Purification and characterization of extreme alkaline, thermostable keratinase, and keratin disulfide reductase produced by Bacillus halodurans PPKS-2 |
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Authors: | Pathange Prakash Senigala K Jayalakshmi Kuruba Sreeramulu |
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Institution: | (1) Department of Biochemistry, Gulbarga University, Gulbarga, 585106, Karnataka, India;(2) Agriculture Research Station, University of Agricultural Sciences, Raichur, Gulbarga, 585103, India; |
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Abstract: | 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 °C. Keratinase-II was found to be more stable at 70 °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% H2O2), 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. |
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