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Biotechnology Letters

, Volume 40, Issue 1, pp 127–133 | Cite as

Cloning and upscale production of monoamine oxidase N (MAO-N D5) by Pichia pastoris

  • Kristína Markošová
  • Andrea Camattari
  • Michal Rosenberg
  • Anton Glieder
  • Nicholas J. Turner
  • Martin RebrošEmail author
Original Research Paper

Abstract

Objective

To clone monoamine oxidase N, that catalyses the selective oxidative deamination or deracemisation of amines into imines, in Pichia pastoris and prove the importance of choosing the proper expression system for its recombinant production.

Results

Monoamine oxidase, originating from Aspergillus niger and subjected to directed evolution (MAO-N D5), was cloned and expressed in Pichia pastoris CBS7435 MutS strain for the first time. Various transformants were screened at microscale level. The production of the clone expressing the most active enzyme was scaled-up to a 1.5 l fermenter and preparation of MAO-N D5 as a crude enzyme extract was optimised. The obstacles in the production of the enzyme in both expression systems, Escherichia coli and P. pastoris, are discussed and demonstrated.

Conclusions

There was an improvement in specific productivity, which was 83 times higher in P. pastoris, clearly proving the importance of choosing the right expression host system for the specific enzymes.

Keywords

Cloning Monoamine oxidase-N D5 Pichia pastoris Upscale 

Notes

Acknowledgements

The research leading to these results has received funding from the European Union Seventh Framework Programme BIONEXGEN under grant agreement no. 266025. This work was co-funded by the Slovak Research and Development Agency under contract no. DO7RP-0042-11 and by the Slovak Grant Agency for Science VEGA 2/0090/16. This work was also supported by ESF COST Chemistry Action “Systems Biocatalysis” (CM1303). Part of this work was supported acib, cofinanced by the Federal Ministry of Science, Research and Economy (BMWFW), the Federal Ministry of Traffic, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol and ZIT—Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG.

Supporting Information

Supplementary material—Materials and methods, Cultivation scale-up and Biotransformation.

Supplementary material

10529_2017_2450_MOESM1_ESM.docx (20 kb)
Supplementary material 1 (DOCX 19 kb)

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Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  • Kristína Markošová
    • 1
  • Andrea Camattari
    • 2
    • 3
  • Michal Rosenberg
    • 1
  • Anton Glieder
    • 3
  • Nicholas J. Turner
    • 4
  • Martin Rebroš
    • 1
    Email author
  1. 1.Faculty of Chemical and Food Technology, Institute of BiotechnologySlovak University of TechnologyBratislavaSlovakia
  2. 2.Biotransformation Innovation Platform (BIP)SingaporeSingapore
  3. 3.Institute of Molecular BiotechnologyNAWI Graz University of TechnologyGrazAustria
  4. 4.School of ChemistryManchester Institute of Biotechnology, University of ManchesterManchesterUK

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