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Current Microbiology

, Volume 75, Issue 5, pp 580–587 | Cite as

Development and Optimization of a High-Throughput Screening Assay for Rapid Evaluation of Lipstatin Production by Streptomyces Strains

  • Michal Híreš
  • Nora Rapavá
  • Martin Šimkovič
  • Ľudovít Varečka
  • Dušan Berkeš
  • Svetlana Kryštofová
Article
  • 401 Downloads

Abstract

Pancreatic lipase inhibitors, such as tetrahydrolipstatin (orlistat), are used in anti-obesity treatments. Orlistat is the only anti-obesity drug approved by the European Medicines Agency (EMA). The drug is synthesized by saturation of lipstatin, a β-lactone compound, isolated from Streptomyces toxytricini and S. virginiae. To identify producers of novel pancreatic lipase inhibitors or microbial strains with improved lipstatin production and higher chemical purity remains still a priority. In this study, a high-throughput screening method to identify Streptomyces strains producing potent pancreatic lipase inhibitors was established. The assay was optimized and validated using S. toxytricini NRRL 15443 and its mutants. Strains grew in 24-well titer plates. Lipstatin levels were assessed directly in culture medium at the end of cultivation by monitoring lipolytic activity in the presence of a chromogenic substrate, 1,2-Di-O-lauryl-rac-glycero-3-glutaric acid 6-methylresorufin ester (DGGR). The lipase activity decreased in response to lipstatin production, and this was demonstrated by accumulation of red-purple methylresorufin, a product of DGGR digestion. The sensitivity of the assay was achieved by adding a lipase of high lipolytic activity and sensitivity to lipstatin to the reaction mixture. In the assay, the fungal lipase from Mucor javanicus was used as an alternative to the human pancreatic lipase. Many fungal lipases preserve high lipolytic activity in extreme conditions and are not colipase dependent. The assay proved to be reliable in differentiation of strains with high and low lipstatin productivity.

Notes

Acknowledgements

We thank Veronika Palušková for help with isolation of strains by UV mutagenesis.

Funding

This study was funded by ITMS 26220220093, Grant of Science and Technology Assistance Agency (Grant No. APVV-0719-12) and KEGA (Grant No. 047STU-4/2016).

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical Approval

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

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2017

Authors and Affiliations

  • Michal Híreš
    • 1
  • Nora Rapavá
    • 1
  • Martin Šimkovič
    • 1
  • Ľudovít Varečka
    • 1
  • Dušan Berkeš
    • 2
  • Svetlana Kryštofová
    • 1
  1. 1.Institute of Biochemistry and MicrobiologySlovak University of TechnologyBratislavaSlovakia
  2. 2.Department of Organic ChemistrySlovak University of TechnologyBratislavaSlovakia

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