The influence of temperature on enzyme selectivity in organic media

Sammanfattning: The influence of different reaction parameters on enzyme selectivity was investigated. The reaction conditions studied were solvent dependence, water activity and reaction temperature. The improved selectivity was observed as higher synthetic yields in a-chymotrypsin and subtilisin catalyzed peptide synthesis respectively, and as higher enantioselectivity in the asymmetric reduction of ketones catalyzed by alcohol dehydrogenase from Thermoanaerobium brockii (TBADH). The synthetic yield in the a-chymotrypsin catalyzed peptide synthesis increased from 84 to 99% when the temperature was decreased from 25 to -20¡C. A decrease in temperature was shown to favour the nucleophile in the competition with water for the acyl-enzyme and as substrate the ester was favoured in the competition with the peptide product. This temperature effect was partly explained by the variation in temperature dependent solvation of water and the nucleophile. The relative decrease of the nucleophile solubility was greater (2.4-fold) than for water, and therefore the ratio of activities can be expected to increase. This will favour synthesis. This effect seems to be general, since similar results were observed for nucleophiles containing either a single amino acid or dipeptides and for reactions performed in different organic solvents. The reaction temperature affect the enantioselectivity, and an increase as well as a decrease of with decreasing temperature has been found. The enantioselectivity is greatly affected by changing the water activity of the reaction medium. It increased until it reached a plateau value at aw 0.84 and decreased slightly with increasing water content above the saturation limit. This effect of water activity seems to be an effect of differences in activation entropy rather than differences in activation enthalpy. However, the enantioselectivity was almost unaffected when the reaction medium was changed from hexane to pure water. This was shown for reduction of five different ketones catalyzed by TBADH.