Purification, characterization, and application of a high activity 3-ketosteroid-Δ1-dehydrogenase from Mycobacterium neoaurum DSM 1381

  • Ruijie Zhang
  • Xuexia Xu
  • Huijin Cao
  • Chenyang Yuan
  • Yuki Yuminaga
  • Suwen Zhao
  • Jiping ShiEmail author
  • Baoguo ZhangEmail author
Biotechnologically relevant enzymes and proteins


Δ1-Dehydrogenation is one of the most important reactions for steroid drug modification. Numerous 3-ketosteroid-Δ1-dehydrogenases (KstDs) catalyzing this reaction were observed in various organisms. However, only a few have been characterized and used for substrate conversion. In this study, a promising enzyme (KstD2) from Mycobacterium neoaurum DSM 1381 was purified and characterized. Interestingly, KstD2 displayed a high activity on a range of substrates, including 17α-hydroxypregn-4-ene-3,20-dione (17α-OH-P); androsta-4,9(11)-diene-3,17-dione (NSC 44826); and 4-androstene-3,17-dione (AD). These reactions were performed under optimal conditions at 40 °C and pH 8.0. Noteworthy, both the activity and stability of the enzyme were sensitive to various metal ions. After optimizing the expression and biocatalyst conditions, up to 1586 U mg−1 intracellular KstD activity on AD could be produced. Furthermore, the associated conversion rate was 99% with 30 g L−1 AD after 8 h. On the other hand, we obtained 99%, 90%, and over 80% of conversion with 20 g L−1 NSC 44826; 10 g L−1 16,17α-epoxyprogesterone; and 20 g L−1 17α-OH-P or canrenone, respectively, after 24 h. Sequence homology and structural analyses indicated that the residue R178 located in a unique short loop among cluster 2 is crucial for substrate recognition which was confirmed by mutagenesis. In summary, this study reports on the first purification and characterization of a KstD from cluster 2. Its remarkable properties deserve more attention to potentially lead to further industrial applications.


3-Ketosteroid-Δ1-dehydrogenase Mycobacterium neoaurum Biocatalysis Steroids Heterogeneous expression 


Conflict of interest

The authors declare that they have no conflict of interest.

Funding information

This research was funded by the grants from the State Key Project of Research and Development Plan (grant number, 2017YFE0112700).

Compliance with ethical standards

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

Supplementary material

253_2019_9988_MOESM1_ESM.pdf (691 kb)
ESM 1 (PDF 690 kb)


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Copyright information

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

Authors and Affiliations

  1. 1.Laboratory of Biorefinery, Shanghai Advanced Research InstituteChinese Academy of SciencesPudongChina
  2. 2.School of Life Science and TechnologyShanghaiTech UniversityShanghaiChina
  3. 3.University of Chinese Academy of SciencesBeijingChina
  4. 4.iHuman InstituteShanghaiTech UniversityShanghaiChina

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