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Biotechnology Letters

, Volume 40, Issue 8, pp 1227–1235 | Cite as

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

  • Sen Huang
  • Feng Shi
Original Research Paper
  • 255 Downloads

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.

Keywords

Biotransformation Catalytic efficiency Error prone-PCR 4-Hydroxyisoleucine l-isoleucine dioxygenase Site-specific mutagenesis 

Notes

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.

Supplementary material

10529_2018_2566_MOESM1_ESM.doc (34 kb)
Supplementary material 1 (DOC 34 kb)

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Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.State Key Laboratory of Food Science and TechnologyJiangnan UniversityWuxiChina
  2. 2.Key Laboratory of Industrial Biotechnology, Ministry of Education, School of BiotechnologyJiangnan UniversityWuxiChina
  3. 3.International Joint Laboratory on Food SafetyJiangnan UniversityWuxiChina

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