Thomas, Katherine Claire; (2001) Process design tools for reductive biocatalysis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Reductive biocatalysis is potentially a very useful tool as it creates a chiral centre. Enzyme catalysed reduction reactions generally require the addition of expensive nicotinamide cofactors in stoichiometric amounts. To reduce the cost of these cofactors they can be recycle by a second substrate or enzyme or by chemical or electrochemical methods. A decisional tool is proposed to assist the choice of the optimal enzymatic cofactor regeneration system for reductive biocatalysis. The tool comprises a series of questions addressing a potential process involving an enzymatic cofactor regeneration system and offers possible solutions to guide an engineer to an economically feasible process. A model system was chosen with which to test the proposed decision tool which was the reduction of 6-bromo-β-tetralone to (S)-6-bromo-β-tetralol by the yeast Trichosporon capitatum. An NADH-specific reductase enzyme from the yeast was isolated to apparent homogeneity with an enantiomeric excess of 99.54%. Purification involved protamine sulphate precipitation, anion exchange chromatography on Q Sepharose FF, affinity chromatography on Amicon Affinity Dyematrex Red A and gel filtration on Superdex 200 HR. The final purification stage resulted in a 54-fold increase in specific activity, to 430U/mg with a yield of 1.34%. The optimum conditions for the operation of a bioconversion using the isolated tetralone reductase enzyme were investigated. The enzyme was immobilised onto Eupergit C in the presence of 0.5M phosphate buffer at pH7 with an optimum enzyme challenge of 0.5U/g beads; both the free and immobilised forms of the enzyme were investigated under process conditions. The optimum pH for activity of the free enzyme was in the range 6-7 and 6-8.5 for the immobilised enzyme. Both forms of the enzyme were at their most stable over this range. The temperature optima, a trade off between optimum activity and stability were found to be 21°C and 31°C for the free and immobilised forms respectively. Optimum solvent regime was 50% hexane for the free enzyme and 50% octanol for the immobilised enzyme. A second enzyme for cofactor regeneration was selected and a combined, two enzyme system was optimised using a semi-automated, high throughput screening method. The regenerative enzyme, formate dehydrogenase, converts formate to CO2 and recycles NADH. The optimum condition of pH (6.8), temperature (26°C) and solvent (30% octanol) were found by factorial experiments using a robotic liquid handling system to produce a semi-automated process. The difficulties associated with operating the robotic system for enzyme reaction characterisation have been investigated with respect to bead handling and accuracy of the system. The main problems identified with bead handling for immobilised enzymes were pipette tips becoming clogged with beads and the bead slurry settling out and therefore not being homogeneous for pipetting. The accuracy of the system is important to investigate for the quantitative use of robotic systems otherwise insufficient data or too much data could be collected which would lead to wasteful redundancy of the data.

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