Different Strategies – One Target: Enantioselective Biocatalysis Using Lipases and Esterases

Professor U. Bornscheuer, Griefswald University, Germany

Professor Bornscheuer described some variations on standard uses of enzymes for dynamic kinetic resolution. For example better results can often be achieved in the resolution of carboxylic acid esters if vinyl esters rather than ethyl esters are used as substrates. The talk also included some work on recombinant pig liver esterases, which showed greatly enhanced activity, compared to commercially available enzymes. Directed evolution was used to modify various esterases in an attempt to produce an enzyme that would give good activity for the resolution of ester (11) to produce (12) a building block for the synthesis of epothilones.

A number of assays were described to allow rapid determination of synthetic activity in mutant organisms in high throughput screens. For example if the hydrolysis of an acetate derivative of an alcohol is being studied, using a hydrolase, one mole of acetic acid is produced for every mole of substrate converted. The acetic acid is then involved in the ATP / AMP cycle leading to the transformation of NAD+ in to NADH which can be quantified spectrophotometrically at 340nm. In the hydrolysis of vinyl esters, acetaldehyde is produced and this can be quantified by derivatisation with 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (13), which is non-fluorescent, but reacts with acetaldehyde to produce hydrazone (14) which is fluorescent.

These assays work efficiently in a variety of organic solvents (except of course those that dissolve the micro titre plate!) and show high sensitivity with the detection limit being in the nanomolar range. The final part of the talk concerned work in progress on the rational design of hydrolases that exhibit activity on tertiary esters such as 3-phenylbut-1-yn-3-yl acetate, 3-methylpent-1-yn-3-yl acetate, and linalyl acetate.