Epoxide hydrolases screening

A great advantage of spectrophotometric assays is that they can be carried out in microtiter plates or as filter paper assays, thus allowing a high sample throughput coupled with small sample volumes. Such systems were used for example for the screening of epoxide hydrolases [34]. A classical example of activity tests is for amylolytic enzymes where starch agar plates are stained with iodine after a certain reaction time. The radius of clear spots is a measure of the reaction rate [35]. 

Active hits were found for every type of substrate screened, including those for which other known microbial epoxide hydrolases were ineffective. For example, hydrolysis of m-stilbenc oxide was not successful with several microbial EHs tested previously.4243 By contrast, several of our new enzymes actively hydrolyzed this substrate and exhibited excellent enantioselectivities (>99% ee). It is important to note that these enzymes were found to be capable of selectively hydrolyzing a wide range of mc.vo-cpoxidcs, including cyclic and acyclic alkyl- and aryl-substituted substrates. 

Microreactor technology offers the possibility to combine synthesis and analysis on one microfluidic chip. A combination of enantioselective biocatalysis and on-chip analysis has recently been reported by Beider et al. [424]. The combination of very fast separations (<1 s) of enantiomers using microchip electrophoresis with enantioselective catalysis allows high-throughput screening of enantioselective catalysts. Various epoxide-hydrolase mutants were screened for the hydrolysis of a specific epoxide to the diol product with direct on-chip analysis of the enantiomeric excess (Scheme 4.112). 

Zocher, F., Enzelberger, M.M., Bomscheuer, U.T., Hauer, B. and Schmid, R.D. (1999) A colorimetric assay suitable for screening epoxide hydrolase activity. Analytica Chimica Acta, 391, 345-351. 

Fig. 2.11 Enzyme-catalyzed desymmetrization of meso-epoxides using epoxide hydrolases of microbial origin that were screened for maximum activity. Note that the relative rate (measured as the turnover frequency, TOE) offormingthe (R,R)-stereoisomer is about 250 times higher than for the (S,S) one.

In spite of the considerable value of epoxide hydrolases for fine chemical synthesis, it was only recently that a detailed search for epoxide hydrolases from microbial sources was undertaken by the groups of Furstoss185, 901 and Faber123, 79, 911, bearing in mind that the use of microbial enzymes allows an (almost) unlimited supply of biocatalyst. The screening was based along the following considerations on the one hand, the catabolism of alkenes often implies the hydrolysis of an epoxide inter-... 

The search for novel microbial epoxide hydrolases through title screening of various fungal and bacterial sources was mainly triggered by chance observations of unexpected side reactions in microbial transformations within two research groups at about the same time. 

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