Purification And Biochemical Characterization Of Two Novel Extracellular Keratinases With Feather-Degradation And Hide-Dehairing Potential

Two novel extracellular keratinases were produced by Actinomadura keratinilytica strain Cpt20. Both enzymes were purified to homogeneity using heat-treatment (60 degrees C for 30 min) and ammonium sulfate salt fractionation (40 %-70 %), followed by anion-exchange chromatography with fast protein liquid chromatography (FPLC) system. The purified keratinases, designated as KERA-71 and KERB-19, are monomeric and named according to their molecular masses of 71 kDa and 19 kDa, respectively, as estimated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), zymography, and high-performance liquid chromatography (HPLC). N-terminal residues of both enzymes exhibited high identity with other Actinomadura keratinases. Their hydrolytic activities were significantly inhibited by phenylmethylsulfonyl fluoride (PMSF) and di-iodopropyl fluorophosphates (DFP), classifying them in the serine proteases family. While KERA-71 was ideally active at 50 degrees C and pH 8, KERB-19 illustrated optimum activity at 40 degrees C and pH 7. The thermo-activity and thermo-stability of both enzymes were improved with 10 mM Ca2+. Interestingly, the KERA-71 displayed broader substrate specificity, higher catalytic efficiency (kcat/Km), and a high degree of hydrolysis (DH) than actinobacterial keratinases, including KERB-19, KERDZ, KERAK-29, Actinase E, and KERAB. Interestingly, both enzymes exhibited effective keratinase activities with high potential, illustrating their possibility to be used in the process of keratincontaining wastes valorization and leather industry.