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Directed evolution and site-specific mutagenesis of l-isoleucine dioxygenase derived from Bacillus weihenstephanensis

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Abstract

Objectives

l-isoleucine dioxygenase (IDO) specifically transforms l-isoleucine (Ile) to 4-hydroxyisoleucine (4-HIL), and 4-HIL is a promising drug for diabetes. To enhance the activity and catalytic efficiency of IDO, we used directed evolution and site-specific mutagenesis.

Results

The IDO gene (ido) derived from Bacillus weihenstephanensis was cloned and expressed in Escherichia coli. Directed evolution using error prone (EP)-PCR and site-specific mutagenesis were conducted. Two improved mutants were obtained after one round of EP-PCR, with IdoN126H exhibiting a 2.8-fold increase in activity. Two improved mutants were obtained through site-specific mutagenesis, with IdoT130K showing a 170% increase in activity. Although the activity of the combined mutant IdoN126H/T130K (0.95 ± 0.08 U/mg) was slightly higher than that of the wild-type Ido, its catalytic efficiency was 2.4-fold and 3.0-fold higher than Ido with Ile and α-ketoglutaric acid as substrates. After biotransformation of Ile by E. coli BL21(DE3) expressing IdoN126H/T130K and Ido, 66.50 ± 0.99 mM and 26.09 ± 1.85 mM 4-HIL was synthesized, respectively, in 24 h.

Conclusion

IdoN126H/T130K had a higher enzyme activity and catalytic efficiency and can therefore be used as a more suitable candidate for 4-HIL production.

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Acknowledgements

The authors thank the “national first-class discipline program of Light Industry Technology and Engineering” (contract no. LITE2018-10) for financial support.

Supporting information

Supplementary Table 1–Primers for amplifying the ido gene and the mutant ido genes.

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Authors and Affiliations

  1. State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China

    Sen Huang & Feng Shi

  2. Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China

    Sen Huang & Feng Shi

  3. International Joint Laboratory on Food Safety, Jiangnan University, Wuxi, 214122, China

    Feng Shi

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  1. Sen Huang
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  2. Feng Shi
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Correspondence to Feng Shi.

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Huang, S., Shi, F. Directed evolution and site-specific mutagenesis of l-isoleucine dioxygenase derived from Bacillus weihenstephanensis. Biotechnol Lett 40, 1227–1235 (2018). https://doi.org/10.1007/s10529-018-2566-8

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  • DOI: https://doi.org/10.1007/s10529-018-2566-8

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