Cloning and characterization of a thermostable detergent-compatible recombinant keratinase fromBacillus pumilusKS12

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 1 8 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 900°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 1,10-0-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.