Cloning and characterization of a thermostable detergent‐compatible recombinant keratinase from Bacillus pumilus KS12

Functional expression of a keratinase from a potential feather‐degrading bacterium, Bacillus pumilus KS12, was achieved in Escherichia coli using pEZZ18 vector. The enzyme was constitutively secreted at 37°C and 300 rpm after 18 H of incubation and was purified to homogeneity using diethylaminoethyl‐Sepharose column. It was completely stable within the pH range of 7.0–10.0 with optima at pH 9.0, and temperature 30°C–90°C with optima at 70°C. It had high thermostability with a t 1/2 of more than 4 H at 70°C, more than 2 H at 80°C, and 30 Min at 90°C. The enzyme was identified as a serine hydrolase as it was completely inhibited by 10 mM phenylmethylsulfonyl fluoride. It retained more than 90% relative activity in the presence of chelating agents such as ethylenediaminetetraacetic acid and 1,10‐o‐phenanthroline. It was also a thiol‐activated enzyme with 3.32‐ and fourfold activation in the presence of 10 mM dithiothreitol and β ‐ mercaptoethanol. The keratinase exhibited high detergent compatibility and oxidation stability with an eight‐ and fivefold enhancement in the presence of triton X‐100 and saponin, respectively. It hydrolyzed a large array of protein substrates with a keratinolytic–caseinolytic ratio of more than 0.5. Amidolytic activity revealed its cleavage on phenylalanine → leucine → alanine– p ‐nitroanilides. In addition, electrospray ionization–mass spectrometry analysis of hydrolyzed insulin B‐chain revealed cleavage between Leu 6 –Cys 7 , Cys 7 –Gly 8 , Tyr 16 –Leu 17 , Cys 19 –Gly 20 , Gly 23 –Phe 24 , Phe 24 –Phe 25 , and Phe 25 –Tyr 26 residues.