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Biotechnol Lett. 2018 Jan;40(1):127-133. doi: 10.1007/s10529-017-2450-y. Epub 2017 Oct 10.

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

Author information

1
Faculty of Chemical and Food Technology, Institute of Biotechnology, Slovak University of Technology, Radlinského 9, 812 37, Bratislava, Slovakia.
2
Biotransformation Innovation Platform (BIP), 61 Biopolis Drive #14-13, Singapore, 138673, Singapore.
3
Institute of Molecular Biotechnology, NAWI Graz University of Technology, Petergasse 14/2, 8010, Graz, Austria.
4
School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK.
5
Faculty of Chemical and Food Technology, Institute of Biotechnology, Slovak University of Technology, Radlinského 9, 812 37, Bratislava, Slovakia. martin.rebros@stuba.sk.

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

PMID:
29019030
DOI:
10.1007/s10529-017-2450-y
[Indexed for MEDLINE]

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