title: A Rapid and High-Throughput Assay for the Estimation of Conversions of Ene-Reductase-Catalysed Reactions.
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.
authors.affiliation: Istituto di Chimica del Riconoscimento Molecolare, CNR, Via Mario Bianco 9, 20131 Milano (Italy).
authors.forename: Maria Chiara
authors.initials: MC
authors.lastname: Forchin
authors.affiliation: Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Politecnico di Milano, Via Mancinelli 7, 20131 Milano (Italy).
authors.forename: Michele
authors.initials: M
authors.lastname: Crotti
authors.affiliation: Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Politecnico di Milano, Via Mancinelli 7, 20131 Milano (Italy).
authors.forename: Francesco G
authors.initials: FG
authors.lastname: Gatti
authors.affiliation: Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Politecnico di Milano, Via Mancinelli 7, 20131 Milano (Italy). fabio.parmeggiani@polimi.it.
authors.forename: Fabio
authors.initials: F
authors.lastname: Parmeggiani
authors.affiliation: Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Politecnico di Milano, Via Mancinelli 7, 20131 Milano (Italy).
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Brenna
authors.affiliation: Istituto di Chimica del Riconoscimento Molecolare, CNR, Via Mario Bianco 9, 20131 Milano (Italy). daniela.monti@icrm.cnr.it.
authors.forename: Daniela
authors.initials: D
authors.lastname: Monti
mesh_terms: D055162:Biocatalysis; D003124:Colorimetry / Q000379:methods*; D005947:Glucose / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism*
chemical_list: D009243:NAD; D009249:NADP; D010088:Oxidoreductases; D005947:Glucose
keywords: biocatalysis; cofactors; high-throughput screening; olefins; reductases; reduction