Improvement of low-temperature caseinolytic activity of a thermophilic subtilase by directed evolution and site-directed mutagenesis

Authors

  • Chuan-Qi Zhong,

    1. State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072; telephone: +86-27-68753583; fax: +86-27-68754833
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  • Shengli Song,

    1. State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072; telephone: +86-27-68753583; fax: +86-27-68754833
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  • Nan Fang,

    1. State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072; telephone: +86-27-68753583; fax: +86-27-68754833
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  • Xiaoliang Liang,

    1. State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072; telephone: +86-27-68753583; fax: +86-27-68754833
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  • Hui Zhu,

    1. State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072; telephone: +86-27-68753583; fax: +86-27-68754833
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  • Xiao-Feng Tang,

    1. State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072; telephone: +86-27-68753583; fax: +86-27-68754833
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  • Bing Tang

    Corresponding author
    1. State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072; telephone: +86-27-68753583; fax: +86-27-68754833
    • State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072; telephone: +86-27-68753583; fax: +86-27-68754833.
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Abstract

By directed evolution and subsequent site-directed mutagenesis, cold-adapted variants of WF146 protease, a thermophilic subtilase, have been successfully engineered. A four-amino acid substitution variant RTN29 displayed a sixfold increase in caseinolytic activity in the temperature range of 15–25°C, a down-shift of optimum temperature by ∼15°C, as well as a decrease in thermostability, indicating it follows the general principle of trade-off between activity and stability. Nevertheless, to some extent RTN29 remained its thermophilic nature, and no loss of activity was observed after heat-treatment at 60°C for 2 h. Notably, RTN29 exhibited a lower hydrolytic activity toward suc-AAPF-pNA, due to an increase in Km and a decrease in kcat, in contrast to other artificially cold-adapted subtilases with increased low-temperature activity toward small synthetic substrates. All mutations (S100P, G108S, D114G, M137T, T153A, and S246N) identified in the cold-adapted variants occurred within or near the substrate-binding region. None of these mutations, however, match the corresponding sites in naturally psychrophilic and other artificially cold-adapted subtilases, implying there are multiple routes to cold adaptation. Homology modeling and structural analysis demonstrated that these mutations led to an increase in mobility of substrate-binding region and a modulation of substrate specificity, which seemed to account for the improvement of the enzyme's catalytic activity toward macromolecular substrates at lower temperatures. Our study may provide valuable information needed to develop enzymes coupling high stability and high low-temperature activity, which are highly desired for industrial use. Biotechnol. Bioeng. 2009; 104: 862–870. © 2009 Wiley Periodicals, Inc.

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