Mapping substrate selectivity of lipases from thermophilic fungi

New methods were adapted to screen, fast and easily, the lipase specificity (topo- or enantio-selectivity) on crude extracellular extracts from thermophilic fungi. Substrate acyl chain length specificity was tested using p-nitrophenyl esters and vinyl esters by the detection of released p-nitrophenolate anions in the first case and protonation of p-nitrophenolate anions (color diminution) in the second case. Enantioselectivity was tested using either the direct reaction rates on individual enantiomers of glycidyl butyrate or on competition between these enantiomers and resorufin esters (-butyrate or -acetate). Among a library of 44 thermophilic fungi, 10 strains were pre-selected (based on their capabilities to produce constitutively extracellular lipases) for further lipase specificity studies. The above methods were applied to lipases from these pre-selected fungi and also to other several lipases preparations from bacterial, fungal and mammalian origin. Remarkably, the method on competition allowed the accurate determination of the enantiomeric ratio (E), since experimental data fitted correctly with the E determined by classical chemical methods. Consequently, these methods can be applicable for screening selectivity in a high number of lipases or esterases from wild isolates or variants generated by directed evolution, using directly in the test, the substrate (i.e. esters) that will be worked out in a given process.