Molecular Characterization Of A Thermostable Sup35NM Prion Protein Degrading Keratinase From Bacillus Pumilus KS12 | 12199
Journal of Biotechnology & Biomaterials
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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?C,
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 ?C and thermostability of >4 h at 70?C. 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?C
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