Bui, Quang Tuong; (2000) Preparation and characterisation of biocompatible magnetic fluids. Doctoral thesis (Ph.D.), University College London.

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Abstract

This thesis describes efforts in the development of an immobilised enzyme biocatalyst for a scaleable biotransformation. The enzyme transketolase, which catalyses the synthesis of carbon-carbon bonds, was chosen as a model. The enzyme has been cloned into E.coli and can be produced on a large scale and is consequently representative of an industrial enzyme. Transketolase has been immobilised onto the commercially available materials Eupergit-C and Amberlite XAD7. With a binding yield of ca 90 % and a retention of activity between 20 % and 60 % depending upon enzyme load in the case of Eupergit-C and a binding yield of 90 % and a retention of 20% activity in the case of Amberlite XAD7. The cofactor thiamine pyrophosphate was successfully introduced to the Eupergit-C immobilised enzyme, by co-immobilising the enzyme and cofactor, to yield a biocatalyst with a specific activity of 840 U.g-1 and a retention of activity of 58 %. Although the cofactor was found to leach from the immobilised holo-enzyme as a consequence excess cofactor had to be maintained within the bioreactor environment. Both immobilised enzymes were characterised and an improvement in the stability of both the Eupergit-C and Amberlite XAD7 preparations against the toxic substrate glycolaldehyde was observed. The half life in the presence of 0.5 M glycolaldehyde was improved from 1 hour to 100 hours and 80 hours in the case of Eupergit-C and Amberlite XAD7 respectively. An economic comparison of Eupergit-C and Amberlite XAD7 was made and Eupergit-C was found to be the more economic support material producing a biocatalyst with a space time yield of 674 kg.L-1 /batch and a calculated enzyme productivity of 100kU/kg product after 20 batches.

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