Optimized oxidoreductases for medium and large scale industrial biotransformations
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Dr Marta Pérez-Boada
E-mail: MPBoada@cib.csic.es
Consejo Superior de Investigaciones Científicas (CSIC)
Biological Research Centre (CIB)
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publications
Total records: 126
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[ 2015 ] Monza E, Lucas F, Camarero S, Alejaldre LC, Martínez AT, Guallar V Insights into Laccase Engineering from Molecular Simulations: Toward a Binding-Focused Strategy J. Phys. Chem. Lett., 6: 1447-1453
[ 2015 ] Ni Y, Fernandez-Fueyo E, Baraibar AG, Ullrich R, Hofrichter M, Yanase H, Alcalde M, van Berkel WJ, Hollmann F Peroxygenase-Catalyzed Oxyfunctionalization Reactions Promoted by the Complete Oxidation of Methanol Angew. Chem. Int. Ed., doi: 10.1002/anie.201507881
[ 2015 ] Pardo I, Camarero S Laccase engineering by rational and evolutionary design Cell Mol. Life Sci., doi: 10.1007/s00018-014-1824-8
[ 2015 ] Pardo I, Camarero S Exploring the Oxidation of Lignin-Derived Phenols by a Library of Laccase Mutants Molecules, 20: 15929-15943
[ 2015 ] Pezzella C, Guarino L, Piscitelli A How to enjoy laccases Cell Mol. Life Sci., 72: 923-940
[ 2015 ] Pham NH, Hollmann F, Kracher D, Preims M, Haltrich D, Ludwig R Engineering an enzymatic regeneration system for NAD(P)H oxidation J. Mol. Cat. B, 120: 38-46
year2014
Ligninolytic peroxidase gene expression by Pleurotus ostreatus: Differential regulation in lignocellulose medium and effect of temperature and pH
Fernandez-Fueyo E, Castanera ER, Ruiz-Dueñas FJ, López-Lucendo MF, Ramírez L, Pisabarro AG, Martínez AT
Fungal Gen. Biol., doi: 10.1016/j.fgb.2014.02.003

Pleurotus ostreatus is an important edible mushroom and a model lignin degrading organism, whose genome contains nine genes of ligninolytic peroxidases, characteristic of white-rot fungi. These genes encode six manganese peroxidase (MnP) and three versatile peroxidase (VP) isoenzymes. Using liquid chromatography coupled to tandem mass spectrometry, secretion of four of these peroxidase isoenzymes (VP1, VP2, MnP2 and MnP6) was confirmed when P. ostreatus grows in a lignocellulose medium at 25°C (three more isoenzymes were identified by only one unique peptide). Then, the effect of environmental parameters on the expression of the above nine genes was studied by reverse transcription-quantitative PCR by changing the incubation temperature and medium pH of P. ostreatus cultures pre-grown under the above conditions (using specific primers and two reference genes for result normalization). The cultures maintained at 25°C (without pH adjustment) provided the highest levels of peroxidase transcripts and the highest total activity on Mn2+ (a substrate of both MnP and VP) and Reactive Black 5 (a VP specific substrate). The global analysis of the expression patterns observed divides peroxidase genes into three main groups according to the level of expression at optimal conditions (vp1/mnp3>vp2/vp3/mnp1/mnp2/mnp6>mnp4/mnp5). Decreasing or increasing the incubation temperature (to 10°C or 37°C) and adjusting the culture pH to acidic or alkaline conditions (pH 3 and 8) generally led to downregulation of most of the peroxidase genes (and decrease of the enzymatic activity), as shown when the transcription levels were referred to those found in the cultures maintained at the initial conditions. Temperature modification produced less dramatic effects than pH modification, with most genes being downregulated during the whole 10°C treatment, while many of them were alternatively upregulated (often 6h after the thermal shock) and downregulated (12h) at 37°C. Interestingly, mnp4 and mnp5 were the only peroxidase genes upregulated under alkaline pH conditions. The differences in the transcription levels of the peroxidase genes when the culture temperature and pH parameters were changed suggest an adaptive expression according to environmental conditions. Finally, the intracellular proteome was analyzed, under the same conditions used in the secretomic analysis, and the protein product of the highly-transcribed gene mnp3 was detected. Therefore, it was concluded that the absence of MnP3 from the secretome of the P. ostreatus lignocellulose cultures was related to impaired secretion.

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