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Directed Evolution and Mutant Characterization of Nitrilase from Rhodococcus rhodochrous tg1-A6

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Abstract

In this paper, a molecularly directed evolution-based approach was applied to modify the nitrilase from Rhodococcus rhodochrous tg1-A6 for improving properties in catalyzing nitriles. In the process of error-prone polymerase chain reaction (PCR) with the wild-type nitrilase gene acting as the template, a library of the randomly mutated nitrilase gene was constructed. Since the pH value of catalyzing solution decreased when glycolonitrile was used as the substrate of nitrilase, a high-throughput strategy based on the color change of a pH-sensitive indicator was established for rapid screening of the mutated nitrilase. After three rounds of random mutation and screening about 5000 clones, a variant (Mut3) with 5.3-fold activity of the wild-type counterpart was obtained. Five amino acid substitutions (D27E, N97K, L246F, D108E, and S111R) were found in the variant Mut3. The properties of three mutated enzymes obtained in the three-round mutation were investigated. In the conversion of glycolonitrile, the variant (Mut2) accumulated the highest concentration of glycolic acid at 10.6 g l−1, a much higher value than the wild type (3.2 g l−1).

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Acknowledgments

This work was supported by the National Science Foundation of China (21276023; 21476025).

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Authors and Affiliations

  1. Department of Biological Science and Engineering, University of Science and Technology Beijing, Beijing, 100083, People’s Republic of China

    Hui Luo & Jinwei Ma

  2. Department of Environmental Engineering, University of Science and Technology Beijing, Beijing, 100083, People’s Republic of China

    Jinwei Ma & Yanhong Chang

  3. Department of Chemical Engineering, Tsinghua University, Beijing, 100084, People’s Republic of China

    Huimin Yu & Zhongyao Shen

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  1. Hui Luo
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  2. Jinwei Ma
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  3. Yanhong Chang
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  4. Huimin Yu
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  5. Zhongyao Shen
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Correspondence to Hui Luo.

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Luo, H., Ma, J., Chang, Y. et al. Directed Evolution and Mutant Characterization of Nitrilase from Rhodococcus rhodochrous tg1-A6. Appl Biochem Biotechnol 178, 1510–1521 (2016). https://doi.org/10.1007/s12010-015-1964-9

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  • DOI: https://doi.org/10.1007/s12010-015-1964-9

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