Structural characterization, catalytic, kinetic and thermodynamic properties of Keratinase from Bacillus pumilus FH9
2017
Abdel-Naby, Mohamed A. | El-Refai, Heba A. | Ibrahim, Mohammad H.A.
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 Km, Vmax, kcat and kcat/Km 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.
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