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Title: Production of novel amine oxidases from microorganisms
Author: Voulgaris, Ioannis
Awarding Body: University of Strathclyde
Current Institution: University of Strathclyde
Date of Award: 2011
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With the advent of molecular biology and genetic transformation the production of high value proteins in recombinant host cells has become an everyday reality. However, the establishment of the optimum conditions for every target protein requires to take into account the nature of the product, and needs experimentation. In this study the over-expression of an industrially important monoamine oxidase was attempted using different expression systems, both homologous and heterologous, and the optimum conditions for the industrial production were established. Amine oxidases exhibit stereoselectivity, and, therefore, can potentially be used for the deracemisation of non-optically pure mixtures of amines leading to optically pure amines of high value in the fine chemical and pharmaceutical areas. Initially, different strains of A.niger were evaluated regarding their ability to produce the industrially important monoamine oxidase (MAO-N), while later optimisation of the culture conditions took place. These were achieved by the use of the appropriate nitrogen source, incorporation of an amine oxidase inhibitor to reduce inducer's breakdown and consequently boost amine oxidase expression, and the induction of the culture at the appropriate time. Over-expression of the genetically modified MAO-5N in E.coli was studied under different experimental set ups with the aim of improving both the activity of the produced enzyme leading to relatively low-cost scalable processes. Towards this aim, substitution of the expensive inducer IPTG with the less expensive lactose took place, while changes in the feed profile were implemented. Moreover, the examination of the combined effects of induction biomass levels and oxygen availability, through the use of air and oxygen enriched air, in the culture led to significant increase of the enzyme activity and gave important insights about the sensitivity of this enzyme under highly oxidative conditions. Undoubtedly, E.coli remains the main workhorse for the production of recombinant proteins, nevertheless, the use of methylotrophic yeasts is gaining significant ground and for that reason a P.pastoris system intracellularly expressing the MAO-5N was investigated. Appropriate monitoring and control of the bioprocess along with optimisation of the carbon flux led to significantly higher volumetric MAO activities and better biotransformation capacity of the whole cells than the previously used E.coli systems. The aforementioned improvements result in more efficient processes, where both the overall productivity is increased with a simultaneous reduction of the cost which ultimately leads to production of cheaper valuable compounds. Moreover, the present study extends our knowledge on the nature and physiology of monoamine oxidase production by microbial organisms and suggest a solution which can be implemented for the industrial manufacture of other oxidase type enzymes.
Supervisor: Speight, Robert ; McNeil, B. ; Arnold, S. Alison ; Harvey, L. M. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral