Prospecting, production and thermodynamic profiling of feather degrading keratinolytic protease from Bacillus subtilis GH2

Abstract

Keratinolytic proteases are increasingly gaining attention across various industries due to their environmentally friendly properties. The main objective of this study was to investigate the kinetic and thermodynamic characteristics of a keratinolytic protease produced by Bacillus subtilis GH2. The 16S rRNA gene sequence of the isolate has been deposited in the GenBank database under accession number OR999897. A purification process involving ammonium sulfate precipitation followed by dialysis effectively enhanced the recovery of the keratinolytic protease, achieving yields of 130.41% and 155.48%, respectively. Enzyme kinetics analysis revealed a Michaelis-Menten constant (K m ) of 1.2 mg/mL. The activation energy (E a ) was determined to be 85.12 kJ/mol. The thermodynamic activation parameters included a Gibbs free energy ( ) of 66.44 kJ/mol, an enthalpy ( ) of -82.53 kJ/mol, and an entropy ( ) of -50.57 J/mol·K. The Gibbs free energy changes for substrate binding ( ) and transition state formation ( were determined to be 0.49 kJ/mol and -11.29 kJ/mol, respectively. The thermal denaturation energy of the keratinase was found to be 162.06 kJ/mol. Collectively, the kinetic and thermodynamic studies demonstrate that the Bacillus subtilis GH2 enzyme efficiently forms the enzyme-substrate (ES) complex and spontaneously converts it to product, supporting its potential for practical applications in feather waste valorization and leather dehairing.