Abstract
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.
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