Optimized oxidoreductases for medium and large scale industrial biotransformations
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
Private area


Total records: 126
Pages:    1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21  

[ 2014 ] Levasseur A, Lomascolo A, Chabrol O, Ruiz-Dueñas FJ, [...] , Martínez AT, [...] , Record E The genome of the white-rot fungus Pycnoporus cinnabarinus: a basidiomycete model with a versatile arsenal for lignocellulosic biomass breakdown BMC Genomics, 15: 486
[ 2014 ] Linde D, Coscolín C, Liers C, Hofrichter M, Martínez AT, Ruiz-Dueñas FJ Heterologous expression and physicochemical characterization of a fungal dye-decolorizing peroxidase from Auricularia auricula-judae Protein Expr. Purif., 103: 28-37
[ 2014 ] Macellaro G, Baratto MC, Piscitelli A, Pezzella C, Fabrizi de Biani F, Palmese A, Piumi F, Record E, Basosi R, Sannia G Effective mutations in a high redox potential laccase from Pleurotus ostreatus Appl. Microbiol. Biotechnol., doi: 10.1007/s00253-013-5491-8
[ 2014 ] Macellaro G, Pezzella C, Cicatiello P, Sannia G, Piscitelli A Fungal Laccases Degradation of Endocrine Disrupting Compounds BioMed Research International, doi: 10.1155/2014/614038
[ 2014 ] Martínez AT, Ruiz-Dueñas FJ, Gutiérrez A, del Río JC, Alcalde M, Liers C, Ullrich R, Hofrichter M, Scheibner K, Kalum L, Vind J, Lund H Search, engineering, and applications of new oxidative biocatalysts Biofuels, Bioprod. Bioref., 8: 819-835
[ 2014 ] Molina-Espeja P, García-Ruiz E, González-Pérez D, Ullrich R, Hofrichter M, Alcalde M Directed evolution of Unspecific Peroxygenase from Agrocybe aegerita Appl. Environ. Microbiol., 80: 3496-3507
Directed evolution of Unspecific Peroxygenase from Agrocybe aegerita
Molina-Espeja P, García-Ruiz E, González-Pérez D, Ullrich R, Hofrichter M, Alcalde M
Appl. Environ. Microbiol., 80: 3496-3507

Unspecific peroxygenase (UPO) represents a new type of heme-thiolate enzyme with self-sufficient mono(per)oxygenase activity and many potential applications in organic synthesis. With a view to taking advantage of these properties, we subjected the Agrocybe aegerita UPO1 encoding gene to directed evolution in Saccharomyces cerevisiae. To promote functional expression, several different signal peptides were fused to the mature protein and the resulting products tested. Over 9,000 clones were screened using an ad-hoc dual-colorimetric assay that assessed both peroxidative and oxygen-transfer activities. After 5 generations of directed evolution combined with hybrid approaches, 9 mutations were introduced that resulted in a 3,250-fold total activity improvement with no alteration in protein stability. A breakdown between secretion and catalytic activity was performed by replacing the native signal peptide of the original parental type with that of the evolved mutant: the evolved leader increased functional expression 27-fold whereas a 18-fold improvement in kcat/Km for oxygen transfer activity was obtained. The evolved UPO1 was active and highly stable in the presence of organic co-solvents. Mutations in the hydrophobic core of the signal peptide contributed to enhance functional expression up to 8 mg/L, while catalytic efficiencies for peroxidative and oxygen transfer reactions were increased by several mutations in the vicinity of the heme-access channel. Overall, the directed evolution platform described is a valuable point of departure for the development of customized UPOs with improved features and for the study of structure-function relationships.

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