Production, biochemical characterization, and kinetic/thermodynamic analysis of a novel keratinase from Bacillus sp. MSGU2024.

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 ∘ 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 max values, 0.102 mM and 0.09 μ 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 ∘ C rise in temperature. The thermodynamic analysis provided key insights, with the activation energy for denaturation ( E d ) measured at 254.6 kJ · mol - 1 . Gibbs free energy ( Δ G ∗ ), entropy ( Δ S ∗ ), and enthalpy ( Δ H ∗ ) 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.