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Directed evolution of the type C feruloyl esterase from Fusarium oxysporum FoFaeC and molecular docking analysis of its improved variants
University of Naples “Federico II”, Naples, Italy.
University of Naples “Federico II”, Naples, Italy.
Université de Toulouse, Toulouse, France.
Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.ORCID iD: 0000-0002-7754-9398
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2019 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 51, p. 14-20Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Elsevier, 2019. Vol. 51, p. 14-20
Keywords [en]
Directed evolution, Feruloyl esterase, Fusarium oxysporum, High-throughput screening, Library
National Category
Bioprocess Technology
Research subject
Biochemical Process Engineering
Identifiers
URN: urn:nbn:se:ltu:diva-72926DOI: 10.1016/j.nbt.2019.01.008ISI: 000461362500003Scopus ID: 2-s2.0-85061183564OAI: oai:DiVA.org:ltu-72926DiVA, id: diva2:1289370
Note

Validerad;2019;Nivå 2;2019-02-18 (svasva)

Available from: 2019-02-18 Created: 2019-02-18 Last updated: 2019-04-12Bibliographically approved

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Antonopoulou, IoChristakopoulos, PaulRova, Ulrika

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