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  • 1.
    Antonopoulou, Io
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Development of biocatalytic processes for selective antioxidant production2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Feruloyl esterases (FAEs, EC 3.1.1.73) represent a subclass of carboxylic acid esterases that under normal conditions catalyze the hydrolysis of the ester bond between hydroxycinnamic acids (ferulic acid, sinapic acid, caffeic acid, p-coumaric acid) and sugar residues in plant cell walls. Based on their specificity towards monoferulates and diferulates, substitutions on the phenolic ring and on their amino acid sequence identity, they have been classified into four types (A-D) while phylogenetic analysis has resulted in classification into thirteen subfamilies (SF1-13). Under low water content, these enzymes are able to catalyze the esterification of hydroxycinnamic acids or the transesterification of their esters (donor) with alcohols or sugars (acceptor) resulting in compounds with modified lipophilicity, having a great potential for use in the tailor-made modification of natural antioxidants for cosmetic, cosmeceutical and pharmaceutical industries. The work described in this thesis focused on the selection,characterization and application of FAEs for the synthesis of bioactive esters with antioxidant activity in non-conventional media. The basis of the current classification systems was investigated in relation with the enzymes’ synthetic and hydrolytic abilities while the developed processes were evaluated for their efficiency and sustainability.

    Paper I was dedicated to the screening and evaluation of the synthetic abilities of 28 fungal FAEs using acceptors of different lipophilicity at fixed conditions in detergentless microemulsions. It was revealed that FAEs classified in phylogenetic subfamilies related to acetyl xylan esterases (SF5 and 6) showed increased transesterification rates and selectivity. In general, FAEs showed preference on more hydrophilic alcohol acceptors and in descending order to glycerol > 1-butanol > prenol. Homology modeling and small molecule docking simulations were employed as tools for the identification of a potential relationship between the predicted surface and active site properties of selected FAEs and the transesterification selectivity.

    Papers II- IV focused on the characterization of eight promising FAEs and the optimization of reaction conditions for the synthesis of two bioactive esters (prenyl ferulate and L-arabinose ferulate) in detergentless microemulsions. The effect of the medium composition, the donor and acceptor concentration, the enzyme load, the pH, the temperature and the agitation on the transesterification yield and selectivity were investigated. It was observed that the acceptor concentration and enzyme load were crucial parameters for selectivity. Fae125 (Type A, SF5) iiexhibited highest prenyl ferulate yield (81.1%) and selectivity (4.685) converting 98.5% of VFA to products after optimization at 60 mM VFA, 1.5 M prenol, 0.04 mg FAE mL-1, 40oC, 24 h, 53.4:43.4:3.2 v/v/v n-hexane: t-butanol: 100 mM MOPS-NaOH pH 8.0. On the other hand, FaeA1 (Type A, SF5) showed highest L-arabinose ferulate yield (52.2 %) and selectivity (1.120) at 80 mM VFA, 55 mM L-arabinose, 0.02 mg FAE mL-1, 50oC, 8 h, 19.8: 74.7: 5.5 v/v/v n-hexane: t-butanol: 100 mM MOPS-NaOH pH 8.0.

    In paper V, the effect of reaction media on the enzyme stability and transesterification yield and selectivity was studied in different solvents for the synthesis of two bioactive esters: prenyl ferulate and L-arabinose ferulate. The best performing enzyme (Fae125) was used in the optimization of reaction conditions in the best solvent (n-hexane) via response surface methodology. Both bioconversions were best described by a two-factor interaction model while optimal conditions were determined as the ones resulting in highest yield and selectivity.Highest prenyl ferulate yield (87.5%) and selectivity (7.616) were observed at 18.56 mM prenol mM-1VFA, 0.04 mg FAE mL-1, 24.5 oC, 24.5 h, 91.8: 8.2 v/v n-hexane: 100 mM sodium acetate pH 4.7. Highest L-arabinose ferulate yield (56.2%) and selectivity (1.284) were observed at 2.96 mM L-arabinose mM-1VFA, 0.02 mg FAE mL-1, 38.9 oC, 12 h, 90.5: 5.0: 4.5 v/v/v n-hexane: dimethyl sulfoxide: 100 mM sodium acetate pH 4.7. The enzyme could be reused for six consecutive reaction cycles maintaining 66.6% of its initial synthetic activity. The developed bioconversions showed exceptional biocatalyst productivities (> 300 g product g-1FAE) and the waste production was within the range of pharmaceutical processes.

    Paper VI focused on the investigation of the basis of the type A classification of a well-studied FAE from Aspergillus niger(AnFaeA) by comparing its activity towards methyl and arabinose hydroxycinnamic acid esters. For this purpose, L-arabinose ferulateand caffeate were synthesized enzymatically. kcat/Kmratios revealed that AnFaeA hydrolyzed arabinose ferulate 1600 times and arabinose caffeate 6.5 times more efficiently than methyl esters. This study demonstrated that short alkyl chain hydroxycinnamate esters which are used nowadays for FAE classification can lead to activity misclassification, while L-arabinose esters could potentially substitute synthetic esters in classification describing more adequately the enzyme specificitiesin the natural environment.

  • 2.
    Antonopoulou, Io
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Use of feruloyl esterases for chemoenzymatic synthesis of bioactive compounds2017Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    Feruloyl esterases (FAEs, EC 3.1.1.73) represent a subclass of carboxylic acid esterases that under normal conditions catalyze the hydrolysis of the ester bond between hydroxycinnamic acids (ferulic acid, sinapic acid, caffeic acid, p-coumaric acid) and arabinose residues in plant cell walls. Based on their specificity towards monoferulates and diferulates, substitutions on the phenolic ring and on their amino acid sequence identity, they have been classified into four types (A-D). The use of FAEs as accessory enzymes for the degradation of lignocellulosic biomass and their synergism with other hemicellulases has been studied for application in many industries, such as the food, the biofuel and the paper pulp industry. In the recent years, the use of FAEs as biosynthetic tools has been underlined. Under low water content, these enzymes are able to catalyze the esterification of hydroxycinnamic acids or the transesterification of their esters resulting in compounds with altered lipophilicity, revealing a great potential for tailor-made modification of natural antioxidants for use in cosmetic, cosmeceutical and pharmaceutical industries.

    The first part of the thesis is focused on the optimization of reaction conditions for the synthesis of two bioactive esters: prenyl ferulate and L-arabinose ferulate using 5 FAEs (FaeA1, FaeA2, FaeB1, FaeB2 and MtFae1a) from Myceliophthora thermophila 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 the agitation. Regarding the synthesis of prenyl ferulate, FaeB2 offered the highest transesterification yield (71.5±0.2%) after 24 h of incubation at 30oC using 60 mM vinyl ferulate (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 hydrolysis was 4-7 fold minimized. Regarding the synthesis of L-arabinose ferulate, FaeA1 offered highest transesterification yield (35.9±2.96%) after 8 h of incubation at 50oC using 80 mM VFA, 55 mM L-arabinose and 0.02 mg FAE/mL in a mixture of 19.8: 74.7: 5.5 v/v/v n-hexane: t-butanol: 100 mM MOPS-NaOH pH 8.0. It was revealed that the type B FAEs from M. thermophila show higher preference to more lipophilic acceptors like prenol, while the type A FaeA1 was more efficient in the synthesis of the more hydrophilic L-arabinose ferulate.

    The second part of the thesis is focused on the investigation of the basis of the type A classification of a well-studied FAE from Aspergillus niger (AnFaeA) by comparing its activity towards methyl and arabinose hydroxycinnamate esters. For this purpose, L-arabinose ferulate and caffeate were synthesized enzymatically. kcat/Km ratios revealed that AnFaeA hydrolyzed arabinose ferulate 1600 times and arabinose caffeate 6.5 times more efficiently than methyl esters. This study demonstrated that short alkyl chain hydroxycinnamate esters which are used nowadays for FAE classification can lead to activity misclassification, while L-arabinose esters could potentially substitute synthetic esters in classification.

  • 3.
    Antonopoulou, Io
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Dilokpimol, Adiphol
    Fungal Physiology, Westerdijk Fungal Biodiversity Institute & Fungal Molecular Physiology, Utrecht University.
    Iancu, Laura
    Dupont Industrial Biosciences.
    Mäkelä, Miia R.
    Department of Microbiology, University of Helsink.
    Varriale, Simona
    Department of Chemical Sciences, University of Naples “Federico II”.
    Cerullo, Gabriella
    Department of Chemical Sciences, University of Naples “Federico II”.
    Hüttner, Silvia
    Department of Biology and Biological Engineering, Division of Industrial Biotechnology, Chalmers University of Technology.
    Uthoff, Stefan
    Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster.
    Jütten, Peter
    Taros Chemicals GmbH & Co KG.
    Piechot, Alexander
    Taros Chemicals GmbH & Co KG.
    Steinbüchel, Alexander
    nstitut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster.
    Olsson, Lisbeth
    Department of Biology and Biological Engineering, Division of Industrial Biotechnology, Chalmers University of Technology.
    Faraco, Vincenza
    Department of Chemical Sciences, University of Naples “Federico II”.
    Hildén, Kristiina
    Department of Microbiology, University of Helsinki.
    de Vries, Ronald
    Fungal Physiology, Westerdijk Fungal Biodiversity Institute & Fungal Molecular Physiology, Utrecht University.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    The Synthetic Potential of Fungal Feruloyl Esterases: A Correlation with Current Classification Systems and Predicted Structural Properties2018In: Catalysts, ISSN 2073-4344, Vol. 8, no 6, article id 242Article in journal (Refereed)
    Abstract [en]

    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.

  • 4.
    Antonopoulou, Io
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Hunt, Cameron
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Cerullo, Gabriella
    Department of Chemical Sciences, University of Naples "Federico II".
    Varriale, Simona
    Department of Chemical Sciences, University of Naples “Federico II”.
    Gerogianni, Alexandra
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Faraco, Vincenza
    Department of Chemical Sciences, University of Naples "Federico II".
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Tailoring the specificity of the type C feruloyl esterase FoFaeC from Fusarium oxysporum towards methyl sinapate by rational redesign based on small molecule docking simulations2018In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 5, article id e0198127Article in journal (Refereed)
    Abstract [en]

    The type C feruloyl esterase FoFaeC from Fusarium oxysporum is a newly discovered enzyme with high potential for use in the hydrolysis of lignocellulosic biomass but it shows low activity towards sinapates. In this work, small molecule docking simulations were employed in order to identify important residues for the binding of the four model methyl esters of hydroxycinnamic acids, methyl ferulate/caffeate/sinapate/p-coumarate, to the predicted structure of FoFaeC. Subsequently rational redesign was applied to the enzyme’ active site in order to improve its specificity towards methyl sinapate. A double mutation (F230H/T202V) was considered to provide hydrophobic environment for stabilization of the methoxy substitution on sinapate and a larger binding pocket. Five mutant clones and the wild type were produced in Pichia pastoris and biochemically characterized. All clones showed improved activity, substrate affinity, catalytic efficiency and turnover rate compared to the wild type against methyl sinapate, with clone P13 showing a 5-fold improvement in catalytic efficiency. Although the affinity of all mutant clones was improved against the four model substrates, the catalytic efficiency and turnover rate decreased for the substrates containing a hydroxyl substitution.

  • 5.
    Antonopoulou, Io
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Iancu, Laura
    Dupont Industrial Biosciences, Wageningen, the Netherlands.
    Jütten, Peter
    Taros Chemicals GmbH & Co KG, Dortmund, Germany.
    Piechot, Alexander
    Taros Chemicals GmbH & Co KG, Dortmund, Germany.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Optimized Enzymatic Synthesis of Feruloyl Derivatives Catalyzed by Three Novel Feruloyl Esterases from Talaromyces wortmannii in Detergentless Microemulsions2018In: Computational and Structural Biotechnology Journal, ISSN 2001-0370, p. 361-369Article in journal (Refereed)
    Abstract [en]

    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.

  • 6.
    Antonopoulou, Io
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Iancu, Laura
    Dupont Industrial Biosciences.
    Jütten, Peter
    Taros Chemicals GmbH & Co KG.
    Piechot, Alexander
    Taros Chemicals GmbH & Co KG.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Screening of novel feruloyl esterases from Talaromyces wortmannii for the development of efficient and sustainable syntheses of feruloyl derivatives2019In: Enzyme and microbial technology, ISSN 0141-0229, E-ISSN 1879-0909, Vol. 120, p. 124-135Article in journal (Refereed)
    Abstract [en]

    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.

  • 7.
    Antonopoulou, Io
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Leonov, Laura
    DuPont Industrial Biosciences.
    Jûtten, Peter
    Taros Chemicals GmbH & Co.
    Cerullo, Gabriella
    Department of Chemical Sciences, University of Naples "Federico II".
    Faraco, Vincenza
    Department of Chemical Sciences, University of Naples "Federico II".
    Papadopoulou, Adamantia
    Institute of Biosciences and Applications NCSR "Demokritos," Laboratory of Cell Proliferation and Aging.
    Kletsas, Dimitris
    Institute of Biosciences and Applications NCSR "Demokritos," Laboratory of Cell Proliferation and Aging.
    Ralli, Marianna
    Korres Natural Products.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Correction to: Optimized synthesis of novel prenyl ferulate performed by feruloyl esterases from Myceliophthora thermophila in microemulsions2018In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 102, no 1, p. 511-511Article in journal (Refereed)
  • 8.
    Antonopoulou, Io
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Leonov, Laura
    DuPont Industrial Biosciences.
    Jûtten, Peter
    Taros Chemicals GmbH & Co.
    Cerullo, Gabriella
    Department of Chemical Sciences, University of Naples "Federico II".
    Faraco, Vincenza
    Department of Chemical Sciences, University of Naples "Federico II".
    Papadopoulou, Adamantia
    Institute of Biosciences and Applications NCSR "Demokritos," Laboratory of Cell Proliferation and Aging.
    Kletsas, Dimitris
    Institute of Biosciences and Applications NCSR "Demokritos," Laboratory of Cell Proliferation and Aging.
    Ralli, Marianna
    Korres Natural Products.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Optimized synthesis of novel prenyl ferulate performed by feruloyl esterases from Myceliophthora thermophila in microemulsions2017In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 101, no 8, p. 3213-3226Article in journal (Refereed)
    Abstract [en]

    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.

  • 9.
    Antonopoulou, Io
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Papadopoulou, Adamantia
    Laboratory of Cell Proliferation & Ageing, Institute of Biosciences & Applications NCSR “Demokritos”, T. Patriarchou Grigoriou & Neapoleos.
    Iancu, Laura
    DuPont Industrial Biosciences.
    Cerullo, Gabriella
    Department of Chemical Sciences, University of Naples "Federico II".
    Ralli, Marianna
    Korres Natural Products.
    Jûtten, Peter
    Taros Chemicals GmbH & Co.
    Piechot, Alexander
    Taros Chemicals GmbH & Co.
    Faraco, Vincenza
    Department of Chemical Sciences, University of Naples "Federico II".
    Kletsas, Dimitris
    Institute of Biosciences and Applications NCSR "Demokritos," Laboratory of Cell Proliferation and Aging.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    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 activities2018In: Process Biochemistry, ISSN 1359-5113, E-ISSN 1873-3298, Vol. 65, p. 100-108Article in journal (Refereed)
    Abstract [en]

    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.

  • 10.
    Antonopoulou, Io
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Spanopoulos, Athanasios
    Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 5 Iroon Polytechniou Str, Zografou Campus, Athens, Greece.
    Matsakas, Leonidas
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Single cell oil and ethanol production by the oleaginous yeast Trichosporon fermentans utilizing dried sweet sorghum stalks2020In: Renewable energy, ISSN 0960-1481, E-ISSN 1879-0682, Vol. 146, p. 1609-1617Article in journal (Refereed)
    Abstract [en]

    The ability of the oleaginous yeast Trichosporon fermentans to efficiently produce lipids when cultivated in dried sweet sorghum was evaluated. First, lipid production was evaluated in synthetic media mimicking the composition of sweet sorghum stalks and optimized based on the nitrogen source and C: N ratio. Under optimum conditions, the lipid production reached 3.66 g/L with 21.91% w/w lipid content by using a mixture of sucrose, glucose and fructose and peptone at C: N ratio 160. Cultivation on pre-saccharified sweet sorghum stalks offered 1.97 g/L, while it was found that sweet sorghum stalks can support yeast growth and lipid production without the need for external nitrogen source addition. At an attempt to increase the carbon source concentration for optimizing lipid production, the Crabtree effect was observed in T. fermentans. To this end, the yeast was evaluated for its potential to produce ethanol under anaerobic conditions in synthetic media and sweet sorghum. The ethanol concentration at 100 g/L glucose was 40.31 g/L, while utilizing sweet sorghum by adding a distinct saccharification step and external nitrogen source offered ethanol concentration equal to 23.5 g/L. To the authors’ knowledge, this is the first time that the Crabtree effect is observed in T. fermentans.

  • 11.
    Antonopoulou, Io
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Varriale, Simona
    Department of Chemical Sciences, University of Naples "Federico II".
    Topakas, Evangelos
    National Technical University of Athens, School of Chemical Engineering, National Technical University of Athens, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Faraco, Voncenza
    Department of Chemical Sciences, University of Naples "Federico II".
    Enzymatic synthesis of bioactive compounds with high potential for cosmeceutical application2016In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 100, no 15, p. 6519-6543Article in journal (Refereed)
    Abstract [en]

    Cosmeceuticals are cosmetic products containing biologically active ingredients purporting to offer a pharmaceutical therapeutic benefit. The active ingredients can be extracted and purified from natural sources (botanicals, herbal extracts, or animals) but can also be obtained biotechnologically by fermentation and cell cultures or by enzymatic synthesis and modification of natural compounds. A cosmeceutical ingredient should possess an attractive property such as anti-oxidant, anti-inflammatory, skin whitening, anti-aging, anti-wrinkling, or photoprotective activity, among others. During the past years, there has been an increased interest on the enzymatic synthesis of bioactive esters and glycosides based on (trans)esterification, (trans)glycosylation, or oxidation reactions. Natural bioactive compounds with exceptional theurapeutic properties and low toxicity may offer a new insight into the design and development of potent and beneficial cosmetics. This review gives an overview of the enzymatic modifications which are performed currently for the synthesis of products with attractive properties for the cosmeceutical industry

  • 12.
    Cerullo, Gabriella
    et al.
    University of Naples “Federico II”, Naples, Italy.
    Varriale, Simona
    University of Naples “Federico II”, Naples, Italy.
    Bozonnet, Sophie
    Université de Toulouse, Toulouse, France.
    Antonopoulou, Io
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Gherbovet, Olga
    Université de Toulouse, Toulouse, France.
    Fauré, Régis
    Université de Toulouse, Toulouse, France.
    Piechot, Alexander
    Taros Chemicals GmbH & Co. KG, Dortmund, Germany.
    Jütten, Peter
    Taros Chemicals GmbH & Co. KG, Dortmund, Germany.
    Brás, Joana L.A.
    NzyTech LDA, Lisbon, Portugal.
    Fontes, Carlos M.G.A.
    NzyTech LDA, Lisbon, Portugal.
    Faraco, Vincenza
    University of Naples “Federico II”, Naples, Italy.
    Directed evolution of the type C feruloyl esterase from Fusarium oxysporum FoFaeC and molecular docking analysis of its improved variants2019In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 51, p. 14-20Article in journal (Refereed)
    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.

  • 13.
    Christakopoulos, Paul
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Antonopoulou, Io
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Topakas, Evangelos
    National Technical University of Athens, School of Chemical Engineering, National Technical University of Athens, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Synthesis of biological active compounds using carbohydrate esterases as biocatalysts2014In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 31, no Supplement, p. S90-S91Article in journal (Refereed)
    Abstract [en]

    Various fungal and bacterial carbohydrate esterases represent appealing biocatalysts that have the ability not only to deconstruct plant biomass but also to modify compounds with a potential use in food, cosmetic and pharmaceutical industries. Feruloyl esterases (FAEs, E.C. 3.1.1.73) have been proved promising candidates for the enzymatic synthesis of antioxidants allowing more flexible process configurations. Among the advantages they provide are use of lower temperatures (50-60 °C) comparing to the counterpart chemical process (150οC), one step production of one product instead of mixtures and no need of by-product and catalyst residues removal in order to produce clean and high quality substances. Glucuronoyl esterase (GE) synthetic ability needs to be explored towards the production of alkyl branched glucuronic acid derivatives which are non-ionic surfactants and have good surface properties, including biodegradability. In addition, due to their tastelessness, non skin-irritation and non toxicity, these bioactive compounds find diverse uses in the cosmetic and pharmaceutical industries.Aim of this work is the development of competitive and eco-friendly bioconversions based on transesterification reactions catalyzed by FAEs and GEs, for the production of molecules with antioxidant activity, such as phenolic fatty and sugar esters. The synthesis of four biological active compounds (prenyl ferulate, prenyl caffeate, 5-O-(trans-feruloyl)-arabinofuranose, and glyceryl ferulate) was evaluated using recombinant FAEs from Myceliopthora thermophila and Fusarium oxysporum, while the synthesis of benzyl D-glucuronate and prenyl-D-glucuronate was evaluated using recombinant GEs from M. thermophila. All reactions were carried out in ternary systems of n-hexane/alcohol/water forming surfactantless microemulsions.

  • 14.
    Hunt, Cameron J
    et al.
    Department of Chemical Engineering, Monash University, Clayton, 3800, Victoria, Australia..
    Antonopoulou, Io
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Tanksale, Akshat
    Department of Chemical Engineering, Monash University, Clayton, 3800, Victoria, Australia..
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Haritos, Victoria S
    Department of Chemical Engineering, Monash University, Clayton, 3800, Victoria, Australia.
    Insights into substrate binding of ferulic acid esterases by arabinose and methyl hydroxycinnamate esters and molecular docking2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, no 1, article id 17315Article in journal (Refereed)
    Abstract [en]

    Ferulic acid esterases (FAE, EC 3.1.1.73) cleave the arabinose hydroxycinnamate ester in plant hemicellulose and other related substrates. FAE are commonly categorised as type A-D based on catalytic activities towards model, short alkyl chain esters of hydroxycinnamates. However, this system correlates poorly with sequence and structural features of the enzymes. In this study, we investigated the basis of the type A categorisation of an FAE from Aspergillus niger, AnFaeA, by comparing its activity toward methyl and arabinose hydroxycinnamate esters. kcat/Km ratios revealed that AnFaeA hydrolysed arabinose ferulate 1600-fold, and arabinose caffeate 6.5 times more efficiently than their methyl ester counterparts. Furthermore, small docking studies showed that while all substrates adopted a catalytic orientation with requisite proximity to the catalytic serine, methyl caffeate and methyl p-coumarate preferentially formed alternative non-catalytic conformations that were energetically favoured. Arabinose ferulate was unable to adopt the alternative conformation while arabinose caffeate preferred the catalytic orientation. This study demonstrates that use of short alkyl chain hydroxycinnnamate esters can result in activity misclassification. The findings of this study provide a basis for developing a robust classification system for FAE and form the basis of sequence-function relationships for this class.

  • 15.
    Karnaouri, Anthi C
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Antonopoulou, Io
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Topakas, Evangelos
    National Technical University of Athens.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Genomic insights into the fungal lignocellulolytic system of Myceliophthora thermophila2014In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 5, article id 5.281Article in journal (Refereed)
    Abstract [en]

    he microbial conversion of solid cellulosic biomass to liquid biofuels may provide a renewable energy source for transportation fuels. Cellulolytic fungi represent a promising group of organisms, as they have evolved complex systems for adaptation to their natural habitat. The filamentous fungus Myceliophthora thermophila constitutes an exceptionally powerful cellulolytic microorganism that synthesizes a complete set of enzymes necessary for the breakdown of plant cell wall. The genome of this fungus has been recently sequenced and annotated, allowing systematic examination and identification of enzymes required for the degradation of lignocellulosic biomass. The genomic analysis revealed the existence of an expanded enzymatic repertoire including numerous cellulases, hemicellulases and enzymes with auxiliary activities, covering the most of the recognized CAZy families. Most of them were predicted to possess a secretion signal and undergo through post translational glycosylation modifications. These data offer a better understanding of activities embedded in fungal lignocellulose decomposition mechanisms and suggest that M. thermophila could be made usable as an industrial production host for cellulolytic and hemicellulolytic enzymes

  • 16.
    Karnaouri, Anthi C.
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Antonopoulou, Io
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Zerva, Anastasia
    Biotechnology Laboratory, Department of Synthesis and Development of Industrial Processes, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Dimarogona, Maria
    Section of Process and Environmental Engineering, Department of Chemical Engineering, University of Patras, Patras, Greece.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering. Biotechnology Laboratory, Department of Synthesis and Development of Industrial Processes, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Thermophilic enzyme systems for efficient conversion of lignocellulose to valuable products: Structural insights and future perspectives for esterases and oxidative catalysts2019In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 279, p. 362-372Article in journal (Refereed)
    Abstract [en]

    Thermophilic enzyme systems are of major importance nowadays in all industrial processes due to their great performance at elevated temperatures. In the present review, an overview of the current knowledge on the properties of thermophilic and thermotolerant carbohydrate esterases and oxidative enzymes with great thermostability is provided, with respect to their potential use in biotechnological applications. A special focus is given to the lytic polysaccharide monooxygenases that are able to oxidatively cleave lignocellulose through the use of oxygen or hydrogen peroxide as co-substrate and a reducing agent as electron donor. Structural characteristics of the enzymes, including active site conformation and surface properties are discussed and correlated with their substrate specificity and thermostability properties.

  • 17.
    Katsimpouras, Constantinos
    et al.
    Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Antonopoulou, Io
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Topakas, Evangelos
    National Technical University of Athens, School of Chemical Engineering, National Technical University of Athens, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Role and Applications of Feruloyl Esterases in Biomass Bioconversion2016In: Microbial Enzymes in Bioconversions of Biomass / [ed] Vijai Kumar Gupta, Springer international , 2016, p. 79-123Chapter in book (Refereed)
    Abstract [en]

    Ferulic acid esterases (FAEs) act synergistically with xylanases to hydrolyze the feruloylated decorations of the hemicellulosic fraction of cell wall material and therefore play a major role in the degradation of plant biomass. In this review, their role in plant biomass degradation, their production, classification, and structural determination are discussed. In addition, the production, physicochemical properties, and molecular biology of the different type of FAEs are presented, giving emphasis in their potential applications utilizing their hydrolytic and synthetic activity. A detailed map of the reaction systems used to date is demonstrated, underpinning the potential of these enzymes as biosynthetic tools in the synthesis of bioactive compounds for use in food and cosmeceutical industries.

  • 18.
    Matsakas, Leonidas
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Antonopoulou, Io
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Evaluation of Myceliopthora thermophila as an enzyme factory for the production of thermophilic cellulolytic enzymes2015In: BioResources, ISSN 1930-2126, E-ISSN 1930-2126, Vol. 10, no 3, p. 5140-5158Article in journal (Refereed)
    Abstract [en]

    Enzymatic hydrolysis is a key step in bioethanol production. Efficient hydrolysis requires a consortium of different enzymes that are able to hydrolyze cellulose and hemicellulose into fermentable sugars. Myceliopthora thermophila is a promising candidate for the production of thermophilic cellulolytic enzymes, the use of which could reduce the cost of ethanol production. The growth conditions of the fungus were optimized in order to achieve increased secretion of extracellular cellulases. Optimal conditions were found to be 7.0% w/v brewer’s spent grain as the carbon source and 0.4% w/v ammonium sulfate as the nitrogen source. The cellulases obtained were characterized for their optimum activity. The optimum temperature and pH for cellulase activity are 65 °C and pH 5.5, respectively. Studies on thermal inactivation of the crude extract showed that the cellulases of M. thermophila are stable for temperatures up to 60 °C. At this temperature the half-life was found to be as high as 27 h. Enzymatic hydrolysis of cellulose resulted in 31.4% hydrolysis yield at 60 °C after 24 h of incubation. Finally, the recalcitrance constant for cellulose and cellulose pretreated with ionic liquids was calculated to be 5.46 and 2.69, respectively.

  • 19.
    Patel, Alok
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Antonopoulou, Io
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Enman, Josefine
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Matsakas, Leonidas
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Lipids detection and quantification in oleaginous microorganisms: an overview of the current state of the art2019In: BMC Chemical Engineering, ISSN 2524-4175, Vol. 1, article id 13Article in journal (Refereed)
    Abstract [en]

    Oleaginous microorganisms are among the most promising feedstocks for the production of lipids for biofuels and oleochemicals. Lipids are synthesized in intracellular compartments in the form of lipid droplets. Therefore, their qualitative and quantitative analysis requires an initial pretreatment step that allows their extraction. Lipid extraction techniques vary with the type of microorganism but, in general, the presence of an outer membrane or cell wall limits their recovery. This review discusses the various types of oleaginous microorganisms, their lipid accumulating capabilities, lipid extraction techniques, and the pretreatment of cellular biomass for enhanced lipid recovery. Conventional methods for lipid quantification include gravimetric and chromatographic approaches; whereas non-conventional methods are based on infrared, Raman, nuclear magnetic resonance, and fluorescence spectroscopic analysis. Recent advances in these methods, their limitations, and fields of application are discussed, with the aim of providing a guide for selecting the best method or combination of methods for lipid quantification.

  • 20.
    Varriale, Simona
    et al.
    Department of Chemical Sciences, Complesso Universitario Monte S. Angelo, University of Naples Federico II, Naples, Italy.
    Cerullo, Gabriella
    Department of Chemical Sciences, Complesso Universitario Monte S. Angelo, University of Naples Federico II, Naples, Italy.
    Antonopoulou, Io
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Tron, Thierry
    Centrale Marseille, CNRS, Aix Marseille Université, Marseille, France.
    Fauré, Régis
    LISBP, CNRS, INRA, INSA, Université de Toulouse, Toulouse, France.
    Jütten, Peter
    Taros Chemicals GmbH & Co. KG, Dortmund, Germany.
    Piechot, Alexander
    Taros Chemicals GmbH & Co. KG, Dortmund, Germany.
    Brás, Joana L. A.
    NzyTech LDA, Estrada Do Paco Do Lumiar Campus Do Lumiar Ed., Portugal.
    Fontes, Carlos M. G. A.
    NzyTech LDA, Estrada Do Paco Do Lumiar Campus Do Lumiar Ed., Portugal.
    Faraco, Vincenza
    Department of Chemical Sciences, Complesso Universitario Monte S. Angelo, University of Naples Federico II, Naples, Italy .
    Evolution of the feruloyl esterase MtFae1a from Myceliophthora thermophila towards improved catalysts for antioxidants synthesis2018In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 102, no 12, p. 5185-5196Article in journal (Refereed)
    Abstract [en]

    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.

  • 21.
    Zerva, Anastasia
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Antonopoulou, Io
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Enman, Josefine
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Iancu, Laura
    DuPont Industrial Biosciences, Wageningen, The Netherlands.
    Jütten, Peter
    Taros Chemicals GmbH & Co. KG, Dortmund, Germany.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Optimization of Transesterification Reactions with CLEA-Immobilized Feruloyl Esterases from Thermothelomyces thermophila and Talaromyces wortmannii2018In: Molecules, ISSN 1420-3049, E-ISSN 1420-3049, Vol. 23, no 9, article id 2403Article in journal (Refereed)
    Abstract [en]

    Feruloyl esterases (FAEs, E.C. 3.1.1.73) are biotechnologically important enzymes with several applications in ferulic acid production from biomass, but also in synthesis of hydroxycinnamic acid derivatives. The use of such biocatalysts in commercial processes can become feasible by their immobilization, providing the advantages of isolation and recycling. In this work, eight feruloyl esterases, immobilized in cross-linked enzyme aggregates (CLEAs) were tested in regard to their transesterification performance, towards the production of prenyl ferulate (PFA) and arabinose ferulate (AFA). After solvent screening, comparison with the activity of respective soluble enzymes, and operational stability tests, FAE125 was selected as the most promising biocatalyst. A central composite design revealed the optimum conditions for each transesterification product, in terms of water content, time, and substrate ratio for both products, and temperature and enzyme load additionally for prenyl ferulate. The optimum product yields obtained were 83.7% for PFA and 58.1% for AFA. FAE125 CLEAs are stable in the optimum conditions of transesterification reactions, maintaining 70% residual activity after five consecutive reactions. Overall, FAE125 CLEAs seem to be able to perform as a robust biocatalyst, offering satisfactory yields and stability, and thus showing significant potential for industrial applications.

  • 22.
    Zerva, Anastasia
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Antonopoulou, Io
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Enman, Josefine
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Iancu, Laura
    DuPont Industrial Biosciences, Nieuwe Kanaal 7-S, 6709 PA Wageningen, The Netherlands.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Cross-Linked Enzyme Aggregates of Feruloyl Esterase Preparations from Thermothelomyces thermophila and Talaromyces wortmannii2018In: Catalysts, ISSN 2073-4344, Vol. 8, no 5, article id 208Article in journal (Refereed)
    Abstract [en]

    Cross-linked enzyme aggregates (CLEA®) technology is a well-established method in the current literature for the low-cost and effective immobilization of several enzymes. The main advantage of this particular method is the simplicity of the process, since it consists of only two steps. However, CLEA immobilization must be carefully designed for each desired enzyme, since the optimum conditions for enzymes can vary significantly, according to their physicochemical properties. In the present study, an investigation of the optimum CLEA immobilization conditions was carried out for eight feruloyl esterase preparations. Feruloyl esterases are a very important enzyme group in the valorization of lignocellulosic biomass, since they act in a synergistic way with other enzymes for the breakdown of plant biomass. Specifically, we investigated the type and concentration of precipitant and the crosslinker concentration, for retaining optimal activity. FAE68 was found to be the most promising enzyme for CLEA immobilization, since in this case, the maximum retained activity, over 98%, was observed. Subsequently, we examined the operational stability and the stability in organic solvents for the obtained CLEA preparations, as well as their structure. Overall, our results support that the maximum activity retaining and the stability properties of the final CLEAs can vary greatly in different FAE preparations. Nevertheless, some of the examined FAEs show a significant potential for further applications in harsh industrial conditions.

1 - 22 of 22
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