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 \(\upmu\) 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 \(\cdot\) mol \(^{-1}\) . Gibbs free energy ( \(\Delta G^*\) ), entropy ( \(\Delta S^*\) ), and enthalpy ( \(\Delta H^*\) ) values ranged from 101.59 to 108.57 kJ \(\cdot\) mol \(^{-1}\) , 436.02 to 443.65 J \(\cdot\) mol \(^{-1}\) \(\cdot\) K \(^{-1}\) , and 249.32 to 249.41 kJ \(\cdot\) mol \(^{-1}\) , respectively. These findings highlight the enzyme’s stability and efficiency, positioning it as a strong candidate for diverse biotechnological applications.
