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Enhancement of keratinolytic activity of a thermophilic subtilase by improving its autolysis resistance and thermostability under reducing conditions

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Abstract

WF146 protease, a thermophilic subtilase from thermophile Bacillus sp. WF146, suffers excessive autolysis in the presence of reducing agents. In this report, two autolytic sites of WF146 protease were modified by site-directed mutagenesis. The introduction of prolines into the autolytic sites increased the autolysis resistance of the enzyme under reducing conditions. The double mutant N63P/A66P displayed a 2.8-fold longer half-life at 80°C and higher hydrolytic activities than wild-type enzyme toward soluble (casein) and insoluble (keratin azure) substrates at high temperatures. In the presence of reducing agents, N63P/A66P was able to degrade feather at 80°C (∼3 h), with hydrolysis efficiency comparable to that of proteinase K at 50°C (∼24 h). Meanwhile, the mutant N63P/A66P had the ability to hydrolyze PrPSc-like prion protein at high temperatures. In virtue of these properties, N63P/A66P is of great interest to be used in recycling of keratinous wastes, such as feather, and disinfection of medical apparatus. In addition, our study may provide useful information needed to explore keratinolytic potential of thermophilic subtilases, even if they are produced by non-keratinolytic microorganisms.

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Acknowledgments

This work was supported in part by the National Grand Fundamental Research Program (973; 2004CG719606), the National Natural Science Foundation (30370018, 30470019, and 30870052), and the Specialized Research Fund for the Doctoral Program of Higher Education (20070486050) of China.

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  1. State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, People’s Republic of China

    Xiaoliang Liang, Yan Bian, Xiao-Feng Tang, Gengfu Xiao & Bing Tang

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  1. Xiaoliang Liang
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  2. Yan Bian
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  3. Xiao-Feng Tang
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  4. Gengfu Xiao
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  5. Bing Tang
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Correspondence to Bing Tang.

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Liang, X., Bian, Y., Tang, XF. et al. Enhancement of keratinolytic activity of a thermophilic subtilase by improving its autolysis resistance and thermostability under reducing conditions. Appl Microbiol Biotechnol 87, 999–1006 (2010). https://doi.org/10.1007/s00253-010-2534-2

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  • DOI: https://doi.org/10.1007/s00253-010-2534-2

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