Rapid screening of hydrolases for the enantioselective conversion of ‘difficult-to-resolve’ substrates

• Published in: Tetrahedron: asymmetry 2000-12, Vol.11 (23), p.4781-4790
• Main Authors: Baumann, Markus, Hauer, Bernhard H, Bornscheuer, Uwe T
• Format: Article
• Language: English
• ISSN: 0957-4166; 1362-511X
• DOI: 10.1016/S0957-4166(00)00465-1

Hydrolases showing high enantioselectivity towards three racemic alcohols (1-methoxy-2-propanol, 3-hydroxy-tetrahydrofuran, 3-butyn-2-ol) and pantolactone were identified by a step-wise screening procedure. Initially, those biocatalysts, which exhibited hydrolytic activity towards the corresponding acetates or butyrates, were selected out of >100 enzymes. Here, rapid screening was performed in a pH-indicator, based format in microtiter plates. Subsequently, enantioselectivity of active hydrolases was determined in small scale reactions (∼1 mg substrate per reaction) by means of gas chromatography using chiral columns. Enzymes exhibiting highest enantioselectivities were then chosen for preparative scale resolution. Using this strategy, at least one suitable hydrolase was found for 3 out of the 4 model compounds examined, allowing efficient kinetic resolution. Moreover, in all cases enantiocomplementary enzymes were identified thus enabling access to both enantiomers of all substrates. More than100 hydrolases were screened in a microtiter plate-based pH-indicator assay to resolve 1– 4. More than 20 useful enzymes were then identified as enantioselective by GC analysis using chiral columns.