Purification and characterization of extreme alkaline, thermostable keratinase, and keratin disulfide reductase produced by Bacillus halodurans PPKS-2.
Two alkaline keratinases-I and II secreted by Bacillus halodurans PPKS-2 were purified and characterized. Both the keratinases were purified using ammonium sulfate, DEAE-Sephadex followed by Sephadex G-200 column chromatography. The purification was 21.5-fold and 11.17% yield for keratinase-I and 23.7-fold with yield 18.46 for keratinase-II and its molecular weights 30 and 66 kDa. Both purified enzymes were relatively stable over a broad pH range 7.0-13.0 and optimally active at pH 11.0 and 60-70 degrees C. Keratinase-II was found to be more stable at 70 degrees C for 3 h and retained 100% of its activity, whereas keratinase-I lost 10% activity. Keratinase-I had high keratin disulfide reductase activity with low keratinase activity whereas keratinase-II had high keratinase activity with low keratin disulfide reductase activity. Keratinase activities of both the enzymes were completely inhibited by PMSF at 1 mM, whereas keratin disulfide reductase activity of keratinase-I was not affected. Enzymes were active and stable in the presence of the surfactants, bleaching agents (20% H(2)O(2)), commercial detergents (1%), and SDS (20%). Both the enzymes were partially sequenced and found that keratinase-I and II had a homology with disulfide reductases and serine type of proteases, respectively.
Prakash
Pathange
P
Department of Biochemistry, Gulbarga University, Gulbarga, 585106, Karnataka, India.
Jayalakshmi
Senigala K
SK
Sreeramulu
Kuruba
K
D000595:Amino Acid Sequence; D001407:Bacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D004220:Disulfides; D009247:NADH, NADPH Oxidoreductases; C014703:disulfide reductase (NADH); D010447:Peptide Hydrolases; C035855:keratinase