Mapping the substrate selectivity of new hydrolases using colorimetric screening: lipases from Bacillus thermocatenulatus and Ophiostoma piliferum, esterases from Pseudomonas fluorescens and Streptomyces diastatochromogenes

Recent advances in biochemistry and molecular biology have simplified the d iscovery and preparation of new hydrolases. Although these hydrolases might solve problems in organic synthesis, measuring their selectivity, especial ly enantioselectivity, remains tedious and time consuming. Recently, we dev eloped a colorimetric screening method to measure the enantioselectivity of hydrolases. Here we apply this rapid screening method to map the substrate selectivity of four new hydrolases: lipases from the thermophilic Bacillus thermocatenulatus (DSM 730, BTL2) and a filamentous fungus Ophiostoma pili ferum (NRRL 18917, OPL) and esterases from two bacteria, Pseudomonas fluore scens (SIK-W1, esterase I, PFE) and Streptomyces diastatochromogenes (Tu 20 , SDE). We screened a general library of 29 substrates and a chiral library of 23 pairs of enantiomers. All four hydrolases catalysed the hydrolysis o f unnatural substrates, but the two lipases accepted a broader range of sub strates than the two esterases. As expected, the two lipases favoured more hydrophobic substrates, while the two esterases showed a preference for sma ller substrates. Several moderately enantioselective reactions were identif ied for the solketal esters: BTL2, butyrate, E=7.9 (R); octanoate, E=4.9 (R ) and 3-bromo-2-methyl propionate methyl esters, PFE, E=12 (S); SDE, E=5.6 (S). OPL showed low enantioselectivity toward all substrates tested. The cu rrent colorimetric screen could not measure the selectivity for several slo w-reacting substrates. Traditional screening identified high enantioselecti vity of BTL2 and PFE toward one of these slow substrates, I-phenylethyl ace tate (E>50), (C) 2001 Elsevier Science Ltd. All rights reserved.