Production, biochemical characterization, and kinetic/thermodynamic analysis of a novel keratinase from Bacillus sp....

Due to their distinct characteristics compared to other proteases, Keratinases present a promising alternative for various industrial applications. This study focused on isolating and screening keratinolytic bacteria from poultry waste-contaminated soil, optimizing keratinase production and thoroughly analyzing the biochemical, physicochemical, and thermodynamic properties of the keratinase produced by the isolated strain, Bacillus sp. MSGU2024. Optimized culture conditions resulted in a 2.9-fold increase in keratinase production compared to the initial unoptimized basal medium. The enzyme exhibited optimal activity at 55 $_{}^{\circ }$ C and pH 8. The keratinase displayed stability in the presence of reducing agents, surfactants, and organic solvents, with stability enhanced by 1.5-2.5 times in the presence of non-ionic detergents such as Tween 20 and Tween 80. Its phenylmethylsulfonyl fluoride (PMSF) inhibition confirmed its classification as a serine protease. The enzyme's $K_m$ and $V_{\text{max}}$ values, 0.102 mM and 0.09 $\mu$ M·min^-1 respectively, indicated high substrate affinity and catalytic efficiency. Furthermore, the enzyme's half-life under varying temperature and pH conditions underscored its robustness. The calculated Z-value revealed that the D-value decreased tenfold with a 5.62 $_{}^{\circ }$ C rise in temperature. The thermodynamic analysis provided key insights, with the activation energy for denaturation ( $E_{\text{d}}$ ) measured at 254.6 kJ $·$ mol $_{}^{-1}$ . Gibbs free energy ( $\Delta G^{\ast }$ ), entropy ( $\Delta S^{\ast }$ ), and enthalpy ( $\Delta H^{\ast }$ ) values ranged from 101.59 to 108.57 kJ $·$ mol $_{}^{-1}$ , 436.02 to 443.65 J $·$ mol $_{}^{-1}$ $·$ K $_{}^{-1}$ , and 249.32 to 249.41 kJ $·$ mol $_{}^{-1}$ , respectively. These findings highlight the enzyme's stability and efficiency, positioning it as a strong candidate for diverse biotechnological applications.