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Substrate Specificity and Thermostability of the Dehairing Alkaline Protease from Bacillus pumilus

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Abstract

An alkaline protease (DHAP) from Bacillus pumilus has shown great potential in hide dehairing. To get better insights on its catalytic properties for application, the substrate specificity and thermostability were investigated using five natural proteins and nine synthetic peptides. The results showed that DHAP could hydrolyze five proteins tested here in different specificity. Collagen, a component of animal skin, was more resistant to hydrolysis than casein, fibrin, and gelatin. Among the synthetic peptides, the enzyme showed activity mainly with tetrapeptide substrates with the catalytic efficiency in order of Phe>Leu>Ala at P1 site, although k m value for AAVA-pN is much lower than that for AAPL-pN and AAPF-pN. With tripeptide substrates, smaller side-chain group (Gly) at P1 site was not hydrolyzed by DHAP. The enzyme showed good thermostability below 60 °C, and lost activity so quickly above 70 °C. The thermostability was largely dependent on metal ion, especially Ca2+, although other ions, like Mg2+, Mn2+, and Co2+, could sustain stability at certain extent within limited time. Cu2+, Fe2+, as well as Al3+, did not support the enzyme to retain activity at 60 °C even in 5 min. In addition, the selected metal ions could coordinate calcium in improvement or destruction of thermostability for DHAP.

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Acknowledgment

This work is supported by the “863” High Tech Program (2006AA02Z221) from the Department of Science and Technology of China to H. F.

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  1. The Sichuan Key Laboratory of Molecular Biology and Biotechnology, College of Life Sciences, Sichuan University, 29, Wangjiang Rd., Chengdu, 610064, Sichuan, China

    Min-Yuan Wan, Hai-Yan Wang, Yi-Zheng Zhang & Hong Feng

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  1. Min-Yuan Wan
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  2. Hai-Yan Wang
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  3. Yi-Zheng Zhang
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  4. Hong Feng
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Correspondence to Hong Feng.

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Wan, MY., Wang, HY., Zhang, YZ. et al. Substrate Specificity and Thermostability of the Dehairing Alkaline Protease from Bacillus pumilus . Appl Biochem Biotechnol 159, 394–403 (2009). https://doi.org/10.1007/s12010-008-8497-4

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