This study focuses on identifying a potent microbe for efficient alkaline protease production, characterizing its properties, and investigating protease gene expression across diverse culture conditions. Stenotrophomonas maltophilia strain GEBN5 is methodically chosen and refined for peak alkaline protease production at 36 h (75.37 U/mL) in basal media employing precise adjustments in temperature, pH, incubation time, and carbon/nitrogen sources, achieving a noteworthy maximum protease activity of 97.03 U/mL in shake flask culture. Alkaline protease gene (StmPr1) up-regulation was explored via Real-time PCR under varied culture conditions (pH, temperature, carbon, and nitrogen sources) in a 2L fermenter at 150 rpm, resulting in peak protease production of 113 ± 0.53 U/mL in batch culture at pH 9.0, 35 °C, with sucrose and casein, respectively. The characterization data demonstrates exceptional stability of the current protease across a broad pH (9.0–11.0) and optimum temperature of 35 °C, highlighting its promising applications in diverse industrial and biotechnological settings. A higher instability complex of protein 28.59 was revealed by in silico analysis, indicating prolonged stability during hydrolysis and a wide range of potential uses. The purified protease demonstrates superior compatibility (45%–88% residual activity) with diverse detergent brands treated for 45 min at 40 °C, indicating its potential as a valuable additive for enhanced washing efficacy. Current research offers a novel strategy to boost protease production, presenting a promising alternative to chemical dehairing in leather industries and solid organic waste treatment. This approach holds the potential for mitigating environmental pollution through bioremediation.
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