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Purification and characterization of keratinase from a new Bacillus subtilis strain

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Abstract

The aim of this study was to purify and characterize a keratinase produced by a new isolated Bacillus subtilis KD-N2 strain. The keratinase produced by the isolate was purified using ammonium sulphate precipitation, Sephadex G-75 and DEAE (diethylaminoethyl)-Sepharose chromatographic techniques. The purified enzyme was shown to have a molecular mass of 30.5 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The optimum pH at 50 °C was 8.5 and the optimum temperature at pH 8.5 was 55 °C. The keratinase was partially inactivated by some metal ions, organic solvents and serine protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Sodium dodecyl sulfate (SDS) and ethylene diamine tetraacetic acid (EDTA) had positive effect on the keratinase activity. Reducing agents including dithiothreitol (DTT), mercaptoethanol, L-cysteine, sodium sulphite, as well as chemicals of SDS, ammonium sulfamate and dimethylsulfoxide (DMSO) stimulated the enzyme activity upon a feather meal substrate. Besides feather keratin, the enzyme is active upon the soluble proteins ovalbumin, bovine serum albumin (BSA), casein and insoluble ones as sheep wool and human hair. Calf hair, silk and collagen could not be hydrolyzed by the keratinase.

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Authors and Affiliations

  1. College of Biology and Environmental Engineering, Zhejiang Shuren University, Hangzhou, 310015, China

    Cheng-gang Cai

  2. College of Food Science and Biosystem Engineering, Zhejiang University, Hangzhou, 310029, China

    Cheng-gang Cai, Jiong-jiong Qi, Yun Yin & Xiao-dong Zheng

  3. Institute of Bioengineering, Zhejiang Sci-Tech University, Hongzhou, 310018, China

    Ji-shuang Chen

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  1. Cheng-gang Cai
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  2. Ji-shuang Chen
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  4. Yun Yin
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  5. Xiao-dong Zheng
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Correspondence to Xiao-dong Zheng.

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Cai, Cg., Chen, Js., Qi, Jj. et al. Purification and characterization of keratinase from a new Bacillus subtilis strain. J. Zhejiang Univ. Sci. B 9, 713–720 (2008). https://doi.org/10.1631/jzus.B0820128

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  • DOI: https://doi.org/10.1631/jzus.B0820128

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