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| 2. |
- Antonopoulou, Io, et al.
(author)
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Optimization of enzymatic synthesis of l-arabinose ferulate catalyzed by feruloyl esterases from Myceliophthora thermophila in detergentless microemulsions and assessment of its antioxidant and cytotoxicity activities
- 2018
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In: Process Biochemistry. - : Elsevier. - 1359-5113 .- 1873-3298. ; 65, s. 100-108
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Journal article (peer-reviewed)abstract
- The feruloyl esterases FaeA1, FaeA2, FaeB1, FaeB2 from Myceliophthora thermophila C1 and MtFae1a from M. thermophila ATCC 42464 were used as biocatalysts for the transesterification of vinyl ferulate (VFA) with l-arabinose in detergentless microemulsions. The effect of parameters such as the microemulsion composition, the substrate concentration, the enzyme load, the pH, the temperature and the agitation was investigated. FaeA1 offered the highest transesterification yield (52.2 ± 4.3%) after 8 h of incubation at 50 °C using 80 mM VFA, 55 mM l-arabinose and 0.02 mg FAE mL−1 in a mixture comprising of 19.8: 74.7: 5.5 v/v/v n-hexane: t-butanol: 100 mM MOPS-NaOH pH 8.0. The ability of l-arabinose ferulate (AFA) to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals was significant (IC50 386.5 μM). AFA was not cytotoxic even at high concentrations (1 mM) however was found to be pro-oxidant at concentrations higher than 20 μM when the antioxidant activity was determined with the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay in human skin fibroblasts.
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| 3. |
- Antonopoulou, Io, 1989-, et al.
(author)
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Optimized Enzymatic Synthesis of Feruloyl Derivatives Catalyzed by Three Novel Feruloyl Esterases from Talaromyces wortmannii in Detergentless Microemulsions
- 2018
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In: Computational and Structural Biotechnology Journal. - : Elsevier. - 2001-0370. ; , s. 361-369
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Journal article (peer-reviewed)abstract
- Three novel feruloyl esterases (Fae125, Fae7262 and Fae68) from Talaromyces wortmanniioverexpressed in the C1 platform were evaluated for the transesterification of vinyl ferulatewith two acceptors of different size and lipophilicity (prenol and L-arabinose) in detergentless microemulsions. The effect of reaction conditions such as the microemulsion composition, the substrate concentration, the enzyme load, the pH, the temperature and the agitation were investigated. The type A Fae125 belonging to the subfamily 5 (SF5) of phylogenetic classification showed highest yields for the synthesis of both products after optimization of reaction conditions: 81.8% for prenyl ferulate and 33.0% for L-arabinose ferulate. After optimization, an 8-fold increase in the yield and a 12-fold increase in selectivity were achieved for the synthesis of prenyl ferulate.
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| 4. |
- Antonopoulou, Io, et al.
(author)
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Optimized synthesis of novel prenyl ferulate performed by feruloyl esterases from Myceliophthora thermophila in microemulsions
- 2017
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In: Applied Microbiology and Biotechnology. - : Springer. - 0175-7598 .- 1432-0614. ; 101:8, s. 3213-3226
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Journal article (peer-reviewed)abstract
- Five feruloyl esterases (FAEs; EC 3.1.1.73), FaeA1, FaeA2, FaeB1, and FaeB2 from Myceliophthora thermophila C1 and MtFae1a from M. thermophila ATCC 42464, were tested for their ability to catalyze the transesterification of vinyl ferulate (VFA) with prenol in detergentless microemulsions. Reaction conditions were optimized investigating parameters such as the medium composition, the substrate concentration, the enzyme load, the pH, the temperature, and agitation. FaeB2 offered the highest transesterification yield (71.5 ± 0.2%) after 24 h of incubation at 30 °C using 60 mM VFA, 1 M prenol, and 0.02 mg FAE/mL in a mixture comprising of 53.4:43.4:3.2 v/v/v n-hexane:t-butanol:100 mM MOPS-NaOH, pH 6.0. At these conditions, the competitive side hydrolysis of VFA was 4.7-fold minimized. The ability of prenyl ferulate (PFA) and its corresponding ferulic acid (FA) to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was significant and similar (IC50 423.39 μM for PFA, 329.9 μM for FA). PFA was not cytotoxic at 0.8–100 μM (IC50 220.23 μM) and reduced intracellular reactive oxygen species (ROS) in human skin fibroblasts at concentrations ranging between 4 and 20 μM as determined with the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay.
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| 5. |
- Antonopoulou, Io, 1989-, et al.
(author)
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Screening of novel feruloyl esterases from Talaromyces wortmannii for the development of efficient and sustainable syntheses of feruloyl derivatives
- 2019
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In: Enzyme and microbial technology. - : Elsevier. - 0141-0229 .- 1879-0909. ; 120, s. 124-135
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Journal article (peer-reviewed)abstract
- The feruloyl esterases Fae125, Fae7262 and Fae68 from Talaromyces wortmannii were screened in 10 different solvent: buffer systems in terms of residual hydrolytic activity and of the ability for the transesterification of vinyl ferulate with prenol or L-arabinose. Among the tested enzymes, the acetyl xylan-related Fae125 belonging to the phylogenetic subfamily 5 showed highest yield and selectivity for both products in alkane: buffer systems (n-hexane or n-octane). Response surface methodology, based on a 5-level and 6-factor central composite design, revealed that the substrate molar ratio and the water content were the most significant variables for the bioconversion yield and selectivity. The effect of agitation, the possibility of DMSO addition and the increase of donor concentration were investigated. After optimization, competitive transesterification yields were obtained for prenyl ferulate (87.5-92.6%) and L-arabinose ferulate (56.2-61.7%) at reduced reaction times (≤ 24 h) resulting in good productivities (> 1 g/L/h, >300 kg product/kg FAE). The enzyme could be recycled for six consecutive cycles retaining 66.6% of the synthetic activity and 100% of the selectivity.
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| 6. |
- Cerullo, Gabriella, et al.
(author)
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Directed evolution of the type C feruloyl esterase from Fusarium oxysporum FoFaeC and molecular docking analysis of its improved variants
- 2019
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In: New Biotechnology. - : Elsevier. - 1871-6784 .- 1876-4347. ; 51, s. 14-20
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Journal article (peer-reviewed)abstract
- 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.
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| 7. |
- Cerullo, Gabriella, et al.
(author)
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The Synthetic Potential of Fungal Feruloyl Esterases: A Correlation with Current Classification Systems and Predicted Structural Properties
- 2018
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In: Catalysts. - : MDPI AG. - 2073-4344. ; 8:242
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Journal article (peer-reviewed)abstract
- Twenty-eight fungal feruloyl esterases (FAEs) were evaluated for their synthetic abilities in a ternary system of n-hexane: t-butanol: 100 mM MOPS-NaOH pH 6.0 forming detergentless microemulsions. Five main derivatives were synthesized, namely prenyl ferulate, prenyl caffeate, butyl ferulate, glyceryl ferulate, and l-arabinose ferulate, offering, in general, higher yields when more hydrophilic alcohol substitutions were used. Acetyl xylan esterase-related FAEs belonging to phylogenetic subfamilies (SF) 5 and 6 showed increased synthetic yields among tested enzymes. In particular, it was shown that FAEs belonging to SF6 generally transesterified aliphatic alcohols more efficiently while SF5 members preferred bulkier l-arabinose. Predicted surface properties and structural characteristics were correlated with the synthetic potential of selected tannase-related, acetyl-xylan-related, and lipase-related FAEs (SF1-2, -6, -7 members) based on homology modeling and small molecular docking simulations.
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| 8. |
- Hüttner, Silvia, 1984, et al.
(author)
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Fungal Glucuronoyl and Feruloyl Esterases for Wood Processing and Phenolic Acid Ester/Sugar Ester Synthesis
- 2015
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In: Biotrans 2015, Vienna, Austria, 26-30 July 2015.
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Conference paper (other academic/artistic)abstract
- Feruloyl esterases (FAEs, E.C. 3.1.1.73, CAZy family CE1) and glucuronoyl esterases(GEs, E.C. 3.1.1.-, CAZy family CE15) are involved in the degradation of plantbiomass by hydrolysing ester linkages in plant cell walls, and thus have potential use inbiofuel production from lignocellulosic materials and in biorefinery applications withthe aim of developing new wood-based compounds [1, 2]. GEs and FAEs are present inthe genomes of a wide range of fungi and bacteria.Under conditions of low water content, these enzymes can also carry out(trans)esterification reactions, making them promising biocatalysts for the modificationof compounds with applications in the food, cosmetic and pharmaceutical industry.Compared to the chemical process, enzymatic synthesis can be carried out under lowerprocess temperatures (50-60°C) and results in fewer side products, thus reducing theenvironmental impact.We characterised new FAE and GE enzymes from mesophilic, thermophilic and coldtolerantfilamentous fungi produced in Pichia pastoris. The enzymes were characterisedfor both their hydrolytic abilities on various model substrates (methyl ferulate, pNPferulate)- for potential applications in deconstruction of lignocellulosic materials andextraction of valuable compounds - as well as for their biosynthetic capacities. Wetested and optimised the FAEs’ transesterification capabilities on ferulate esters in a 1-butanol-buffer system, with the aim of using the most promising candidates for theproduction of antioxidant compounds with improved hydrophobic or hydrophilicproperties, such as prenyl ferulate, prenyl caffeate, glyceryl ferulate and 5-O-(transferuloyl)-arabinofuranose.
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| 9. |
- Hüttner, Silvia, 1984, et al.
(author)
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Synthesis of antioxidants with free and immobilised fungal feruloyl esterases
- 2016
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In: European Symposium on Biochemical Engineering Sciences, 11-14 Sep 2016, Dublin, Ireland.
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Conference paper (other academic/artistic)abstract
- Feruloyl esterases (FAEs, E.C. 3.1.1.73, CAZy family CE1) are enzymes that are secreted by a wide range of fungi and bacteria as part of the enzymes hydrolysing plant biomass. Under conditions of low water content, FAEs can also carry out (trans)esterification reactions. Thus, their potential use as biocatalysts for the production of antioxidants with applications in food, cosmetic and pharmaceutical industries has been investigated in recent years. We characterised the biosynthetic potential of four new FAE enzymes from a thermophilic fungus. We focused on optimizing reaction conditions for the synthesis of ferulate esters with improved hydrophobic or hydrophilic properties; prenyl ferulate and 5-O-(trans-feruloyl)-arabinofuranose, respectively. In addition to using free enzymes, we also immobilised them on the mesoporous silica material SBA-15 with pore sizes ranging from 7 to 10 nm, to improve the esterification-to-hydrolysis ratio of the enzymes. It has been shown previously that immobilisation renders enzymes more resilient to adverse conditions and increases their productive life time [1]. Furthermore, immobilisation may also result in a decrease of unwanted side reactions (hydrolysis of transesterification) [2]. In agreement with that, we achieved a higher product yield with immobilised enzymes compared to free enzymes. The immobilised biocatalysts are also more easily re-usable for several production cycles, thus lowering production costs.
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| 10. |
- Varriale, Simona, et al.
(author)
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Evolution of the feruloyl esterase MtFae1a from Myceliophthora thermophila towards improved catalysts for antioxidants synthesis
- 2018
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In: Applied Microbiology and Biotechnology. - : Springer. - 0175-7598 .- 1432-0614. ; 102:12, s. 5185-5196
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Journal article (peer-reviewed)abstract
- 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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