Structural characterization, catalytic, kinetic and thermodynamic properties of Keratinase from Bacillus pumilus FH9

Bacillus pumilus FH9 keratinase was purified to homogeneity with a 59.9% yield through a series of three steps. The purified enzyme was a monomeric protein with a molecular mass around 50kDa and containing 7.3% carbohydrates. The pure B. pumilus FH9 keratinase was optimally active at pH 9.0 and 60°C. The calculated activation energy for keratin hydrolysis was 24.52kJmol^-1 and its temperature quotient (Q₁₀) was 1.19. The calculated values of thermodynamic parameters for keratin hydrolysis were as follows: ΔH*=21.75kJmol^-1, ΔG*=65.86kJmol^-1 ΔS*=-132.46Jmol^-1K^-1, (ΔG*_E-S)=4.74kJmol^-1 and ΔG*_E-T=-11.254kJmol^-1. The pure keratinase exhibited K_m, V_max, k_cat and k_cat/K_m of 5.55mg/ml keratin, 5882Umgprotein^-1 323.54s^-1 and 58.28 (s^-1/mgml^-1). The calculated half-life time at 50, 60, 70 and 80°C was 90.69, 59.1, 16.62 and 9.48min, respectively. Similarly, the thermodynamic parameters for irreversible thermal inactivation at temperature ranging from 50 to 80°C were determined. The pure enzyme was stimulated by Ca²⁺ and Mg²⁺. However, Zn²⁺, EDTA, Co²⁺ and Hg²⁺ significantly inhibited the enzyme activity. The purified enzyme was able to hydrolyze different substrates showing its higher proteolytic activity on casein, bovine serum albumin, and collagen, followed by feather, horn and wool.