonstrated vigorous biofilm-disrupting activity against Pseudomonas aeruginosa MTCC 741. These results suggest that this alkaline serine protease is a promising candidate for industrial and therapeutic applications.