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.
A Rapid and High-Throughput Assay for the Estimation of Conversions of Ene-Reductase-Catalysed Reactions
Forchin Maria Chiara;Monti Daniela
2015
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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
