Research in to enzyme catalysis has enormously enriched the success of biological organic synthesis; particularly in the last decade, remarkable progress in biological catalysis has taken place leading to the application of enzymes to a wider extent in industrial processes. The benefits of establishing enzymes as industrial catalysts are founded on the need to develop a 'clean' and 'pure' technology which carry out isomer and region selective reactions; generate or transform pure isomers compared with racemic mixtures and produce pure compounds in comparison with mixtures of by-products often arising from conventional chemical synthesis. With the onset of molecular biology and genetics, the manufacturing of high value proteins in recombinant host cells has become commonplace; nevertheless, there needs to be a better understanding of the optimum conditions for the production of every recombinant protein. In this study, the over expression of an industrially important amine oxidase - D-Amino Acid Oxidase (DAAO) - was attempted using two different expression systems: Escherichia coli and Pichia pastoris in order to establish the optimum conditions for industrial production. Amine oxidases exhibit stereoselectivity; ergo they can theoretically be used for the deracemisation of nonopticallypure mixtures of amines leading to optically pure amines of high value.Furthermore, DAAO is involved in the production of 7-aminocephalosporanic acid (7-ACA) from cephalosporin c (CPC), which can be used in the synthesis of novel cephalosporin antibiotic derivatives. In order to accelerate development of these new antibiotics DAAO must be readily available. Overexpression of DAAO in E. coli was studied under varying experimental conditions in order to ascertain the best conditions for enzymatic activity whilst simultaneously investigating the variations between both defined and complex media to understand suitability for scale-up.Although E. coli is still regarded as the 'go to' organisms for initial laboratory scale up the methylotrophic yeast P. pastoris is considered an industrial workhorse for the production of recombinant proteins and thus the expression of DAAO was also investigated to ascertain how enzyme activities are affected by this expression system. Furthermore, the supplementation of media with different nutrient sources of varying complexity was investigated, as was the implementation of co-feeding inorder to improve carbon flux during recombinant protein production. Finally, the oxygen availability was investigated in order to gain important understandings on its effects on microbial physiology and DAAO activity.
|Date of Award||12 Oct 2016|
- University Of Strathclyde
|Sponsors||University of Strathclyde & BBSRC (Biotech & Biological Sciences Research Council)|
|Supervisor||Brian McNeil (Supervisor) & Linda Harvey (Supervisor)|