QUANTITATIVE SCREENING OF HYDROLASE LIBRARIES USING PH INDICATORS - IDENTIFYING ACTIVE AND ENANTIOSELECTIVE HYDROLASES

The slowest step in finding a selective hydrolase for synthesis is oft en the screening step. Researchers must run small test reactions and m easure the amounts of stereoisomers formed by HPLC, GC, or NMR, We hav e developed a colorimetric method to speed up this screening. We quant itatively detect ester hydrolysis using a pH indicator, 4-nitrophenol. We estimate the selectivity by measuring the initial rates of hydroly sis for pure stereoisomers separately. To demonstrate the utility of t his method, we screened seventy-two commercial enzymes for enantiosele ctive hydrolysis of racemic solketal butyrate, an important chiral bui lding block. First, we eliminated the twenty hydrolases that did not c atalyze hydrolysis of either enantiomer. Next, we measured initial rat es of hydrolysis of the pure enantiomers of solketal butyrate. For hor se-liver esterase, these initial rates differed by a factor of twelve. Subsequent GC experiments confirmed an enantiomeric ratio of fifteen for this hydrolase, Although this enantioselectivity is moderate, it i s the highest enantioselectivity reported for a hydrolysis of solketal esters.