Colorimetric assays for quantitative analysis and screening of epoxide hydrolase activity.
Focusing on directed evolution to tailor enzymes as usable biocatalysts for fine chemistry, we have studied in detail several colorimetric assays for quantitative analysis of epoxide hydrolase (EH) activity. In particular, two assays have been optimized to characterize variants issued from the directed evolution of the EH from Aspergillus niger. Assays described in this paper are sufficiently reliable for quantitative screening of EH activity in microtiter plates and are low cost alternatives to GC or MS analysis. Moreover, they are usable for various epoxides and not restricted to a type of substrate, such as those amenable to assay by UV absorbancy. They can be used to assay EH activity on any epoxide and to directly assay enantioselectivity when both (R) and (S) substrates are available. The advantages and drawbacks of these two methods to assay EH activity of a large number of natural samples are summarized.
Cedrone
F
F
Faculté des Sciences de Luminy, Université de la Méditerranée UMR CNRS 6111, 163 avenue de Luminy - case 901, 13288 Marseille cedex 9, France.
Bhatnagar
T
T
Baratti
Jacques C
JC
D001234:Aspergillus niger / Q000201:enzymology; D003124:Colorimetry / Q000379:methods; D004851:Epoxide Hydrolases / Q000032:analysis* / Q000378:metabolism; D004852:Epoxy Compounds / Q000737:chemistry / Q000378:metabolism*; D005026:Ethylene Glycols / Q000737:chemistry; D011725:Pyridines / Q000737:chemistry; D015203:Reproducibility of Results; D013053:Spectrophotometry; D013237:Stereoisomerism
D004852:Epoxy Compounds; D005026:Ethylene Glycols; D011725:Pyridines; C008633:4-(4-nitrobenzyl)pyridine; C031474:styrene glycol; C013690:styrene oxide; D004851:Epoxide Hydrolases