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 degrees C-90 degrees C with optima at 70 degrees C. It had high thermostability with a t(1/2) of more than 4 H at 70 C, more than 2 H at 80 degrees C, and 30 Min at 90 degrees 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 p-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 -> atanine-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. (C) 2011 International Union of Biochemistry and Molecular Biology, Inc. Volume 58, Number 2, March/April 2011, Pages 109-118. E-mail: ranigupta15@yahoo.com