Identification of organic solvent tolerant carboxylic ester hydrolases for organic synthesis.

Biocatalysis has emerged as an important tool in synthetic organic chemistry enabling the chemical industry to execute reactions with high regio- or enantioselectivity and under usually mild reaction conditions while avoiding toxic waste. Target substrates and products of reactions catalyzed by carboxylic ester hydrolases are often poorly water-soluble and require organic solvents, enzymes are evolved by nature to be active in cells, i.e. in aqueous rather than organic solvents. Therefore, biocatalysts withstanding organic solvents are urgently needed. Current strategies to identify such enzymes rely on laborious tests carried out by incubation in different organic solvents and determination of residual activities. Here, we describe a simple assay useful to screen large libraries of carboxylic ester hydrolases for resistance and activity in water miscible organic solvents. We have screened a set of 26 enzymes, most of them identified in this study, with four different water miscible organic solvents. The triglyceride tributyrin was used as a substrate and fatty acids released by enzymatic hydrolysis were detected by a pH shift indicated by the indicator dye nitrazine yellow. With this strategy, we succeeded to identify a novel highly organic solvent tolerant esterase from Pseudomonas aestusnigri. In addition, the newly identified enzymes were tested with sterically demanding substrates, as common in pharmaceutical intermediates, and two enzymes from Alcanivorax borkumensis were identified which outcompeted the gold standard ester hydrolase CalB from Candida antarctica. [ABSTRACT FROM AUTHOR]