Shorrock, V.J.; (2006) An NADH dependent reductase for isolated enzyme and whole cell catalysis. Doctoral thesis , University of London.

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Abstract

Isolated enzymes and whole cell biocatalysts can both be applied for the synthesis of chiral hydroxy compounds. It is hypothesized that whole cells can easily be employed for such reactions using simple technology which is robust. This is because whole cells contain all the necessary enzymes and metabolic pathways for cofactor regeneration. This also means that the enzymes and their cofactors are well-protected within their natural cell environment. In contrast, it is hypothesized that isolated enzymes require complicated and expensive purification procedures. They also require the stoichiometric addition of cofactors (or methods employed for their regeneration), and are susceptible to inactivation since they are isolated from their natural cell environment. The aim of this thesis was to systematically compare a whole cell biocatalyst (Trichosporon capitatum (MY 1890)) and an NADH dependent isolated reductase (tetralone reductase) in the synthesis of a chiral alcohol (6-bromo-P-tetralol). The comparison was carried out to ascertain which type biocatalyst is preferred, and also to establish whether the general hypotheses (as stated above) are true with respect to each biocatalyst. Comparison of the isolated enzyme and whole cell biocatalyst showed that there were significant differences with respect to each of the systems. These included differences in: the biocatalytic purity, the reaction methodology, the system efficiency, and the effects on the biocatalyst from the addition of substrate and solvent. The isolated enzyme methods were much more complicated than the whole cell methods, from the preparation of the isolated enzyme through to the bioreduction. This was because a novel protein purification process needed to be set up and a cofactor regeneration system was required. However, the isolated enzyme system showed higher substrate conversions than the whole cell system. At 1 g/L, a conversion of 86% after 420min was achieved, whereas the whole cell system exhibited a conversion of 79% after 450min. It was hypothesized that the whole cell system suffered from lower conversions due to the substrate and product accumulating inside the cell membrane and disrupting cell metabolism. In the same configuration, the whole cell system also suffered from lower reaction rates which were attributed to mass transfer limitations through the cell membrane. The addition of a solvent enhanced whole cell biocatalytic reaction rates, but only at low substrate concentrations. The isolated enzyme system was susceptible to inactivation, and increased solvent concentrations caused a detrimental affect on the reaction rates and conversions. This was most likely due to the solvent causing an irreversible change in the active site conformation. The similarities and differences of employing an NADH dependent reductase and a whole cell biocatalyst for the production of a chiral alcohol are discussed in this thesis.

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