A fast and sensitive colorimetric assay (FRED, fast and reliable ene-reductases detection) that allows the estn. of levels of conversion of ene-reductase (ER)-catalyzed 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 detd. colorimetrically by a multienzymic system. The FRED assay offers a wide range of possible applications, from enzyme fingerprinting and kinetic anal., to primary screening of enzyme libraries and optimization of ERs' performances under different reaction conditions.
A Rapid and High-Throughput Assay for the Estimation of Conversions of Ene-Reductase-Catalysed Reactions
CROTTI, MICHELE;GATTI, FRANCESCO GILBERTO;PARMEGGIANI, FABIO;BRENNA, MARIA ELISABETTA;
2015-01-01
Abstract
A fast and sensitive colorimetric assay (FRED, fast and reliable ene-reductases detection) that allows the estn. of levels of conversion of ene-reductase (ER)-catalyzed 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 detd. colorimetrically by a multienzymic system. The FRED assay offers a wide range of possible applications, from enzyme fingerprinting and kinetic anal., to primary screening of enzyme libraries and optimization of ERs' performances under different reaction conditions.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
