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Enhancing the Thermostability of a Novel β-agarase AgaB through Directed Evolution

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Abstract

To increase the thermostability of β-agarase AgaB by directed evolution, the mutant gene libraries were generated by error-prone polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) shuffling. Mutants with high thermostability were screened by a simple method based on agarase-degrading agar to generate a clear zone on the agar plate. A mutant S2 was obtained through two rounds of error-prone PCR and a single round of DNA shuffling and selection. It has higher thermostability and slightly increased catalytic activity than wild-type AgaB. Melting temperature (T m) of S2, as determined by circular dichroism, is 4.6 °C higher than that of wild-type AgaB, and the half-life of S2 is 350 min at 40 °C, which is 18.4-fold longer than that of the wild-type enzyme. Saturation mutagenesis and hydrophobic cluster analysis indicated that hydrophobic interaction might be the key factor that enhances the enzyme stability.

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Acknowledgments

This work was supported by grants from the National Basic Research Program of China (973 Program; 2003CB716400) and the National High Technology Research and Development Program of China (863 Program; 2004AA625020). We thank Dr. Brian A. Steer (Diversa) for helpful discussion.

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Authors and Affiliations

  1. The Department of Molecular Biology, School of Medicine and Pharmacy, Ocean University of China, Qingdao, 266003, China

    Chao Shi, Xinzhi Lu, Cuiping Ma, Yiming Ma, Xiaoyan Fu & Wengong Yu

  2. College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao, 266042, China

    Chao Shi & Cuiping Ma

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  1. Chao Shi
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  2. Xinzhi Lu
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  3. Cuiping Ma
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  4. Yiming Ma
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  5. Xiaoyan Fu
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  6. Wengong Yu
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Correspondence to Wengong Yu.

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Shi, C., Lu, X., Ma, C. et al. Enhancing the Thermostability of a Novel β-agarase AgaB through Directed Evolution. Appl Biochem Biotechnol 151, 51–59 (2008). https://doi.org/10.1007/s12010-008-8169-4

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  • DOI: https://doi.org/10.1007/s12010-008-8169-4

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