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Applied Microbiology and Biotechnology

, Volume 103, Issue 6, pp 2665–2674 | Cite as

Enhanced activity and substrate tolerance of 7α-hydroxysteroid dehydrogenase by directed evolution for 7-ketolithocholic acid production

  • Bin HuangEmail author
  • Qiang Zhao
  • Jing-hui Zhou
  • Gang Xu
Biotechnologically relevant enzymes and proteins
  • 241 Downloads

Abstract

7-Ketolithocholic acid (7-KLCA) is an important intermediate for the synthesis of ursodeoxycholic acid (UDCA). UDCA is the main effective component of bear bile powder that is used in traditional Chinese medicine for the treatment of human cholesterol gallstones. 7α-Hydroxysteroid dehydrogenase (7α-HSDH) is the key enzyme used in the industrial production of 7-KLCA. Unfortunately, the natural 7α-HSDHs reported have difficulty meeting the requirements of industrial application, due to their poor activities and strong substrate inhibition. In this study, a directed evolution strategy combined with high-throughput screening was applied to improve the catalytic efficiency and tolerance of high substrate concentrations of NADP+-dependent 7α-HSDH from Clostridium absonum. Compared with the wild type, the best mutant (7α-3) showed 5.5-fold higher specific activity and exhibited 10-fold higher and 14-fold higher catalytic efficiency toward chenodeoxycholic acid (CDCA) and NADP+, respectively. Moreover, 7α-3 also displayed significantly enhanced tolerance in the presence of high concentrations of substrate compared to the wild type. Owing to its improved catalytic efficiency and enhanced substrate tolerance, 7α-3 could efficiently biosynthesize 7-KLCA with a substrate loading of 100 mM, resulting in 99% yield of 7-KLCA at 2 h, in contrast to only 85% yield of 7-KLCA achieved for the wild type at 16 h.

Keywords

7-Ketolithocholic acid 7α-Hydroxysteroid dehydrogenase Directed evolution Substrate tolerance NADP+ regeneration Biosynthesis 

Notes

Acknowledgements

This work was supported by the National Science and Technology Major Special Independent Project of China (No. 2017ZX07402003). We are grateful to Prof. Bochu Wang from Chongqing University, for providing us with the wild-type CA 7α-HSDH gene.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2019_9668_MOESM1_ESM.pdf (538 kb)
ESM 1 (PDF 537 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Hunan Flag Bio-tech Co., Ltd.ChangshaChina
  2. 2.College of Life Science and TechnologyCentral South University of Forestry and TechnologyChangshaChina

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