Premium
A Rapid and High‐Throughput Assay for the Estimation of Conversions of Ene‐Reductase‐Catalysed Reactions
Author(s) -
Forchin Maria Chiara,
Crotti Michele,
Gatti Francesco G.,
Parmeggiani Fabio,
Brenna Elisabetta,
Monti Daniela
Publication year - 2015
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201500219
Subject(s) - substrate (aquarium) , cofactor , nad+ kinase , chemistry , ene reaction , reductase , enzyme , dehydrogenase , microtiter plate , chromatography , high throughput screening , olefin fiber , biochemistry , combinatorial chemistry , catalysis , stereochemistry , biology , ecology
A fast and sensitive colorimetric assay (FRED, fast and reliable ene‐reductases detection) that allows the estimation of levels of conversion of ene‐reductase (ER)‐catalysed reactions has been developed. The activated olefin is reduced by ER at the expense of NAD(P)H cofactor, whose regeneration is carried out in situ by the glucose/glucose dehydrogenase system. Subsequently, the consumption of the co‐substrate glucose is determined colorimetrically by a multienzymatic system. The FRED assay offers a wide range of possible applications, from enzyme fingerprinting and kinetic analysis, to primary screening of enzyme libraries and optimisation of ERs' performances under different reaction conditions.