Evolution of the feruloyl esterase MtFae1a from Myceliophthora thermophila towards improved catalysts for antioxidants synthesis
Varriale, Simona | Cerullo, Gabriella | Antonopoulou, Io | Christakopoulos, Paul | Rova, Ulrika | Tron, Thierry | Fauré, Régis | Jütten, Peter | Piechot, Alexander | Brás, Joana | Fontes, Carlos | Faraco, Vincenza | 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) ; Aix Marseille Université (AMU)-École Centrale de Marseille (ECM)-Institut de Chimie - CNRS Chimie (INC-CNRS)-Centre National de la Recherche Scientifique (CNRS) | Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP) ; 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)
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Показать больше [+] Меньше [-]Английский. 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.
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