A colorimetric assay suitable for screening epoxide hydrolase activity

A UV/VIS spectrophotometric microtiter-plate and a filter-paper based assay using 4-(p-nitrobenzyl)pyridine (NBP) were developed to determine epoxide hydrolytic activity by measuring the decrease of the epoxide concentration. Both systems were applied for screening an expression gene bank of Rhodococcus sp. NCIMB 11216. As a reference, whole cells from Rhodococcus sp. NCIMB 11216 and Beauveria sulfurescens ATCC 7159 exhibiting epoxide hydrolase activity were used. The microtiter-plate system was also evaluated for different epoxides and performed in a laboratory robotic system for high throughput screening. The microtiter-plate assay showed a high sensitivity for the detection of small concentrations of epoxides (0.1-1 mg/well) such as styrene oxide, ethyl phenylglycidate, n-hexane oxide and indene oxide. The filter paper assay was further optimized for styrene oxide. Both assays were suitable to screen within libraries of epoxide hydrolases without interference with other enzymes such as esterases, lipases or proteases. The assay should allow to screen large libraries obtained by directed evolution, strain collections and (expression) gene banks for epoxide hydrolytic activity or to monitor the purification process of an epoxide hydrolase. (C) 1999 Elsevier Science B.V. All rights reserved.