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  • 1.
    Agnantiari, G.
    et al.
    National Technical University of Athens.
    Christakopoulos, Paul
    Kekos, D.
    National Technical University of Athens.
    Macris, B.J.
    National Technical University of Athens.
    A Purified α-galactosidase from aspergillus niger with enhanced kinetic characteristics1991In: Acta Biotechnologica, ISSN 0138-4988, E-ISSN 1521-3846, Vol. 11, no 5, p. 479-484Article in journal (Refereed)
    Abstract [en]

    Extracellular α-galactosidase from Aspergillus niger was purified 128-fold over the crude extract by gel filtration, ion exchange chromatography and chromatofocusing. Certain substrates and end products affected enzyme activity. Among the former p-nitrophenyl-α-galactopyranoside (PNPG) inhibited the enzyme at 1.4 mM while melibiose did not inhibit α-galactosidase at concentrations up to 50 mM. Enzymic end products such as glucose did not inhibit the enzyme at concentrations up to 100 mM while galactose exhibited a competitive inhibition with a Ki = 1.29 mM. The kinetic characteristics of the enzyme compared favourably to other microbial α-galactosidases and make it suitable for food process applications.

  • 2.
    Anasontzis, George E
    et al.
    Industrial Biotechnology, Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden; Wallenberg Wood Science Center, Chalmers University of Technology, Gothenburg, Sweden.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Challenges in ethanol production with Fusarium oxysporum through consolidated bioprocessing2014In: Bioengineered, ISSN 2165-5979, E-ISSN 2165-5987, Vol. 5, no 6, p. 393-395Article in journal (Other (popular science, discussion, etc.))
    Abstract [en]

    Fusarium oxysporum has been reported as being able to both produce the enzymes necessary to degrade lignocellulosic biomass to sugars and also ferment the monosaccharides to ethanol under anaerobic or microaerobic conditions. However, in order to become an economically feasible alternative to other ethanol-producing microorganisms, a better understanding of its physiology, metabolic pathways, and bottlenecks is required, together with an improvement in its efficiency and robustness. In this report, we describe the challenges for the future and give additional justification for our recent publication.

  • 3.
    Anasontzis, George E.
    et al.
    National and Kapodistrian University of Athens, Chalmers University of Technology, Department of Chemical and Biological Engineering, Microbial Biotechnology Unit, Sector of Botany, Department of Biology, National and Kapodistrian University of Athens, Zografou.
    Kourtoglou, Elisavet
    National Technical University of Athens, BIOtechMASS Unit, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Villas-Boâs, Silas G
    Centre for Microbial Innovation, School of Biological Sciences, The University of Auckland, Technical University of Denmark.
    Hatzinikolaou, Dimitris G.
    Department of Chemical Engineering, National Technical University of Athens.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Metabolic Engineering of Fusarium oxysporum to Improve Its Ethanol-Producing Capability2016In: Frontiers in Microbiology, E-ISSN 1664-302X, Vol. 7, article id 632Article in journal (Refereed)
    Abstract [en]

    Fusarium oxysporum is one of the few filamentous fungi capable of fermenting ethanol directly from plant cell wall biomass. It has the enzymatic toolbox necessary to break down biomass to its monosaccharides and, under anaerobic and microaerobic conditions, ferments them to ethanol. Although these traits could enable its use in consolidated processes and thus bypass some of the bottlenecks encountered in ethanol production from lignocellulosic material when Saccharomyces cerevisiae is used-namely its inability to degrade lignocellulose and to consume pentoses-two major disadvantages of F. oxysporum compared to the yeast-its low growth rate and low ethanol productivity-hinder the further development of this process. We had previously identified phosphoglucomutase and transaldolase, two major enzymes of glucose catabolism and the pentose phosphate pathway, as possible bottlenecks in the metabolism of the fungus and we had reported the effect of their constitutive production on the growth characteristics of the fungus. In this study, we investigated the effect of their constitutive production on ethanol productivity under anaerobic conditions. We report an increase in ethanol yield and a concomitant decrease in acetic acid production. Metabolomics analysis revealed that the genetic modifications applied did not simply accelerate the metabolic rate of the microorganism; they also affected the relative concentrations of the various metabolites suggesting an increased channeling toward the chorismate pathway, an activation of the γ-aminobutyric acid shunt, and an excess in NADPH regeneration

  • 4.
    Anasontzis, George
    et al.
    Chalmers University of Technology, Department of Chemical and Biological Engineering.
    Kourtoglou, Elisavet
    BIOtechMASS Unit, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Mamma, Diomi
    BIOtechMASS Unit, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Villas-Boâs, Silas G
    Centre for Microbial Innovation, School of Biological Sciences, The University of Auckland.
    Hatzinikolaou, Dimitris
    Microbial Biotechnology Unit, Sector of Botany, Department of Biology, National and Kapodistrian University of Athens.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Constitutive homologous expression of phosphoglucomutase and transaldolase increases the metabolic flux of Fusarium oxysporum2014In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 13, article id 43Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Fusarium oxysporum is among the few filamentous fungi that have been reported of being able to directly ferment biomass to ethanol in a consolidated bioprocess. Understanding its metabolic pathways and their limitations can provide some insights on the genetic modifications required to enhance its growth and subsequent fermentation capability. In this study, we investigated the hypothesis reported previously that phosphoglucomutase and transaldolase are metabolic bottlenecks in the glycolysis and pentose phosphate pathway of the F. oxysporum metabolism.RESULTS: Both enzymes were homologously overexpressed in F. oxysporum F3 using the gpdA promoter of Aspergillus nidulans for constitutive expression. Transformants were screened for their phosphoglucomutase and transaldolase genes expression levels with northern blot. The selected transformant exhibited high mRNA levels for both genes, as well as higher specific activities of the corresponding enzymes, compared to the wild type. It also displayed more than 20 and 15% higher specific growth rate upon aerobic growth on glucose and xylose, respectively, as carbon sources and 30% higher xylose to biomass yield. The determination of the relative intracellular amino and non-amino organic acid concentrations at the end of growth revealed higher abundance of most determined metabolites between 1.5- and 3-times in the recombinant strain compared to the wild type. Lower abundance of the determined metabolites of the Krebs cycle and an 68-fold more glutamate were observed at the end of the cultivation, when xylose was used as carbon source.CONCLUSIONS: Homologous overexpression of phosphoglucomutase and transaldolase in F. oxysporum was shown to enhance the growth characteristics of the strain in both xylose and glucose in aerobic conditions. The intracellular metabolites profile indicated how the changes in the metabolome could have resulted in the observed growth characteristics.

  • 5.
    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, E-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.

  • 6.
    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, 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.

  • 7.
    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, E-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.

  • 8.
    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.

  • 9.
    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.

  • 10.
    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.

  • 11.
    Antonopoulou, Io
    et al.
    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.
    CO2 to Methanol: A Highly Efficient Enzyme Cascade2022In: Multienzymatic Assemblies: Methods and Protocols / [ed] Haralambos Stamatis, Springer Nature, 2022, 1, p. 317-344Chapter in book (Other academic)
    Abstract [en]

    Carbon dioxide (CO2) has been increasingly regarded not only as a greenhouse gas but also as a valuable feedstock for carbon-based chemicals. In particular, biological approaches have drawn attention as models for the production of value-added products, as CO2 conversion serves many natural processes. Enzymatic CO2 reduction in vitro is a very promising route to produce fossil free and bio-based fuel alternatives, such as methanol. In this chapter, the advances in constructing competitive multi-enzymatic systems for the reduction of CO2 to methanol are discussed. Different integrated methods are presented, aiming to address technological challenges, such as the cost effectiveness, need for material regeneration and reuse and improving product yields of the process.

  • 12.
    Antonopoulou, Io
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Sapountzaki, Eleftheria
    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.
    Ferulic Acid From Plant Biomass: A Phytochemical With Promising Antiviral Properties2022In: Frontiers in Nutrition, E-ISSN 2296-861X, Vol. 8, article id 777576Article, review/survey (Refereed)
    Abstract [en]

    Plant biomass is a magnificent renewable resource for phytochemicals that carry bioactive properties. Ferulic acid (FA) is a hydroxycinnamic acid that is found widespread in plant cell walls, mainly esterified to polysaccharides. It is well known of its strong antioxidant activity, together with numerous properties, such as antimicrobial, anti-inflammatory and neuroprotective effects. This review article provides insights into the potential for valorization of FA as a potent antiviral agent. Its pharmacokinetic properties (absorption, metabolism, distribution and excretion) and the proposed mechanisms that are purported to provide antiviral activity are presented. Novel strategies on extraction and derivatization routes, for enhancing even further the antiviral activity of FA and potentially favor its metabolism, distribution and residence time in the human body, are discussed. These routes may lead to novel high-added value biorefinery pathways to utilize plant biomass toward the production of nutraceuticals as functional foods with attractive bioactive properties, such as enhancing immunity toward viral infections.

  • 13.
    Antonopoulou, Io
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Sapountzaki, Eleftheria
    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.
    Inhibition of the main protease of SARS-CoV-2 (Mpro) by repurposing/designing drug-like substances and utilizing nature’s toolbox of bioactive compounds2022In: Computational and Structural Biotechnology Journal, ISSN 2001-0370, Vol. 20, p. 1306-1344Article, review/survey (Refereed)
    Abstract [en]

    The emergence of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has resulted in a long pandemic, with numerous cases and victims worldwide and enormous consequences on social and economic life. Although vaccinations have proceeded and provide a valuable shield against the virus, the approved drugs are limited and it is crucial that further ways to combat infection are developed, that can also act against potential mutations. The main protease (Mpro) of the virus is an appealing target for the development of inhibitors, due to its importance in the viral life cycle and its high conservation among different coronaviruses. Several compounds have shown inhibitory potential against Mpro, both in silico and in vitro, with few of them also having entered clinical trials. These candidates include: known drugs that have been repurposed, molecules specifically designed based on the natural substrate of the protease or on structural moieties that have shown high binding affinity to the protease active site, as well as naturally derived compounds, either isolated or in plant extracts. The aim of this work is to collectively present the results of research regarding Mpro inhibitors to date, focusing on the function of the compounds founded by in silico simulations and further explored by in vitro and in vivo assays. Creating an extended portfolio of promising compounds that may block viral replication by inhibiting Mpro and by understanding involved structure–activity relationships, could provide a basis for the development of effective solutions against SARS-CoV-2 and future related outbreaks.

  • 14.
    Antonopoulou, Io
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Sapountzaki, Eleftheria
    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.
    The Inhibitory Potential of Ferulic Acid Derivatives against the SARS-CoV-2 Main Protease: Molecular Docking, Molecular Dynamics, and ADMET Evaluation2022In: Biomedicines, E-ISSN 2227-9059, Vol. 10, no 8, article id 1787Article in journal (Refereed)
    Abstract [en]

    The main protease (Mpro) of SARS-CoV-2 is an appealing target for the development of antiviral compounds, due to its critical role in the viral life cycle and its high conservation among different coronaviruses and the continuously emerging mutants of SARS-CoV-2. Ferulic acid (FA) is a phytochemical with several health benefits that is abundant in plant biomass and has been used as a basis for the enzymatic or chemical synthesis of derivatives with improved properties, including antiviral activity against a range of viruses. This study tested 54 reported FA derivatives for their inhibitory potential against Mpro by in silico simulations. Molecular docking was performed using Autodock Vina, resulting in comparable or better binding affinities for 14 compounds compared to the known inhibitors N3 and GC376. ADMET analysis showed limited bioavailability but significantly improved the solubility for the enzymatically synthesized hits while better bioavailability and druglikeness properties but higher toxicity were observed for the chemically synthesized ones. MD simulations confirmed the stability of the complexes of the most promising compounds with Mpro, highlighting FA rutinoside and compound e27 as the best candidates from each derivative category. View Full-TextKeywords: SARS-CoV-2; Mpro; enzyme inhibition; ferulic acid; molecular docking; molecular dynamics; ADMET

  • 15.
    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

  • 16.
    Bajracharya, Suman
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Krige, Adolf
    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.
    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.
    Advances in cathode designs and reactor configurations of microbial electrosynthesis systems to facilitate gas electro-fermentation2022In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 354, article id 127178Article in journal (Refereed)
    Abstract [en]

    In gas fermentation, a range of chemolithoautotrophs fix single-carbon (C1) gases (CO2 and CO) when H2 or other reductants are available. Microbial electrosynthesis (MES) enables CO2 reduction by generating H2 or reducing equivalents with the sole input of renewable electricity. A combined approach as gas electro-fermentation is attractive for the sustainable production of biofuels and biochemicals utilizing C1 gases. Various platform compounds such as acetate, butyrate, caproate, ethanol, butanol and bioplastics can be produced. However, technological challenges pertaining to the microbe-material interactions such as poor gas-liquid mass transfer, low biomass and biofilm coverage on cathode, low productivities still exist. We are presenting a review on latest developments in MES focusing on the configuration and design of cathodes that can address the challenges and support the gas electro-fermentation. Overall, the opportunities for advancing CO and CO2-based biochemicals and biofuels production in MES with suitable cathode/reactor design are prospected.

  • 17.
    Bajracharya, Suman
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Krige, Adolf
    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.
    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.
    Dual cathode configuration and headspace gas recirculation for enhancing microbial electrosynthesis using Sporomusa ovata2022In: Chemosphere, ISSN 0045-6535, E-ISSN 1879-1298, Vol. 287, Part 3, article id 132188Article in journal (Refereed)
    Abstract [en]

    High-rate production of acetate and other value-added products from the reduction of CO2 in microbial electrosynthesis (MES) using acetogens can be achieved with high reducing power where H2 appears as a key electron mediator. H2 evolution using metal cathodes can enhance the availability of H2 to support high-rate microbial reduction of CO2. Due to the low solubility of H2, the availability of H2 remains limited to the bacteria. In this study, we investigated the performances of Sporomusa ovata for CO2 reduction when dual cathodes were used together in an MES, one was regular carbon cathode, and the other was a titanium mesh that allows higher hydrogen evolution. The dual cathode configuration was investigated in two sets of MES, one set had the usual S. ovata inoculated graphite rod, and another set had a synthetic biofilm-imprinted carbon cloth. Additionally, the headspace gas in MES was recirculated to increase the H2 availability to the bacteria in suspension. High-rate CO2 reduction was observed at −0.9 V vs Ag/AgCl with dual cathode configuration as compared to single cathodes. High titers of acetate (up to ∼11 g/L) with maximum instantaneous rates of 0.68–0.7 g/L/d at −0.9 V vs Ag/AgCl were observed, which are higher than the production rates reported in literatures for S. ovata using MES with surface modified cathodes. A high H2 availability supported the high-rate acetate production from CO2 with diminished electricity input.

  • 18.
    Bajracharya, Suman
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Krige, Adolf
    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.
    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.
    Microbial Electrosynthesis Using 3D Bioprinting of Sporomusa ovata on Copper, Stainless-Steel, and Titanium Cathodes for CO2 Reduction2023In: Fermentation, E-ISSN 2311-5637, Vol. 10, no 1, article id 34Article in journal (Refereed)
    Abstract [en]

    Acetate can be produced from carbon dioxide (CO2) and electricity using bacteria at the cathode of microbial electrosynthesis (MES). This process relies on electrolytically-produced hydrogen (H2). However, the low solubility of H2 can limit the process. Using metal cathodes to generate H2 at a high rate can improve MES. Immobilizing bacteria on the metal cathode can further proliferate the H2 availability to the bacteria. In this study, we investigated the performances of 3D bioprinting of Sporomusa ovata on three metal meshes—copper (Cu), stainless steel (SS), and titanium (Ti), when used individually as a cathode in MES. Bacterial cells were immobilized on the metal using a 3D bioprinter with alginate hydrogel ink. The bioprinted Ti mesh exhibited higher acetate production (53 ± 19 g/m2/d) at −0.8 V vs. Ag/AgCl as compared to other metal cathodes. More than 9 g/L of acetate was achieved with bioprinted Ti, and the least amount was obtained with bioprinted Cu. Although all three metals are known for catalyzing H2 evolution, the lower biocompatibility and chemical stability of Cu hampered its performance. Stable and biocompatible Ti supported the bioprinted S. ovata effectively. Bioprinting of synthetic biofilm on H2-evolving metal cathodes can provide high-performing and robust biocathodes for further application of MES.

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  • 19.
    Bajracharya, Suman
    et al.
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Sarkar, Omprakash
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Krige, Adolf
    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.
    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.
    Chapter 12 - Advances in gas fermentation processes2022In: Current Developments in Biotechnology and Bioengineering: Advances in Bioprocess Engineering / [ed] Sirohi, Ranjna; Pandey, Ashok; Taherzadeh, Mohammad J.; Larroche, Christian, Elsevier, 2022, p. 321-351Chapter in book (Other academic)
    Abstract [en]

    Microbial metabolism enables the sustainable synthesis of fuels and chemicals from gaseous substrates (H2, CO, and CO2), thus drastically diminishing the carbon load in the atmosphere. Various value-added biochemicals and biofuels, such as acetate, methane, ethanol, butanol, butyrate, caproate, and bioplastics, have been produced during the conversion of syngas or H2/CO2, using a variety of microorganisms as biocatalysts. Gas fermentation processes using acetogenic and methanogenic organisms are being extensively investigated. This chapter provides an overview of microbial CO and CO2 conversion technology, with an emphasis on recent developments and integration with renewable electricity for the generation of H2 or other forms of electron donors. A discussion on technological challenges in gas fermentation addresses issues, such as poor mass transfer, low microbial biomass, and low productivity. It also presents possible solutions based on the latest advances in bioelectrochemical processes including microbial gas electrofermentation. Finally, the chapter includes a sustainability analysis of the process and includes a brief update on commercially established companies operating gas fermentation systems. Overall, an integrated approach combining gas fermentation and renewable electricity offers an opportunity for the development of CO and CO2- based biochemical and biofuel production at commercial scale.

  • 20.
    Bennett, Neil A.
    et al.
    Institute of Food Research.
    Ryan, James
    Institute of Food Research.
    Biely, Peter
    Slovak Academy of Sciences, Bratislava.
    Vrsanska, Maria
    Slovak Academy of Sciences, Bratislava.
    Kremnicky, Lubomir
    Slovak Academy of Sciences, Bratislava.
    Macris, Basil J.
    National Technical University of Athens.
    Kekos, Dimitris
    National Technical University of Athens.
    Christakopoulos, Paul
    Katapodis, Petros
    National Technical University of Athens.
    Claeyssens, Marc
    University of Ghent.
    Nerinckx, Wim
    University of Ghent.
    Ntauma, Patricia
    University of Ghent.
    Bhat, Mahalingeshwara K.
    Institute of Food Research.
    Biochemical and catalytic properties of an endoxylanase purified from the culture filtrate of Thermomyces lanuginosus ATCC 468821998In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 306, no 3, p. 445-455Article in journal (Refereed)
    Abstract [en]

    An endoxylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) from the culture filtrates of T. lanuginosus ATCC 46882 was purified to homogeneity by DEAE-Sepharose and Bio-Gel P-30 column chromatographies. The purified endoxylanase had a specific activity of 888.8 mumol min-1 mg-1 protein and accounted for approximately 30% of the total protein secreted by this fungus. The molecular mass of native (non-denatured) and denatured endoxylanase were 26.3 and 25.7 kD as, respectively. Endoxylanase had a pI of 3.7 and was optimally active between pH 6.0-6.5 and at 75 degrees C. The enzyme showed > 50% of its original activity between pH 5.5-9.0 and at 85 degrees C. The pH and temperature stability studies revealed that this endoxylanase was almost completely stable between pH 5.0-9.0 and up to 60 degrees C for 5 h and at pH 10.0 up to 55 degrees C for 5 h. Thin-layer chromatography (TLC) analysis showed that endoxylanase released mainly xylose (Xyl) and xylobiose (Xyl2) from beechwood 4-O-methyl-D-glucuronoxylan, O-acetyl-4-O-methyl-D-glucuronoxylan and rhodymenan (a beta-(1-->3)-beta(1-->4)-xylan). Also, the enzyme released an acidic xylo-oligosaccharide from 4-O-methyl-D-glucuronoxylan, and an isomeric xylotetraose and an isomeric xylopentaose from rhodymenan. The enzyme hydrolysed [1-3H]-xylo-oligosaccharides in an endofashion, but the hydrolysis of [1-3H]-xylotriose appeared to proceed via transglycosylation. since the xylobiose was the predominant product. Endoxylanase was not active on pNPX and pNPC at 40 and 100 mM for up to 6 h, but showed some activity toward pNPX at 100 mM after 20-24 h. The results suggested that the endoxylanase from T. lanuginosus belongs to family 11.

  • 21.
    Bhattacharyya, Shubhankar
    et al.
    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.
    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.
    Melt Stable Functionalized Organosolv and Kraft Lignin Thermoplastic2020In: Processes, ISSN 2227-9717, Vol. 8, no 9, article id 1108Article in journal (Refereed)
    Abstract [en]

    A shift towards an economically viable biomass biorefinery concept requires the use of all biomass fractions (cellulose, hemicellulose, and lignin) for the production of high added-value products. As lignin is often underutilized, the establishment of lignin valorization routes is highly important. In-house produced organosolv as well as commercial Kraft lignin were used in this study. The aim of the current work was to make a comparative study of thermoplastic biomaterials from two different types of lignins. Native lignins were alkylate with two different alkyl iodides to produce ether-functionalized lignins. Successful etherification was verified by FT-IR spectroscopy, changes in the molecular weight of lignin, as well as 13C and 1H Nuclear Magnetic Resonance (NMR). The thermal stability of etherified lignin samples was considerably improved with the T2% of organosolv to increase from 143 °C to up to 213 °C and of Kraft lignin from 133 °C to up to 168 °C, and glass transition temperature was observed. The present study shows that etherification of both organosolv and Kraft lignin with alkyl halides can produce lignin thermoplastic biomaterials with low glass transition temperature. The length of the alkyl chain affects thermal stability as well as other thermal properties.

  • 22.
    Bonturi, Nemailla
    et al.
    Department of Materials and Bioprocess Engineering, School of Chemical Engineering, State University of Campinas.
    Matsakas, Leonidas
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Nilsson, Robert
    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.
    Miranda, Everson Alves
    Department of Materials and Bioprocess Engineering, School of Chemical Engineering, State University of Campinas.
    Berglund, Kris
    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.
    Single Cell Oil Producing Yeasts Lipomyces starkeyi and Rhodosporidium toruloides: Selection of Extraction Strategies and Biodiesel Property Prediction2015In: Energies, E-ISSN 1996-1073, Vol. 8, no 6, p. 5040-5052Article in journal (Refereed)
    Abstract [en]

    Single cell oils (SCOs) are considered potential raw material for the production of biodiesel. Rhodosporidium sp. and Lipomyces sp. are good candidates for SCO production. Lipid extractability differs according to yeast species and literature on the most suitable method for each oleaginous yeast species is scarce. This work aimed to investigate the efficiency of the most cited strategies for extracting lipids from intact and pretreated cells of Rhodosporidium toruloides and Lipomyces starkeyi. Lipid extractions were conducted using hexane or combinations of chloroform and methanol. The Folch method resulted in the highest lipid yields for both yeasts (42% for R. toruloides and 48% for L. starkeyi). Also, this method eliminates the cell pretreatment step. The Bligh and Dyer method underestimated the lipid content in the tested strains (25% for R. toruloides and 34% for L. starkeyi). Lipid extractability increased after acid pretreatment for the Pedersen, hexane, and Bligh and Dyer methods. For R. toruloides unexpected fatty acid methyl esters (FAME) composition were found for some lipid extraction strategies tested. Therefore, this work provides useful information for analytical and process development aiming at biodiesel production from the SCO of these two yeast species.

  • 23. Caridis, K A
    et al.
    Christakopoulos, Paul
    Macris, B J
    Control of catalase production and purity by altering certain nutritional factors of Alternaria alternata growth medium1991In: Biotechnology letters, ISSN 0141-5492, E-ISSN 1573-6776, Vol. 13, no 1, p. 35-38Article in journal (Refereed)
    Abstract [en]

    Both activity level of catalase and presence of glucose oxidase as an impurity were controlled by the type and concentration of nitrogen and carbon source in the culture medium of Alternaria alternata. It was possible to produce glucose oxidase-free catalase at activity levels competing favourably with those reported for other catalase hyperproducing microorganisms.

  • 24. Caridis, Konstantina-Anna
    et al.
    Christakopoulos, Paul
    Macris, Basil J.
    National Technical University of Athens.
    Simultaneous production of glucose oxidase and catalase by Alternaria alternata1991In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 34, no 6, p. 794-797Article in journal (Refereed)
    Abstract [en]

    A number of factors affecting simultaneous production of cell-bound glucose oxidase and catalase by the fungus Alternaria alternata have been investigated. Consecutive optimization of the type and concentration of nitrogen and carbon source, the initial pH and growth temperature resulted in a simultaneous increase in glucose oxidase and catalase by 780% and 68% respectively. Two second-order equations, describing the combined effect of pH and temperature on the activity of each enzyme, revealed that glucose oxidase had its optima at pH 7.9 and 32.3°C and catalase at pH 8.5 and 18.1°C. Under certain growth conditions, yields as high as 23.5 and 18,100 units/g carbon source for glucose oxidase and catalase, respectively, were simultaneously obtained.

  • 25.
    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.

  • 26.
    Chang, Jo-Shu
    et al.
    Research Centre for Smart Sustainable Circular Economy, Tunghai 407, Taiwan; Department of Chemical and Materials Engineering, College of Engineering, Tunghai University, Taichung 407, Taiwan; Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan.
    Show, Pau Loke
    Department of Chemical and Environmental Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, Broga Road, 43500 Semenyih, Selangor Darul Ehsan, Malaysia.
    Lee, Duu-Jong
    Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Recent advances in lignocellulosic biomass refinery2022In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 347, article id 126735Article in journal (Other academic)
  • 27.
    Charavgi, Maria-Despoina
    et al.
    Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, 48 Vassileos Constantinou Avenue, 11635 Athens, Greece; School of Chemical Engineering, National Technical University of Athens, Iroon Polytechniou Street, Zografou Campus, 15700 Athens, Greece.
    Dimarogona, Maria
    Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, 48 Vassileos Constantinou Avenue, 11635 Athens, Greece; School of Chemical Engineering, National Technical University of Athens, Iroon Polytechniou Street, Zografou Campus, 15700 Athens, Greece.
    Topakas, Evangelos
    School of Chemical Engineering, National Technical University of Athens, Iroon Polytechniou Street, Zografou Campus, 15700 Athens, Greece.
    Christakopoulos, Paul
    School of Chemical Engineering, National Technical University of Athens, Iroon Polytechniou Street, Zografou Campus, 15700 Athens, Greece.
    Chrysina, Evangelia D.
    Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, 48 Vassileos Constantinou Avenue, 11635 Athens, Greece.
    The structure of a novel glucuronoyl esterase from Myceliophthora thermophila gives new insights into its role as a potential biocatalyst2013In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 69, no 1, p. 63-73Article in journal (Refereed)
    Abstract [en]

    The increasing demand for the development of efficient biocatalysts is a consequence of their broad industrial applications. Typical difficulties that are encountered during their exploitation in a variety of processes are interconnected with factors such as temperature, pH, product inhibitors etc. To eliminate these, research has been directed towards the identification of new enzymes that would comply with the required standards. To this end, the recently discovered glucuronoyl esterases (GEs) are an enigmatic family within the carbohydrate esterase (CE) family. Structures of the thermophilic StGE2 esterase from Myceliophthora thermophila (synonym Sporotrichum thermophile), a member of the CE15 family, and its S213A mutant were determined at 1.55 and 1.9 Å resolution, respectively. The first crystal structure of the S213A mutant in complex with a substrate analogue, methyl 4-O-methyl-[beta]-D-glucopyranuronate, was determined at 2.35 Å resolution. All of the three-dimensional protein structures have an [alpha]/[beta]-hydrolase fold with a three-layer [alpha][beta][alpha]-sandwich architecture and a Rossmann topology and comprise one molecule per asymmetric unit. These are the first crystal structures of a thermophilic GE both in an unliganded form and bound to a substrate analogue, thus unravelling the organization of the catalytic triad residues and their neighbours lining the active site. The knowledge derived offers novel insights into the key structural elements that drive the hydrolysis of glucuronic acid esters.

  • 28.
    Charisteidis, Ioannis
    et al.
    Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece.
    Lazaridis, Polykarpos
    Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece.
    Fotopoulos, Apostolos
    Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece.
    Pachatouridou, Eleni
    Chemical Process and Energy Resources Institute, Centre for Research and Technology-Hellas (CPERI/CERTH), Thessaloniki, Greece.
    Matsakas, Leonidas
    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.
    Triantafyllidis, Konstantinos
    Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece.
    Catalytic Fast Pyrolysis of Lignin Isolated by Hybrid Organosolv—Steam Explosion Pretreatment of Hardwood and Softwood Biomass for the Production of Phenolics and Aromatics2019In: Catalysts, E-ISSN 2073-4344, Vol. 9, no 11, article id 935Article in journal (Refereed)
    Abstract [en]

    Lignin, one of the three main structural biopolymers of lignocellulosic biomass, is the most abundant natural source of aromatics with a great valorization potential towards the production of fuels, chemicals, and polymers. Although kraft lignin and lignosulphonates, as byproducts of the pulp/paper industry, are available in vast amounts, other types of lignins, such as the organosolv or the hydrolysis lignin, are becoming increasingly important, as they are side-streams of new biorefinery processes aiming at the (bio)catalytic valorization of biomass sugars. Within this context, in this work, we studied the thermal (non-catalytic) and catalytic fast pyrolysis of softwood (spruce) and hardwood (birch) lignins, isolated by a hybrid organosolv–steam explosion biomass pretreatment method in order to investigate the effect of lignin origin/composition on product yields and lignin bio-oil composition. The catalysts studied were conventional microporous ZSM-5 (Zeolite Socony Mobil–5) zeolites and hierarchical ZSM-5 zeolites with intracrystal mesopores (i.e., 9 and 45 nm) or nano-sized ZSM-5 with a high external surface. All ZSM-5 zeolites were active in converting the initially produced via thermal pyrolysis alkoxy-phenols (i.e., of guaiacyl and syringyl/guaiacyl type for spruce and birch lignin, respectively) towards BTX (benzene, toluene, xylene) aromatics, alkyl-phenols and polycyclic aromatic hydrocarbons (PAHs, mainly naphthalenes), with the mesoporous ZSM-5 exhibiting higher dealkoxylation reactivity and being significantly more selective towards mono-aromatics compared to the conventional ZSM-5, for both spruce and birch lignin.

  • 29.
    Cheilas, T
    et al.
    Department of Chemical Engineering, Biotechnology Laboratory, National Technical University of Athens.
    Stoupis, T
    Department of Chemical Engineering, Biotechnology Laboratory, National Technical University of Athens.
    Christakopoulos, Paul
    Katapodis, P
    Department of Chemical Engineering, Biotechnology Laboratory, National Technical University of Athens.
    Mamma, D
    Department of Chemical Engineering, Biotechnology Laboratory, National Technical University of Athens.
    Hatzinikolaou, D.G
    Department of Chemical Engineering, Biotechnology Laboratory, National Technical University of Athens.
    Kekos, D
    Department of Chemical Engineering, Biotechnology Laboratory, National Technical University of Athens.
    Macris, B.J
    Department of Chemical Engineering, Biotechnology Laboratory, National Technical University of Athens.
    Hemicellulolytic activity of Fusarium oxysporum grown on sugar beet pulp. Production of extracellular arabinanase2000In: Process Biochemistry, ISSN 1359-5113, E-ISSN 1873-3298, Vol. 35, no 6, p. 557-561Article in journal (Refereed)
    Abstract [en]

    Fusarium oxysporum F3 exhibited hemicellulolytic enzymic activity when grown on sugar beet pulp, a by-product of the sugar industry. The growth medium was specifically optimised for enhanced production of extracellular arabinanase. The optimum medium contained sugar beet pulp (4%, w/v) and corn steep liquor (6%, v/v) as carbon and nitrogen sources, respectively. Arabinanase activity as high as 0.25 U/ml of culture was obtained, which compared favourably to those reported for other microorganisms. Optimal arabinanase activity was observed at pH 6-7 and 50 °C. Investigation of the degradation of the main components of sugar beet pulp showed that arabinose containing polysaccharides and pectin were first degraded, followed by the glucose-containing polysaccharides.

  • 30.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Project: Bioconversion of Recalcitrant Polysaccharides by Novel Oxidative Biocatalysts for the production of ethanol and other novel products (SWEDISH ENERGY AGENCY)2014Other (Other (popular science, discussion, etc.))
  • 31.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Project: Efficient bioconversion of forest biomass insoluble polyesters with a potential use in lignocellulosic feedstock biorefineries (FORMAS)2014Other (Other (popular science, discussion, etc.))
  • 32.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Project: Optimized esterase biocatalysts for cost-effective industrial production (EU-FP7)2014Other (Other (popular science, discussion, etc.))
  • 33.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Project: SOUnd-DRIven BIOtechnology (VR)2014Other (Other (popular science, discussion, etc.))
  • 34.
    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.

  • 35. Christakopoulos, Paul
    et al.
    Bhat, Mahalingeshwara K.
    Enzymatic synthesis of trisaccharides and alkyl β-d-glucosides by the transglycosylation reaction of β-glucosidase from Fusarium oxysporum1994In: International Journal of Biological Macromolecules, ISSN 0141-8130, E-ISSN 1879-0003, Vol. 16, no 6, p. 331-334Article in journal (Refereed)
    Abstract [en]

    Purified β-glucosidase from Fusarium oxysporum catalyses hydrolysis and transglycosylation reactions. By utilizing the transglycosylation reaction, trisaccharides and alkyl β-d-glucosides were synthesized under optimal conditions in the presence of various disaccharides and alcohols. The yields of trisaccharides and alkyl β-d-glucosides were 22–37% and 10–33% of the total sugar, respectively. The enzyme retained 70–80% of its original activity in the presence of 25% (w/v) methanol, ethanol and propanol. Thus, β-glucosidase from F. oxysporum appears to be an ideal enzyme for the synthesis of useful trisaccharides and alkyl β-d-glucosides.

  • 36. Christakopoulos, Paul
    et al.
    Goodenough, Peter W.
    Institute of Food Research, Reading.
    Kekos, Dimitris
    National Technical University of Athens.
    Macris, Basil J.
    National Technical University of Athens.
    Claeyssens, Marc
    University of Ghent.
    Bhat, M.K.
    Institute of Food Research, Reading.
    Purification and Characterisation of an Extracellular β-Glucosidase with Transglycosylation and Exo-glucosidase Activities from Fusarium oxysporum1994In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 224, no 2, p. 379-385Article in journal (Refereed)
    Abstract [en]

    An extracellular β-glucosidase from Fusarium oxysporum was purified to homogeneity by gel-filtration and ion-exchange chromatographies. The enzyme, a monomeric protein of 110 kDa, was maximally active at pH 5.0–6.0 and at 60°C. It hydrolysed 1→4-linked aryl-β-glucosides and 1→4-linked, 1→3-linked and 1→6–linked β-glucosides. The apparent Km and kcat values for p -nitrophenyl β-d-glucopyranoside (4-NpGlcp) and cellobiose were 0.093 (Km), 1.07 mM (kcat) and 1802 (Km), 461.5 min-1 (kcat), respectively. Glucose and gluconolactone inhibited the enzyme competitively with Ki values of 2.05 mM and 3.03 μM, respectively. Alcohols activated the enzyme; butanol showed maximum effect (2.2-fold at 0.5 M) while methanol increased the activity by 1.4-fold at 1 M. The enzyme catalysed the synthesis of methylglucosides, ethylglucoside and propylglucosides, as well as trisaccharides in the presence of different alcohols and disaccharides, respectively. In addition, the enzyme hydrolysed the unsubstituted and methylumbelliferyl cello-oligosaccharides [MeUmb(Glc)n] but the rate of hydrolysis decreased with increasing chain length. Analysis of products released from MeUmb(Glc)n as a function of time revealed that the enzyme attacked these substrates in a stepwise manner and from both ends. Thus, β-glucosidase from F. oxysporum, with the above interesting properties, could be of commercial interest.

  • 37. Christakopoulos, Paul
    et al.
    Hatzinikolaou, Dimitris G.
    National Technical University of Athens.
    Fountoukidis, George
    National Technical University of Athens.
    Kekos, Dimitris
    National Technical University of Athens.
    Claeyssens, Marc
    University of Ghent.
    Macris, Basil J.
    National Technical University of Athens.
    Purification and mode of action of an alkali-resistant endo-1,4-β-glucanase from Bacillus pumilus1999In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 364, no 1, p. 61-66Article in journal (Refereed)
    Abstract [en]

    Alkaline endo-1,4-β-d-glucanase was secreted byBacillus pumilusgrown in submerged culture on a combination of oat spelt xylan and corn starch as carbon sources. The enzyme was purified to homogeneity by Sephacryl S-200 and Q-Sepharose column chromatography. The protein corresponded to molecular mass and pIvalues of 67 kDa and 3.7, respectively. The enzyme was optimally active at pH 7.0–8.0 and 60°C and retained 50% of its optimum activity at pH 12. The most notable characteristic of the endoglucanase was its high stability up to pH 12 for 20 h at 30°C. The enzyme hydrolyzed carboxymethylcellulose (CMC) and cello-oligosaccharides but was inactive on cellobiose, cellotriose, Avicel, xylan, 4-nitrophenyl-β-d-glucoside, 4-nitrophenyl-β-d-cellobioside, and 4-nitrophenyl-β-d-xyloside. Analysis of reaction mixtures by HPLC revealed that the enzyme produced almost exclusively cellotriose when acted on CMC and appeared to hydrolyze cello-oligosaccharides by successively releasing cellotriose. The use of 4-methylumbelliferyl cello-oligosaccharides and the determination of bond cleavage frequency revealed that the enzyme preferentially hydrolyzed the third glycosidic bond adjacent to the glycon. The enzyme mediated a decrease in the viscosity of CMC associated with a release of only small amounts of reducing sugar. The enzyme activity was not inhibited by metal ions, surfactants, and chelating agents used as components of laundry detergents.

  • 38. Christakopoulos, Paul
    et al.
    Katapodis, P
    Laboratory of Biotechnology, Department of Chemical Engineering, National Technical University of Athens.
    Hatzinikolaou, D
    Laboratory of Biotechnology, Department of Chemical Engineering, National Technical University of Athens.
    Kekos, D
    Laboratory of Biotechnology, Department of Chemical Engineering, National Technical University of Athens.
    Macris, B.J
    Laboratory of Biotechnology, Department of Chemical Engineering, National Technical University of Athens.
    Purification and characterization of an extracellular α-L- arabinofuranosidase from Fusarium oxysporum2000In: Applied Biochemistry and Biotechnology, ISSN 0273-2289, E-ISSN 1559-0291, Vol. 87, no 2, p. 127-133Article in journal (Refereed)
    Abstract [en]

    An α-L-arabinofuranosidase from Fusarium oxysporum F3 was purified to homogeneity by a two-step ion exchange intercalated by a gel filtration chromatography. The enzyme had a molecular mass of 66 kDa and was optimally active at pH 6.0 and 60°C. It hydrolyzed aryl α-L-arabinofuranosides and cleaved arabinosyl side chains from arabinoxylan and arabinan. There was a marked synergistic effect between the α-L-arabinofuranosidase and an endo-(1→4)-β-D-xylanase produced by F. oxysporum in the extensive hydrolysis of arabinoxylan.

  • 39. Christakopoulos, Paul
    et al.
    Katapodis, P.
    National Technical University of Athens.
    Kalogeris, E.
    National Technical University of Athens.
    Kekos, D.
    National Technical University of Athens.
    Macris, B.J.
    National Technical University of Athens.
    Stamatis, H.
    University of Ioannina.
    Skaltsa, H.
    School of Pharmacy, Panepistimiopolis, Zografou.
    Antimicrobial activity of acidic xylo-oligosaccharides produced by family 10 and 11 endoxylanases2003In: International Journal of Biological Macromolecules, ISSN 0141-8130, E-ISSN 1879-0003, Vol. 31, no 4-5, p. 171-175Article in journal (Refereed)
    Abstract [en]

    Acidic oligosaccharides were obtained from birchwood xylan by treatment with a Thermoascus aurantiacus family 10 and a Sporotrichum thermophile family 11 endoxylanases. The main difference between the products liberated by xylanases of family 10 and 11 concerned the length of the products containing 4-O-methyl-d-glucuronic acid. The xylanase from T. aurantiacus liberate from glucuronoxylan an aldotetrauronic acid as the shortest acidic fragment in contrast with the enzyme from S. thermophile, which liberated an aldopentauronic acid. Acidic xylooligosaccharides were separated from the hydrolysate by anion-exchange and size-exclusion chromatography (SEC) and the primary structure was determined by 13C NMR spectroscopy. The acidic xylo-oligosaccharides were tested against three Gram-positive and three Gram-negative aerobically grown bacteria, as well as against Helicobacterpylori. Aldopentauronic acid was proved more active against the Gram-positive bacteria and against H. pylori.

  • 40. Christakopoulos, Paul
    et al.
    Kekos, D.
    National Technical University of Athens.
    Kolisis, F.N.
    National Technical University of Athens.
    Macris, B.J.
    National Technical University of Athens.
    Controlling simultaneous production of endoglucanase and beta-glucosidase by Fusarium oxysporum in submerged culture1995In: Biotechnology letters, ISSN 0141-5492, E-ISSN 1573-6776, Vol. 17, no 8, p. 883-888Article in journal (Refereed)
    Abstract [en]

    The simultaneous production of endoglucanase and β-glucosidase by Fusarium oxysporum was investigated in submerged culture. Consecutive optimization of growth conditions resulted in the correction of large activity differences, observed during production of enzymes, and substantially enhanced low enzyme yields. At optimum growth conditions yields as high as 1650 and 232 U per g of carbon source of endoglucanase and β-glucosidase were obtained respectively competing favourably with those reported for microorganisms grown on the same carbon source. The most important kinetic characteristics of the enzymes were the high temperature optima of endoglucanase (60°C) and β-glucosidase (65°C) and the exceptionally high thermostability of endoglucanase. The latter enzyme retained 50% of the activity at pH 5.0 after approximately 6.5 h at 70°C

  • 41. Christakopoulos, Paul
    et al.
    Kekos, D.
    Macris, B.J.
    Bhat, M.K.
    Institute of Food Research, Reading.
    Multiple forms of endo-1,4-β-D-glucanase in the extracellular cellulase system of Fusarium oxysporum1995In: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 23, no 4, p. 586S-Article in journal (Refereed)
  • 42. Christakopoulos, Paul
    et al.
    Kekos, D.
    National Technical University of Athens.
    Macris, B.J.
    National Technical University of Athens.
    Claeyssen, M.
    Universiteit of Gent.
    Bhat, M.K.
    Institute of Food Research, Reading.
    Purification and mode of action of a low molecular mass endo-1,4-β-d-glucanase from Fusarium oxysporum1995In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 39, no 1, p. 85-93Article in journal (Refereed)
    Abstract [en]

    A low molecular mass (23.2 kDa) endo-1,4-β-d-glucanase from Fusarium oxysporum was purified to homogeneity by gel-filtration and ion-exchange chromatographies. The enzyme was optimally active at pH 6.0 and at 50 ° C. It had a pI value of 8.6 and was stable at 55 ° C for 1 h. It hydrolyzed carboxymethylcellulose, cello-oligosaccharides (Glcn) and 4-methylumbelliferylcello-oligosaccharides but did not hydrolyze cellobiose, p-nitrophenyl β-o-glucoside, p-nitrophenyl β-d-xyloside, Avicel, filter paper and xylan. Analysis of reaction mixtures by high pressure liquid chromatography revealed that this enzyme cleaved preferentially the internal glycoside bonds of higher cello-oligosaccharides. The enzyme also catalyzed the formation of transfer products in the presence of cellotriose, cellotetraose and 4-methylumbelliferylglucoside (MeUmbGlc).

  • 43. Christakopoulos, Paul
    et al.
    Kekos, D.
    National Technical University of Athens.
    Macris, B.J.
    National Technical University of Athens.
    Claeyssens, M.
    University of Ghent.
    Bhat, M.K.
    Institute of Food Research, Reading.
    Purification and characterization of a less randomly acting endo-1,4-beta-D-glucanase from the culture filtrates of Fusarium oxysporum1995In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 316, no 1, p. 428-433Article in journal (Refereed)
    Abstract [en]

    An extracellular endo-1,4-β-D-glucanase from Fusarium oxysporum was purified by affinity chromatography and gel filtration. The enzyme purified in this way was homogeneous when judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing-polyacrylamide gel electrophoresis. The protein corresponded to a molecular mass and pI value of 41.7 kDa and 6.4, respectively. It was optimally active at pH 4.5 and at 55°C. The enzyme hydrolyzed carboxymethylcellulose (CMC) and unsubstituted and substituted cello-oligosaccharides but was inactive on Avicel, filter paper, xylan, cellobiose, p-nitrophenyl-β-D-glucoside, and p-nitrophenyl-β-D-xyloside. However, the enzyme effected only a small change in viscosity of CMC per unit increase of reducing sugar. When cellotriose, cellotetraose, and cellopentaose were used as substrates, the enzyme released mainly cellobiose. Use of 4-methylumbelliferyl cello-oligosaccharides and the determination of bond cleavage frequency revealed that the enzyme preferentially hydrolyzed the glycosidic bond adjacent to 4-methylumbelliferone. Thus, the purified enzyme appeared to be a less randomly acting endoglucanase.

  • 44. Christakopoulos, Paul
    et al.
    Kekos, Dimitris
    National Technical University of Athens.
    Macris, Basil J.
    National Technical University of Athens.
    Claeyssens, Marc
    University of Ghent.
    Bhat, Mahalingeshwara K.
    Institute of Food Research.
    Purification and characterisation of a major xylanase with cellulase and transferase activities from Fusarium oxysporum1996In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 289, p. 91-104Article in journal (Refereed)
    Abstract [en]

    A major xylanase from Fusarium oxysporum was purified to homogeneity by gel filtration, affinity, and ion-exchange chromatographies. It has a molecular mass of 60.2 kDa and pl of 6.6 and was optimally active at pH 7.4 and at 50 °C. The enzyme was stable over the pH range 5.8–8.2 at 40 °C for 24 h and lost 45% of its original activity at pH 9.0 under the identical conditions. The enzyme rapidly hydrolysed xylans from oat spelts (husks) and birchwood, but the activities on carboxymethylcellulose (CMC), filter paper, and Avicel were very low. Determination of kcat/Km revealed that the enzyme hydrolysed oat spelts and birchwood xylans, 15–30 times more efficiently than CMC. In a 24 h incubation, at pH 7.0 and 9.0, the enzyme hydrolysed oat spelts and birchwood xylans by 75 and 65%, respectively. However, at pH 7.0, the enzyme released almost equal amounts of xylose and xylobiose from both xylans, whereas at pH 9.0, the concentration of xylobiose was twice as muchi as that of xylose and xylotriose. Xylanase attacked preferentially the internal glycosidic bonds of xylo- and 4-methylumbelliferyl cello-oligosaccharides [MeUmb(Glc)n]. The enzyme catalysed transglycosylation reaction with xylotriose, xylotetraose, and xylopentaose as donors and 4-methylumbelliferyl β-d-glucoside (MeUmbGlc) as an acceptor.

  • 45. Christakopoulos, Paul
    et al.
    Kekos, Dimitris
    National Technical University of Athens.
    Macris, Basil J.
    National Technical University of Athens.
    Goodenough, Peter W.
    Institute of Food Research, Reading.
    Bhat, Mahalingeshwara K.
    Institute of Food Research, Reading.
    Optimization of β-glucosidase catalysed synthesis of trisaccharides from cellobiose and gentiobiose1994In: Biotechnology letters, ISSN 0141-5492, E-ISSN 1573-6776, Vol. 16, no 6, p. 587-592Article in journal (Refereed)
    Abstract [en]

    Purified β-Glucosidase from Fusarium oxysporum catalysed the hydrolysis and transglycosylation reactions in the presence of cellobiose and gentiobiose. The product of the latter reaction was mainly a triose. The time of incubation, pH and substrate concentration for transglycosylation reaction were optimised. Under optimal conditions, the concentration of glucose and triose reached approximately 15–20 % of the initial substrate concentration. These results suggested that β-glucosidase from F.oxysporum is an ideal enzyme for the synthesis of triose in reasonable quantities.

  • 46. Christakopoulos, Paul
    et al.
    Koullas, D.P.
    National Technical University of Athens.
    Kekos, D.
    National Technical University of Athens.
    Koukios, E.G.
    National Technical University of Athens.
    Macris, B.J.
    National Technical University of Athens.
    Direct conversion of straw to ethanol by Fusarium oxysporum: Effect of cellulose crystallinity1991In: Enzyme and microbial technology, ISSN 0141-0229, E-ISSN 1879-0909, Vol. 13, no 3, p. 272-274Article in journal (Refereed)
    Abstract [en]

    Wheat straw was successfully fermented to ethanol by Fusarium oxysporum F3 in a one-step process. Cellulose crystallinity was found to be a major factor in the bioconversion process. Ethanol yields increased linearly with decreasing crystallinity index. Approximately 80% of straw carbohydrates were converted directly to ethanol with a yield of 0.28 g ethanol/g−1 of straw when the crystallinity index was reduced to 23.6%.

  • 47. Christakopoulos, Paul
    et al.
    Koullas, D.P.
    National Technical University of Athens.
    Kekos, D.
    National Technical University of Athens.
    Koukios, E.G.
    National Technical University of Athens.
    Macris, B.J.
    National Technical University of Athens.
    Direct ethanol conversion of pretreated straw by Fusarium oxysporum1991In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 35, no 3, p. 297-300Article in journal (Refereed)
    Abstract [en]

    Factors affecting the direct conversion of alkali pretreated straw to ethanol by Fusarium oxysporum F3 were investigated and the alkali level used for pretreatment and the degree of delignification of straw were found to be the most important. A linear correlation between ethanol yield and both the degree of straw delignification and the alkali level was observed. At optimum delignified straw concentration (4% w/v), a maximum ethanol yield of 0·275 g ethanol g−1 of straw was obtained corresponding to 67·8% of the theoretical yield.

  • 48. Christakopoulos, Paul
    et al.
    Kourentzi, Ekaterini
    National Technical University of Athens.
    Hatzinikolaou, Dimitris G.
    National Technical University of Athens.
    Claeyssens, Mark
    University of Ghent.
    Kekos, Dimitris
    National Technical University of Athens.
    Macris, Basil J.
    National Technical University of Athens.
    Enhancement of pH-stability of a low molecular mass endoglucanase from Fusarium oxysporum by protein pegylation1998In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 314, no 1-2, p. 95-99Article in journal (Refereed)
    Abstract [en]

    The stability of the low molecular mass endoglucanase (23.2 kDa) from Fusarium oxysporum at alkaline pH was enhanced by chemical modification. Two distinct types of amino acid-specific modifiers were used. The first, either cyanuric chloride activated polyethylene glycol (CC–PEG) or polyethylene glycol succinimidyl succinate active ester (SS–PEG), react (more or less specifically) with protein amino groups. The second type, maleimide polyethylene glycol (Mal–PEG), is specific for cysteinyl residues. The enzyme lost almost all of its activity when modified with CC–PEG, whereas no inactivation was observed with SS–PEG and Mal–PEG. The modified endoglucanase showed remarkably enhanced alkaline pH stability. When acting upon cello-oligosaccharides and 4-methylumbelliferyl cello-oligosaccharides, the enzyme preferentially cleaved the internal glycosidic bonds. The modified enzymes mediated a decrease in the viscosity of carboxymethyl cellulose (CMC) associated with the release of only small amounts of reducing sugar. Thus, the modified enzyme retains the endo character of the native enzyme

  • 49. Christakopoulos, Paul
    et al.
    Li, Lian-Wu
    National Technical University of Athens.
    Kekos, Dimitris
    National Technical University of Athens.
    Macris, Basil J.
    National Technical University of Athens.
    Direct conversion of sorghum carbohydrates to ethanol by a mixed microbial culture1993In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 45, no 2, p. 89-92Article in journal (Refereed)
    Abstract [en]

    The carbohydrates of sweet sorghum were directly converted to ethanol by a mixed culture of Fusarium oxysporum F3 and Saccharomyces cerevisiae 2541. A number of factors affecting this bioconversion was studied. Optimum ethanol yields of 33·2 g/100 g of total sorghum carbohydrates, corresponding to 10·3 g/100 g of fresh stalks, were obtained. These values represented 68·6% of the theoretical yield based on total polysaccharides and exceeded that based on oligosaccharides of sorghum by 53·7%. The results demonstrated that more than half of the sorghum polysaccharides were directly fermented to ethanol, thus making the process worthy of further investigation.

  • 50. Christakopoulos, Paul
    et al.
    Macris, B.J.
    National Technical University of Athens.
    Kekos, D.
    National Technical University of Athens.
    Direct fermentation of cellulose to ethanol by Fusarium oxysporum1989In: Enzyme and microbial technology, ISSN 0141-0229, E-ISSN 1879-0909, Vol. 11, no 4, p. 236-239Article in journal (Refereed)
    Abstract [en]

    The cellulase hyperproducing strain F3 of Fusarium oxysporum fermented glucose, xylose, cellobiose, and cellulose directly to ethanol. Conversion of cellulose to ethanol was markedly affected by the pH of both aerated preculture and nonaerated fermentation. Optimum values of cellulose conversion to ethanol were obtained when aerated and nonaerated processes were carried out at pH 5.5 and 6, respectively. Maximum ethanol concentrations of 9.6 and 14.5 g l−1, corresponding to 89.2 and 53.2% of the theoretical yield, were obtained when the fungus was grown under nonaerated conditions at 34°C for 6 days in a medium containing 20 and 50 g l−1cellulose, respectively.

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