Optimized oxidoreductases for medium and large scale industrial biotransformations
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Project Secretariat
Dr Marta Pérez-Boada
E-mail: MPBoada@cib.csic.es
Consejo Superior de Investigaciones Científicas (CSIC)
Biological Research Centre (CIB)
Calle Ramiro de Maeztu 9, E-28040 Madrid, Spain
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publications
Total records: 122
Pages:   1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21  

[ 2018 ] Carro J, Fernandez-Fueyo E, Fernández-Alonso C, Cañada J, Ullrich R, Hofrichter M, Alcalde M, Ferreira P, Martínez AT Self-sustained enzymatic cascade for the production of 2,5-furandicarboxylic acid from 5-methoxymethylfurfural Biotechnol. Biofuels, 11: 86-96
[ 2018 ] Carro J, Ferreira P, Martínez AT, Gadda G Stepwise Hydrogen Atom and Proton Transfers in Dioxygen Reduction by Aryl-Alcohol Oxidase Biochemistry, doi: 10.1021/acs.biochem.8b00106
[ 2018 ] Ewing TA, van Noord A, Paul CE, van Berkel WJ A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases Molecules, 23: 164-182
[ 2018 ] Gygli G, de Vries RP, van Berkel WJ On the origin of vanillyl alcohol oxidases Fungal Gen. Biol., 116: 24-32
[ 2018 ] Leonhardt S, Büttner E, Gebauer AM, Hofrichter M, Kellner H Draft Genome Sequence of the Sordariomycete Lecythophora (Coniochaeta) hoffmannii CBS 245.38 Genome Announc., 6
[ 2018 ] Martínez AT, Camarero S, Ruiz-Dueñas FJ, Martínez MJ Biological Lignin Degradation Lignin Valorization: Emerging Approaches. Ed. Gregg Beckham. RSC: 199-225
year2017
High-Throughput Screening Assay for Laccase Engineering toward Lignosulfonate Valorization
Rodríguez-Escribano D, de Salas F, Pardo I, Camarero S
Int. J. Mol. Sci., 18: 1793-1803

Lignin valorization is a pending issue for the integrated conversion of lignocellulose in consumer goods. Lignosulfonates (LS) are the main technical lignins commercialized today. However, their molecular weight should be enlarged to meet application requirements as additives or dispersing agents. Oxidation of lignosulfonates with fungal oxidoreductases, such as laccases, can increase the molecular weight of lignosulfonates by the cross-linking of lignin phenols. To advance in this direction, we describe here the development of a high-throughput screening (HTS) assay for the directed evolution of laccases, with lignosulfonate as substrate and the Folin–Ciocalteau reagent (FCR), to detect the decrease in phenolic content produced upon polymerization of lignosulfonate by the enzyme. Once the reaction conditions were adjusted to the 96-well-plate format, the enzyme for validating the assay was selected from a battery of high-redox-potential laccase variants functionally expressed in S. cerevisiae (the preferred host for the directed evolution of fungal oxidoreductases). The colorimetric response (absorbance at 760 nm) correlated with laccase activity secreted by the yeast. The HTS assay was reproducible (coefficient of variation (CV) = 15%) and sensitive enough to detect subtle differences in activity among yeast clones expressing a laccase mutant library obtained by error-prone PCR (epPCR). The method is therefore feasible for screening thousands of clones during the precise engineering of laccases toward valorization of lignosulfonates.

Official webpage of indox [ industrialoxidoreductases ]. Optimized oxidoreductases for medium and large scale industrial biotransformations. This project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under Grant Agreement nº: FP7-KBBE-2013-7-613549. © indox 2013. Developed by garcíarincón