Quantitative screening of hydrolase libraries using pH indicators: Identifying active and enantioselective hydrolases

Lana E. Janes, A. Christina Löwendahl, Romas J. Kazlauskas

Research output: Contribution to journalArticle

149 Scopus citations

Abstract

The slowest step in finding a selective hydrolase for synthesis is often the screening step. Researchers must run small test reactions and measure the amounts of stereoisomers formed by HPLC, GC, or NMR. We have developed a colorimetric method to speed up this screening. We quantitatively detect ester hydrolysis using a pH indicator, 4-nitrophenol. We estimate the selectivity by measuring the initial rates of hydrolysis for pure stereoisomers separately. To demonstrate the utility of this method, we screened seventy-two commercial enzymes for enantioselective hydrolysis of racemic solketal butyrate, an important chiral building block. First, we eliminated the twenty hydrolases that did not catalyze hydrolysis of either enantiomer. Next, we measured initial rates of hydrolysis of the pure enantiomers of solketal butyrate. For horse-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 is the highest enantioselectivity reported for a hydrolysis of solketal esters.

Original languageEnglish (US)
Pages (from-to)2324-2331
Number of pages8
JournalChemistry - A European Journal
Volume4
Issue number11
DOIs
StatePublished - Jan 1 1998

Keywords

  • Enantiomeric resolution
  • Enzyme catalysis
  • Lipases
  • Screening
  • Synthetic methods

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