91桃色

Abstract

A feather degrading strain of Bacillus pumilus KS12 elaborated a thermostable 27 kDa keratinase in presence of chicken feather. The broth was able to degrade Sup35NM, a yeast prion-like protein from Saccharomyces cerevisiae. Peptide fingerprinting and N-terminal analysis of keratinase led to identification of its ORF from genomic database of B. pumilus SAFR032. Sequence analysis indicated that the mature protein showed 76 % identity to that of natto-kinase of Bacillus subtilis and 75% identity to that of subtilisin Carlsberg from Bacillus licheniformis. The keratinase gene rK27 was cloned in E. coli DH5α-pSTREPHIS1525 (shuttle vecor) and was expressed and secreted into Bacillus subtilis WB980-pSTREPHIS1525 under the control of xylose promoter (PxylA). Keratinase was produced by B. subtilis of 3,821 U/mg in the extracellular broth after 24 h of fermentation at 37癈, 200 rpm under 0.5 % xylose induction at 0.3 OD600nm. The enzyme was concentrated by ultrafiltration and purified through affinity chromatography using nickel (Ni2+-NTA) resin with 3.63 purity fold and 80 % recovery. The biochemical studies revealed maximum activity at pH 9 and 70 癈 and thermostability of >4 h at 70癈. The enzyme hydrolyzed a large array of protein substrates both soluble and insoluble substrates including keratin with K:C ratio >0.5. Specificity of rK27 for amidolytic substrates revealed the cleavage of phenylalanine- >leucine- >alanine- residues. ESI-MS analysis of insulin B-chain hydrolysis revealed seven cleavage sites after cysteine and hydrophobic amino acid residues. Keratinase was immobilized by covalent crosslinking onto glutaraldehyde (2.5% v/v) activated chitin beads. Immobilized enzyme exhibited better pH and temperature stability. Comparative degradative potential for Sup35NM was checked by Western blotting using antibody against N-domain (infective domain). It showed that only 100 μg of immobilized keratinase was required to degrade 50 μg Sup35NM protein in contrast to 890 μg of free keratinase after 60 min at pH 7 and 37癈

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