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 Rebroš

Research output: Contribution to journalArticle

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.

LanguageEnglish
Article numberhttps://doi.org/10.1007/s10529-017-2450-y
JournalBiotechnology letters
Early online date10 Oct 2017
DOIs
StatusPublished - 2017

Fingerprint

Pichia
Monoamine Oxidase
Organism Cloning
Enzymes
Clone Cells
Deamination
Imines
Aspergillus niger
Complex Mixtures
Amines
Escherichia coli

Keywords

  • Journal Article

Cooperations

  • NAWI Graz

Cite this

Markošová, K., Camattari, A., Rosenberg, M., Glieder, A., Turner, N. J., & Rebroš, M. (2017). Cloning and upscale production of monoamine oxidase N (MAO-N D5) by Pichia pastoris. Biotechnology letters, [https://doi.org/10.1007/s10529-017-2450-y]. DOI: 10.1007/s10529-017-2450-y

Cloning and upscale production of monoamine oxidase N (MAO-N D5) by Pichia pastoris. / Markošová, Kristína; Camattari, Andrea; Rosenberg, Michal; Glieder, Anton; Turner, Nicholas J; Rebroš, Martin.

In: Biotechnology letters, 2017.

Research output: Contribution to journalArticle

Markošová K, Camattari A, Rosenberg M, Glieder A, Turner NJ, Rebroš M. Cloning and upscale production of monoamine oxidase N (MAO-N D5) by Pichia pastoris. Biotechnology letters. 2017. https://doi.org/10.1007/s10529-017-2450-y. Available from, DOI: 10.1007/s10529-017-2450-y
Markošová, Kristína ; Camattari, Andrea ; Rosenberg, Michal ; Glieder, Anton ; Turner, Nicholas J ; Rebroš, Martin. / Cloning and upscale production of monoamine oxidase N (MAO-N D5) by Pichia pastoris. In: Biotechnology letters. 2017
@article{c61d34ba5d2b43239ea1d92f3655630d,
title = "Cloning and upscale production of monoamine oxidase N (MAO-N D5) by Pichia pastoris",
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 = "Journal Article",
author = "Krist{\'i}na Markošov{\'a} and Andrea Camattari and Michal Rosenberg and Anton Glieder and Turner, {Nicholas J} and Martin Rebroš",
year = "2017",
doi = "10.1007/s10529-017-2450-y",
language = "English",
journal = "Biotechnology letters",
issn = "0141-5492",
publisher = "Springer Netherlands",

}

TY - JOUR

T1 - Cloning and upscale production of monoamine oxidase N (MAO-N D5) by Pichia pastoris

AU - Markošová,Kristína

AU - Camattari,Andrea

AU - Rosenberg,Michal

AU - Glieder,Anton

AU - Turner,Nicholas J

AU - Rebroš,Martin

PY - 2017

Y1 - 2017

N2 - 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.

AB - 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.

KW - Journal Article

U2 - 10.1007/s10529-017-2450-y

DO - 10.1007/s10529-017-2450-y

M3 - Article

JO - Biotechnology letters

T2 - Biotechnology letters

JF - Biotechnology letters

SN - 0141-5492

M1 - https://doi.org/10.1007/s10529-017-2450-y

ER -