Harnessing Bacillus velezensis for enhanced protease biosynthesis: optimization, purification, and kinetic profiling.

Microbial proteases represent critical biocatalysts for industrial applications, yet optimizing their production remains challenging. This study isolated and characterized a high-yield protease-producing bacterium from environmental sources and systematically optimized production parameters. Among 124 bacterial isolates screened from six environmental sources, poultry waste exhibited the highest microbial diversity, yielding 63 morphologically diverse isolates. The most potent proteolytic isolate, P15, was identified through 16 S rRNA gene sequencing as Bacillus velezensis AZH3S (GenBank accession PX417380), sharing 99% sequence similarity with B. amyloliquefaciens. Initial screening of eight culture media identified glucose-casein-yeast extract medium as optimal, producing 442 U/mL protease activity. Time-course analysis revealed maximum enzyme production at 24 h (420 U/mL), with yield coefficients increasing to 480 U/g biomass in late stationary phase. Carbon and nitrogen source optimization demonstrated that starch (470 U/mL) and peptone (480 U/mL) were superior substrates. Response surface methodology employing Box-Behnken design across five variables (starch, peptone, pH, temperature, agitation speed) generated a highly predictive model (R² = 0.99) identifying agitation speed as the most influential parameter. Optimized conditions (9.84 g/L starch, 5.46 g/L peptone, pH 7.5, 33.1 °C, 244 rpm) achieved 1160.3 ± 9.20U/mL protease activity, representing a 2.80-fold improvement over initial production. Three-step purification achieved 5.82-fold enrichment with 32.0% yield. The purified enzyme exhibited a molecular mass of approximately 20 kDa with exceptional substrate affinity (Km = 0.207 mM, Vmax = 39.8 µmol/min), following classical Michaelis-Menten kinetics.These findings establish a robust bioprocess for industrial-scale protease production with significant biotechnological potential.