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

doi:10.1007/s10529-017-2450-y

Cited by 6 publications

(3 citation statements)

References 17 publications

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“…In contrast to the mitochondrial MAO, the Aspergillus niger enzyme, MAO N is more tractable for protein engineering and large-scale production although the FAD is not covalently bound to the enzyme [92][93][94]. MAO N is suitable for chiral catalysis and use of whole cells avoids the limitation of FAD supply [95,96].…”

Section: Is There Potential For Applied Use Of Mao?mentioning

confidence: 99%

Questions in the Chemical Enzymology of MAO

Ramsay

Albreht

2021

Chemistry

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We have structure, a wealth of kinetic data, thousands of chemical ligands and clinical information for the effects of a range of drugs on monoamine oxidase activity in vivo. We have comparative information from various species and mutations on kinetics and effects of inhibition. Nevertheless, there are what seem like simple questions still to be answered. This article presents a brief summary of existing experimental evidence the background and poses questions that remain intriguing for chemists and biochemists researching the chemical enzymology of and drug design for monoamine oxidases (FAD-containing EC 4.1.3.4).

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Section: Is There Potential For Applied Use Of Mao?mentioning

confidence: 99%

Questions in the Chemical Enzymology of MAO

Ramsay

Albreht

2021

Chemistry

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“…A possible way to circumvent to this issue derives from the cloning and expression of MAO-N D5 in an eukaryotic system, Pichia pastoris, achieving an 83-fold increase in enzyme production and subsequent 203-fold increase in total activity, by comparison with E. coli …”

Section: Mao From Aspergillus Niger: Successful Engineering In Amine ...mentioning

confidence: 99%

Monoamine Oxidase: Tunable Activity for Amine Resolution and Functionalization

Batista

Galman²,

Pinto³

et al. 2018

ACS Catal.

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Monoamine oxidases (MAO) are biocatalysts for the oxidation of a wide range of different amines including α-chiral amines. Their high selectivity and activity, along with the environmental advantages inherent to enzymatic synthesis, place MAOs in the spotlight for future application in industrial biocatalytic processes. To date, these enzymes have been used in both amine resolution and amine functionalization. MAO from Micrococcus luteus was employed in the multienzymatic synthesis of benzylisoquinoline alkaloids, and MAO from Aspergillus niger (MAO-N) was used in deracemization experiments. MAO-N was also applied to several biobio and biochemo cascades for amine functionalization, exploring the increased reactivity of the imine/iminium species. MAO-N has been extensively engineered to alter the size and electronic properties of its active site, creating variants capable of oxidizing a broad range of α-aliphatic and aromatic amines. This Review provides an in-depth analysis of current research in the biocatalytic applications of MAOs, coupled with available data on the limitations and challenges that still hinder their industrial application. It also highlights the importance of chiral amines and the biochemical importance of human MAO in metabolism. Finally, the development of alternative amine oxidases, such as CHAO or HLNO/HDNO, is briefly surveyed, along with a discussion on possible future developments on this field.

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“…As a result, the yield of FGF-2 protein in this study was lower than other expression systems in previous studies of P. pastoris 9,21 , where the culture condition was optimized for methanol concentration, pH, and induction time. Therefore, the efficiency of the P. pastoris strain in this study could be improved by optimized shake-flask condition 21 and be further enhanced in up-scale production, if dissolved oxygen, pH, temperature and carbon source feeding strategy could be controlled 19,25 . In short, P. pastoris might be a promising strain for high yield production of FGF-2 protein.…”

Section: Discussionmentioning

confidence: 99%

Secretory expression of the recombinant FGF-2 protein in Pichia pastoris carrying multiple copies of target gene

et al. 2020

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Introduction: Fibroblast growth factor-2 (FGF-2) is a multifunctional protein that plays an important role in the regulation of proliferation, differentiation and migration of a variety of cells. The recombinant human FGF-2 (rhFGF-2) is currently used in stem cell culture, medicine and cosmetic products. In this study, we aim to produce secreted rhFGF-2 protein from a Pichia pastoris strain containing multiple copies of the fgf-2 gene to eliminate the disadvantages of intracellular expression systems. Methods: The recombinant Pichia pastoris carrying the fgf-2 gene was cloned by using homologous cloning method. The recombinant strains were screened by PCR reactions using specific primers for the target gene and the AOX1 promoter sequence. Moreover, the copy number of the fgf-2 gene inserted into the P. pastoris genome was identified by semi-quantitative PCR method. The secreted rhFGF-2 protein was collected in the induced BMMY medium at a final methanol concentration of 0.5%, and purified by one-step heparin affinity chromatography. The quantity and biological activity of the purified protein were determined by competitive ELISA method and MTT assay on NIH-3T3 cell line, respectively. Results: Various recombinant P. pastoris clones carrying different copy numbers of the fgf-2 gene were obtained and categorized into 3 groups: the low copy strains (4-5 copies), medium copy strains (8-11 copies), and high copy strains (more than 20 copies). Among those strains, the 4-copy one produced the rhFGF-2 protein at the highest expression level. After purification, the purity of rhFGF-2 protein reached 98.8%, and the recovery yield was 179.2 µg of protein from 200 mL of flask culture (equivalent to 850 µg/L). The purified rhFGF-2 protein showed similar biological activity on NIH-3T3 cell line with the commercial FGF-2 protein. Conclusion: The recombinant FGF-2 protein was successfully secretory expressed from Pichia pastoris, and successfully purified by only one-step chromatography.

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

Abstract: 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.

Cited by 6 publications

References 17 publications

Questions in the Chemical Enzymology of MAO

Questions in the Chemical Enzymology of MAO

Monoamine Oxidase: Tunable Activity for Amine Resolution and Functionalization

Secretory expression of the recombinant FGF-2 protein in Pichia pastoris carrying multiple copies of target gene

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