Development of a high throughput screening tool for biotransformations utilising a thermophilic L-aminoacylase enzyme

Micro-reactors containing a monolith-immobilised thermophilic L-aminoacylase, from Thermococcus litoralis, have been developed for use in biotransformation reactions and a study has been carried out to investigate the stereospecificity and stability of file immobilised enzyme. The potential to use the developed micro-reactors as a tool for rapid screening of enzyme specificity was demonstrated, confirming that the L-aminoacylase showed a similar substrate specificity to that previously reported of the free enzyme. From this baseline, the technique was employed as a tool to evaluate potential unreported Substrates with N-benzoyl- (L-threonine, L-leucine and L-arginine) and N-acetyl- (D,L-serine, D,L-leucine, L-tyrosine and L-lysine) protecting groups. The order of preferred Substrates was found to be Phe > Thr > Len > Arg for N-benzoyl substrates and Phe >> Set > Leu > Met > Tyr > Trp for N-acetyl substrates. It was found that by using the micro-reactor a significantly smaller quantity of enzyme and substrates was required. It was shown that the micro-reactors were still operational in the presence of selected organic solvents, such as ethanol, methanol, acetone, dimethylformamide (DMF) and dimethylsulfoxide (DMSO). The results indicated that a combination of a Small amount of an appropriate solvent (5% DMSO) and a higher reaction temperature Could be employed in biotransformations where substrate solubility was an issue. (C) 2009 Elsevier B.V. All rights reserved.