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2 results on '"Brás JLA"'

1. Directed evolution of the type C feruloyl esterase from Fusarium oxysporum FoFaeC and molecular docking analysis of its improved variants

Author

Peter Jütten, Joana L. A. Brás, Io Antonopoulou, Ulrika Rova, Simona Varriale, Sophie Bozonnet, Gabriella Cerullo, Paul Christakopoulos, Olga Gherbovet, Vincenza Faraco, Régis Fauré, Alexander Piechot, Carlos M. G. A. Fontes, Department of Chemical Sciences, University of Naples Federico II, Napoli, Italy, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Luleå University of Technology (LUT), Taros Chemicals GmbH & Co. KG, NZYTech Genes & Enzymes, Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), European Project: 613868,EC:FP7:KBBE,FP7-KBBE-2013-7-single-stage,OPTIBIOCAT(2013), University of Naples Federico II = Università degli studi di Napoli Federico II, Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Cerullo, G, Varriale, S, Bozonnet, S, Antonopoulou, I, Christakopoulos, P, Rova, U, Gherbovet, O, Fauré, R, Piechot, A, Jütten, P, Brás, Jla, Fontes, Cmga, and Faraco, V.

Subjects
0106 biological sciences, Library, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, bioconversion, High-throughput screening, Bioengineering, Biotechnologies, 01 natural sciences, Polymerase Chain Reaction, 03 medical and health sciences, Fusarium, Feruloyl esterase, 010608 biotechnology, Fusarium oxysporum, Molecular Biology, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, biology, Chemistry, Molecular Docking Analysis, General Medicine, biology.organism_classification, Directed evolution, Molecular Docking Simulation, Directed evolutionHigh-throughput screeningFusarium oxysporumLibraryFeruloyl esterase, Enzyme, Biochemistry, Directed Molecular Evolution, Carboxylic Ester Hydrolases, Biotechnology
Abstract

International audience; The need to develop competitive and eco-friendly processes in the cosmetic industry leads to the search for new enzymes with improved properties for industrial bioconversions in this sector. In the present study, a complete methodology to generate, express and screen diversity for the type C feruloyl esterase from Fusarium oxysporium FoFaeC was set up in a high-throughput fashion. A library of around 30,000 random mutants of FoFaeC was generated by error prone PCR of fofaec cDNA and expressed in Yarrowia lipolytica. Screening for enzymatic activity towards the substrates 5-bromo-4-chloroindol-3-yl and 4-nitrocatechol-1-yl ferulates allowed the selection of 96 enzyme variants endowed with improved enzymatic activity that were then characterized for thermo- and solvent- tolerance. The five best mutants in terms of higher activity, thermo- and solvent- tolerance were selected for analysis of substrate specificity. Variant L432I was shown to be able to hydrolyze all the tested substrates, except methyl sinapate, with higher activity than wild type FoFaeC towards methyl p-coumarate, methyl ferulate and methyl caffeate. Moreover, the E455D variant was found to maintain completely its hydrolytic activity after two hour incubation at 55 °C, whereas the L284Q/V405I variant showed both higher thermo- and solvent- tolerance than wild type FoFaeC. Small molecule docking simulations were applied to the five novel selected variants in order to examine the binding pattern of substrates used for enzyme characterization of wild type FoFaeC and the evolved variants.

Published
2019
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2. Evolution of the feruloyl esterase MtFae1a from Myceliophthora thermophila towards improved catalysts for antioxidants synthesis

Author

Joana L. A. Brás, Paul Christakopoulos, Carlos M. G. A. Fontes, Thierry Tron, Vincenza Faraco, Simona Varriale, Ulrika Rova, Régis Fauré, Io Antonopoulou, Alexander Piechot, Peter Jütten, Gabriella Cerullo, Department of Civil Environmental and Natural Resources Engineering, Division of Sustainable Process Engineering, Luleå University of Technology (LUT), Institut des Sciences Moléculaires de Marseille (ISM2), Centre National de la Recherche Scientifique (CNRS)-École Centrale de Marseille (ECM)-Aix Marseille Université (AMU), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Aix Marseille Université (AMU)-École Centrale de Marseille (ECM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), European Project: 613868,EC:FP7:KBBE,FP7-KBBE-2013-7-single-stage,OPTIBIOCAT(2013), Varriale, S, Cerullo, G, Antonopoulou, I, Christakopoulos, P, Rova, U, Tron, Thierry, Fauré, R, Jütten, P, Piechot, A, Brás, Jla, Fontes, Cmga, and Faraco, V

Subjects
0301 basic medicine, High-throughput screening, Saccharomyces cerevisiae, Sordariales, [CHIM.INOR]Chemical Sciences/Inorganic chemistry, Applied Microbiology and Biotechnology, high-throughput screening, Antioxidants, Evolution, Molecular, 03 medical and health sciences, Feruloyl esterase, feruloyl esterase, directed evolution, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], ComputingMilieux_MISCELLANEOUS, chemistry.chemical_classification, biology, Chemistry, library, General Medicine, [CHIM.CATA]Chemical Sciences/Catalysis, biology.organism_classification, Hydroxycinnamic acid, Directed evolution, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], 030104 developmental biology, Enzyme, Biochemistry, Docking (molecular), Carboxylic Ester Hydrolases, Myceliophthora thermophila, Biotechnology, Protein Binding
Abstract

International audience; The chemical syntheses currently employed for industrial purposes, including in the manufacture of cosmetics, present limitations such as unwanted side reactions and the need for harsh chemical reaction conditions. In order to overcome these drawbacks, novel enzymes are developed to catalyze the targeted bioconversions. In the present study, a methodology for the construction and the automated screening of evolved variants library of a Type B feruloyl esterase from Myceliophthora thermophila (MtFae1a) was developed and applied to generation of 30,000 mutants and their screening for selecting the variants with higher activity than the wild-type enzyme. The library was generated by error-prone PCR of mtfae1a cDNA and expressed in Saccharomyces cerevisiae. Screening for extracellular enzymatic activity towards 4-nitrocatechol-1-yl ferulate, a new substrate developed ad hoc for high-throughput assays of feruloyl esterases, led to the selection of 30 improved enzyme variants. The best four variants and the wild-type MtFae1a were investigated in docking experiments with hydroxycinnamic acid esters using a model of 3D structure of MtFae1a. These variants were also used as biocatalysts in transesterification reactions leading to different target products in detergentless microemulsions and showed enhanced synthetic activities, although the screening strategy had been based on improved hydrolytic activity.

Published
2018
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