Hussain, W.; (2007) Characterisation and selection of room temperature ionic liquids as co-solvents for redox biocatalysis. Doctoral thesis , University of London.

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Abstract

The use of biocatalysis to create chiral centres for chemical pharmaceuticals is rapidly becoming a standard industrial technology. Often, the compounds of interest are poorly soluble in aqueous solvents leading to the use of organic co- solvents to deliver substrate into the environment of the biocatalyst. Unfortunately these co-solvents can impact negatively on the stability of the biocatalyst. Recently, room temperature ionic liquids have been attracting interest as potential replacements for organic solvents in such reactions as they offer lower toxicity and an increased rate of reaction. This thesis describes an original experimental study into the use of ionic liquids with biocatalytic redox reactions. Initially, the stereospecific reduction of 6-Br-p-tetralone to its corresponding alcohol (5)-6-Br-P-tetralol was carried out by the yeast Trichosporon capitatum MY 1890 and by the bacterium Rhodococcus erythropolis MA7213 using a range of ionic liquids chosen on the diversity of their composition. The decrease in cell viability of both types of whole cell biocatalyst upon exposure to ionic liquids was found to be intermediate to that determined for cells residing purely in fermentation media and cells residing in a two-phase mixture of media and organic solvent (toluene). For T. capitatum MY1890 bioconversions, the ionic liquid Emim TOS gave a reaction profile comparable to that observed in the previously studied water-ethanol system, in terms of overall rate of reaction (0.2g(prod)L"1hr"1) and conversion (100% w/w). For bioconversions carried out with R. erythropolis MA7213, through use of 20% v/v Emim TOS as a co- solvent the conversion yield doubled and a four-fold increase in initial rate was found compared to the standard ethanol co-solvent. Subsequent flow cytometric analysis of the R. erythropolis MA7213 in these ionic liquids showed that the stability of the biocatalyst was increased in ionic liquids, such as Emim TOS and Oc3MeN BTA accounting for the increased rates of reaction and overall conversion observed. The potential for multi parameter flow cytometry to be used as a tool for identifying biocompatible ionic liquids was demonstrated through the improved oxidation of 1-indanone to 3,4- dihydrocoumarin by cyclo-hexanone mono-oxygenase expressed by E. coli TOP10 pQR239 in the presence of 20% v/v Emim TOS with 30% w/w overall conversion compared to only 20% w/w overall conversion in the presence of 10% v/v ethanol. The ability to perform isolated enzyme catalysed conversions in ionic liquids was also investigated. The asymmetric reduction of 6-Br-P-Tetralone to its corresponding (S) alcohol was found to be mediated by alcohol dehydrogenase isolated from R. erythropolis (ADH RE) and proceeded more favourably in both a miscible and immiscible ionic liquid compared to the organic solvent systems identified, with initial rate increases of more than two-fold. The ionic liquid BMP BTI acting as a co-solvent (20% v/v) yielded 100% conversion of 50 gL' 1 substrate in less than 12 hours (an increase of 25 times over the maximum yield from whole cells). This was attributed to a combined effect of increased enzyme stability in the ionic liquid and more efficient mass transfer of substrate from the ionic liquid phase to buffer compared to toluene. The asymmetric reduction of 4'-Br-2,2,2-trifluoroacetophenone to ()-4'-Br-2,2,2-trifluoroacetophenyl alcohol was also carried out using the ADH RE and was found to be limited to 10 gL"1 conversion due to toxicity of the substrate on the enzyme in the presence of toluene (10% v/v). Using 10% v/v BMP BTI as a co-solvent led to an enzyme half life of almost 300 hours at 30 C and complete conversion of 50gL_1 substrate in less than 10 hours. The selection of a suitable ionic liquid for a particular bioconversion appears to rest primarily on biocatalyst stability, but is also dependent on solubility and mass transfer issues of substrate from the ionic liquid into the aqueous phase. The potential of a high throughput ionic liquid selection strategy based on these measurements was shown through the use of automated pipetting platforms, miniature bioreactors with online monitoring, and microwell compatible devices for flow cytometry and spectrometry for biocatalyst stability studies. The potential for ionic liquids to be employed in industrial redox bioconversions with both whole cell and isolated enzyme biocatalysts has thus been demonstrated.

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