Optimized oxidoreductases for medium and large scale industrial biotransformations
CLOSE
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
Phone: 34 918373112
Fax: 34 915360432
Mobile: 34 650080476
CLOSE
Private area
User:


Password:

publications
Total records: 112
Pages:   1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19  

[ 2017 ] Acebes S, Ruiz-Dueñas FJ, Toubes M, Saez-Jimenez V, Pérez-Boada M, Lucas F, Martínez AT, Guallar V Mapping the Long-Range Electron Transfer Route in Ligninolytic Peroxidases J. Phys. Chem. B, 121: 3946-3954
[ 2017 ] Alcalde M When directed evolution met ancestral enzyme resurrection Microbial Biotechnol., 10: 22-24
[ 2017 ] Ayuso-Fernández I, Martínez AT, Ruiz-Dueñas FJ Experimental recreation of the evolution of lignin-degrading enzymes from the Jurassic to date Biotechnol. Biofuels, 10: 67
[ 2017 ] Carro J, Martínez A, Medina M, Martínez AT, Ferreira P Protein dynamics promote hydride tunnelling in substrate oxidation by aryl-alcohol oxidase Phys. Chem. Chem. Phys., doi: 10.1039/C7CP05904C
[ 2017 ] González-Pérez D, Alcalde M The making of versatile peroxidase by directed evolution Biocatalysis and Biotransformation, doi: 10.1080/10242422.2017.1363190
[ 2017 ] Martínez AT, Ruiz-Dueñas FJ, Camarero S, Serrano A, Linde D, Lund H, Vind J, Tovborg M, Herold-Majumdar OM, Hofrichter M, Liers C, Ullrich R, Scheibner K, Sannia G, Piscitelli A, Sener ME, Kılıç S, van Berkel WJ, Guallar V, et al. Oxidoreductases on their way to industrial biotransformations Biotechnol. Adv., doi: 10.1016/j.biotechadv.2017.06.003
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