Expression, purification, and functional analysis of a recombinant Bacillus tequilensis strain YIV keratinase for green bioprocessing of keratin biomass.

Abstract

Keratinases are specialized proteases capable of degrading recalcitrant keratin and have attracted considerable interest for environmentally sustainable bioprocessing. In this study, a keratinase gene from Bacillus tequilensis strain YIV, isolated from tannery waste in Kasur, Pakistan, was cloned, expressed, and characterized. The 1064 bp gene was inserted into pET-28a(+) and expressed in Escherichia coli BL21-CodonPlus (DE3). Recombinant enzyme production was improved by optimizing IPTG concentration, cultivation medium, agitation rate, inoculum size, and nutrient sources. The purified 38 kDa keratinase, obtained by Ni-NTA affinity chromatography, exhibited a specific activity of 15.63 U/mg, with optimum activity at pH 8.0 and 40 °C. Apparent kinetic analysis using keratin azure yielded an apparent Kₘ of 5.6 mg/mL, an apparent Vmax of 2.06 U/mL, and an apparent kcat of 21 s-1. Arrhenius analysis indicated an activation energy (Ea) of 15.1 kJ/mol and an apparent activation enthalpy (ΔH‡) of 12.6 kJ/mol at 40 °C. The enzyme retained activity in the presence of EDTA, whereas PMSF caused 94% inhibition, consistent with serine-protease activity. It also exhibited tolerance toward selected metal ions, organic solvents, and moderate salt concentrations. In application studies, the enzyme improved detergent-assisted washing and facilitated dehairing of goat hides under mild conditions, while histological and SEM analyses supported effective surface modification with minimal disruption of dermal architecture. These findings support the potential application of the recombinant keratinase in keratin-waste valorization, detergent formulations, and environmentally sustainable leather processing.