TY - JOUR
T1 - Mapping the substrate selectivity of new hydrolases using colorimetric screening
T2 - Lipases from Bacillus thermocatenulatus and Ophiostoma piliferum, esterases from Pseudomonas fluorescens and Streptomyces diastatochromogenes
AU - Liu, Andrew Man Fai
AU - Somers, Neil A.
AU - Kazlauskas, Romas J.
AU - Brush, Terry S.
AU - Zocher, Frank
AU - Enzelberger, Markus M.
AU - Bornscheuer, Uwe T.
AU - Horsman, Geoff P.
AU - Mezzetti, Alessandra
AU - Schmidt-Dannert, Claudia
AU - Schmid, Rolf D.
N1 - Funding Information:
We thank NSERC (Canada) for financial support. We thank Professor Ted A. Meighen (McGill Biochemistry) for cultures of E. coli JM105 and DH5α, Mr. Nadim Saadeh for running the mass spectra, Dr. Lana Janes for helpful discussions and Ms. Ebru Togan-Tekin for help with the scale up reaction of BTL2.
PY - 2001/3/19
Y1 - 2001/3/19
N2 - Recent advances in biochemistry and molecular biology have simplified the discovery and preparation of new hydrolases. Although these hydrolases might solve problems in organic synthesis, measuring their selectivity, especially enantioselectivity, remains tedious and time consuming. Recently, we developed 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 piliferum (NRRL 18917, OPL) and esterases from two bacteria, Pseudomonas fluorescens (SIK-W1, esterase I, PFE) and Streptomyces diastatochromogenes (Tü 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 of unnatural substrates, but the two lipases accepted a broader range of substrates than the two esterases. As expected, the two lipases favoured more hydrophobic substrates, while the two esterases showed a preference for smaller substrates. Several moderately enantioselective reactions were identified 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 current colorimetric screen could not measure the selectivity for several slow-reacting substrates. Traditional screening identified high enantioselectivity of BTL2 and PFE toward one of these slow substrates, 1-phenylethyl acetate (E>50).
AB - Recent advances in biochemistry and molecular biology have simplified the discovery and preparation of new hydrolases. Although these hydrolases might solve problems in organic synthesis, measuring their selectivity, especially enantioselectivity, remains tedious and time consuming. Recently, we developed 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 piliferum (NRRL 18917, OPL) and esterases from two bacteria, Pseudomonas fluorescens (SIK-W1, esterase I, PFE) and Streptomyces diastatochromogenes (Tü 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 of unnatural substrates, but the two lipases accepted a broader range of substrates than the two esterases. As expected, the two lipases favoured more hydrophobic substrates, while the two esterases showed a preference for smaller substrates. Several moderately enantioselective reactions were identified 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 current colorimetric screen could not measure the selectivity for several slow-reacting substrates. Traditional screening identified high enantioselectivity of BTL2 and PFE toward one of these slow substrates, 1-phenylethyl acetate (E>50).
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U2 - 10.1016/S0957-4166(01)00072-6
DO - 10.1016/S0957-4166(01)00072-6
M3 - Article
AN - SCOPUS:0035911359
SN - 0957-4166
VL - 12
SP - 545
EP - 556
JO - Tetrahedron Asymmetry
JF - Tetrahedron Asymmetry
IS - 4
ER -