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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
    et al.
    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, ISSN 1664-302X, 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, 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, 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.

  • 7.
    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 derivatives2018In: Enzyme and microbial technology, ISSN 0141-0229, E-ISSN 1879-0909Article 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.

  • 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.
    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)
  • 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.
    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.
    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.

  • 13.
    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, ISSN 1996-1073, 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.

  • 14. 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.

  • 15. 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.

  • 16.
    Charavgi, Maria-Despoina
    et al.
    National Technical University of Athens.
    Dimarogona, Maria
    National Technical University of Athens.
    Topakas, Evangelos
    National Technical University of Athens.
    Christakopoulos, Paul
    Chrysina, Evangelia D.
    National Hellenic Research Foundation, Athens.
    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.

  • 17.
    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.

  • 18.
    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.))
  • 19.
    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.))
  • 20.
    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.))
  • 21.
    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.))
  • 22.
    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.

  • 23. 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.

  • 24. 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.

  • 25. 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.

  • 26. 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.

  • 27. 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.

  • 28. 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

  • 29. 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)
  • 30. 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).

  • 31. 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.

  • 32. 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.

  • 33. 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.

  • 34. Christakopoulos, Paul
    et al.
    Koullas, Dimitrios P.
    Department of Chemical Engineering, National Technical University of Athens.
    Kekos, Dimitris
    Department of Chemical Engineering, National Technical University of Athens.
    Macris, Basil J
    Department of Chemical Engineering, National Technical University of Athens.
    Koukios, Emmanuel G.
    Department of Chemical Engineering, National Technical University of Athens.
    Correlating the effect of pretreatment on the enzymatic hydrolysis of straw1992In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 39, no 1, p. 113-116Article in journal (Refereed)
    Abstract [en]

    Avicell, Alkali-treated straw cellulose (ATSC), and wheat straw were ball-milled to reduce crystallinity; wheat straw was delignified by hot (120°C) sodium hydroxide solutions of various concentrations. The physically and chemically pretreated cellulosic materials were hydrolyzed by the cellulases of Fusarium oxysporum strain F3. Enzymic hydrolysis data were fitted by the hyperbolic correlation of Holtzapple, which involves two kinetic parameters, the maximum conversion (x(max)), and the enzymic hydrolysis time corresponding to 50% of x(max) (t( 1/2 )). An empirical correlation between x(max) and cellulose crystallinity, lignin content, and degree of delignification has been found under our experimental conditions. Complete cellulose hydrolysis is shown to be possible at less than 60% crystallinity indices or less than 10% lignin content. Avicell, Alkali-treated straw cellulose (ATSC), and wheat straw were ball-milled to reduce crystallinity; wheat straw was delignified by hot (120°C) sodium hydroxide solutions of various concentrations. The physically and chemically pretreated cellulosic materials were hydrolyzed by the cellulases of Fusarium oxysporum strain F3. Enzymic hydrolysis data were fitted by the hyperbolic correlation of Holtzapple, which involves two kinetic parameters, the maximum conversion (xmax), and the enzymic hydrolysis time corresponding to 50% of Xmax (t1/2). An empirical correlation between Xmax and cellulose crystallinity, lignin content, and degree of delignification has been found under our experimental conditions. Complete cellulose hydrolysis is shown to be possible at less than 60% crystallinity indices or less than 10% lignin content.

  • 35. 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%.

  • 36. 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.

  • 37. 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

  • 38. 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.

  • 39. 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.

  • 40. Christakopoulos, Paul
    et al.
    Macris, B.J.
    Kekos, D.
    National Technical University of Athens.
    Exceptionally thermostable α- and β-galactosidase from Aspergillus niger separated in one step1990In: Process Biochemistry, ISSN 1359-5113, E-ISSN 1873-3298, Vol. 25, no 6, p. 210-212Article in journal (Refereed)
    Abstract [en]

    Extracellular alpha- and-beta-galactosidases from a strain of Aspergillus niger were separated and purified in one step by cation exchange chromatography. Both enzymes had acidic pH (3.5-4.0) and high temperature (65-degrees-C) optima and an exceptionally high thermostability. Thus, -alpha-galactosidase had an activity half-time of 104 min at 60-degrees-C whereas at the same temperature the respective value for-beta-galactosidase was 835 min. At optimum conditions of activity the apparent K(m) values of alpha- and beta-galactosidase were 0.44mM and 1.1mM respectively. Both the high temperature optima and thermostability properties of the enzymes make them particularly suitable for high temperature processes.

  • 41. Christakopoulos, Paul
    et al.
    Macris, B.J.
    Kekos, D.
    On the mechanism of direct conversion of cellulose to ethanol by Fusarium oxysporum: Effect of cellulase and β-glucosidase1990In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 33, no 1, p. 18-20Article in journal (Refereed)
    Abstract [en]

    The effects of the three main enzymes involved in cellulose saccharification, namely cellobiohydrolase, carboxymethylcellulase and beta-glucosidase, on the direct conversion of cellulose to ethanol by Fusarium oxysporum F3 were investigated. Ethanol production was not affected when the activity of the former two enzymes was varied within a wide range. By contrast, beta-glucosidase markedly affected ethanol production showing an optimum level of 0.7-0.8 unit/ml growth medium. A significant decrease of cellulose bioconversion time to ethanol was obtained when beta-glucosidase activity was adjusted to this optimal level at the beginning of the fermentation process.

  • 42. Christakopoulos, Paul
    et al.
    Mamma, D.
    National Technical University of Athens.
    Kekos, D.
    National Technical University of Athens.
    Macris, B.J.
    National Technical University of Athens.
    Enhanced acetyl esterase production by Fusarium oxysporum1999In: World Journal of Microbiology & Biotechnology, ISSN 0959-3993, E-ISSN 1573-0972, Vol. 15, no 4, p. 443-446Article in journal (Refereed)
    Abstract [en]

    Production of acetyl esterase (EC 3.1.1.6) by Fusarium oxysporum strain F3 was enhanced by optimization of growth conditions. Under optimal conditions, activities as high as 0.89 U/ml of culture medium were obtained. The culture filtrate was equally active on p-nitrophenyl acetate and acetylxylan. The enzyme produced 71% deacetylation of acetylxylan in 2 h at 40 ∘C. Activity was optimized at pH6.5 and at 55 ∘C. The respective Km values for p-nitrophenyl acetate and acetylxylan were 0.25 mM and 1.05% (w/v) and the Vm values were 0.65 and 0.43 μmol acetate/min/mg protein.

  • 43. Christakopoulos, Paul
    et al.
    Mamma, D.
    National Technical University of Athens.
    Nerinckx, W.
    University of Ghent.
    Kekos, D.
    National Technical University of Athens.
    Macris, B.
    National Technical University of Athens.
    Claeyssens, M.
    University of Ghent.
    Production and partial characterization of xylanase from Fusarium oxysporum1996In: Bioresource Technology, ISSN 0960-8524, E-ISSN 1873-2976, Vol. 58, no 2, p. 115-119Article in journal (Refereed)
    Abstract [en]

    Production of xylanase by Fusarium oxysporum strain F3 was enhanced by optimization of initial pH of the culture medium, the type and concentration of nitrogen and carbon source, and the growth temperature. Under these conditions, yields as high as 245 U/ml of culture medium were obtained. The most important characteristic of the enzyme is its high pH stability. It retained 80 and 66% of the activity at pH 9.0 after 24 h at 4 and 30°C, respectively. Chromogenic (fluorogenic) 4-methylumbelliferyl-β-glycosides of xylose (MUX) and xylobiose (MUX2) were used to characterize xylanase multienzyme components, after separation by isoelectric focusing. The zymogram indicated one major, one minor xylanase and one active β-xylosidase exhibiting pI values of 9.5, 6.5 and 3.8, respectively

  • 44. Christakopoulos, Paul
    et al.
    Nerinckx, B.
    Klarskov, K.
    Kekos, D.
    Macris, B.
    Beeumen, J. Van
    Claeyssens, M.
    Purification and characterization of two low molecular mass endo1-4-beta-xylanases from Fusarium oxysporum1995In: Proceedings, Forum for Applied Biotechnology: Faculty of Agricultural and Applied Biological Sciences, Gent, 27 - 29 September 1995, 1995Conference paper (Refereed)
  • 45. Christakopoulos, Paul
    et al.
    Nerinckx, W.
    University of Ghent.
    Kekos, D.
    National Technical University of Athens.
    Macris, B.
    National Technical University of Athens.
    Claeyssens, M.
    University of Ghent.
    Purification and characterization of two low molecular mass alkaline xylanases from Fusarium oxysporum F31996In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 51, no 2, p. 181-189Article in journal (Refereed)
    Abstract [en]

    Two low molecular mass endo-1,4-β-d-xylanases from Fusarium oxysporum were purified to homogeneity by gel-filtration and ion-exchange chromatography. They exhibit molecular masses of 20.8 (xylanase I) and 23.5 (xylanase II) kDa, and isoelectric points of 9.5 and 8.45–8.70, respectively. Both xylanases display remarkable pH (9.0) stability. At 40 to 55 °C xylanase II is more thermostable than xylanase I but less active on xylan. In contrast to xylanase I, xylanase II is able to hydrolyze 1-O-4-methylumbelliferyl-(β-d-glucopyranosyl)-β-d-xylopyranoside (muxg). Neither of these enzymes hydrolyze xylotriose. They bind on crystalline cellulose but not on insoluble xylan. Analysis of reaction mixtures by high pressure liquid chromatography revealed that both enzymes cleave preferentially the internal glycosidic bonds of xylopentaose and oat spelts xylan. Thus the purified enzymes appeared to be true endo-β-1,4-xylanases. The amino terminal sequences of xylanases I and II show no homology. Xylanase I shows high similarity with alkaline low molecular mass xylanases of family G/11.

  • 46. Christakopoulos, Paul
    et al.
    Nerinckx, W.
    University of Ghent.
    Samyn, B.
    University of Ghent.
    Kekos, D.
    National Technical University of Athens.
    Macris, B.
    National Technical University of Athens.
    Beeumen, J. van
    University of Ghent.
    Claeyssens, M.
    University of Ghent.
    Functional characterization of a cellulose binding xylanase from Fusarium oxysporum1996In: Biotechnology letters, ISSN 0141-5492, E-ISSN 1573-6776, Vol. 18, no 3, p. 349-354Article in journal (Refereed)
    Abstract [en]

    An extracellular endoxylanase from Fusarium oxysporum binds onto crystalline cellulose. A small peptide (~ 2kDa) could be isolated after partial proteolysis of the native protein. It consists of 18 amino acids, is located in the C-terminal region of the protein and corresponds functionally to a cellulose binding domain (CBD), the first one to be reported in a fungal xylanase. The amino acid sequence of this peptide shows no homology with any known CBD

  • 47. Christakopoulos, Paul
    et al.
    Nerinckx, Wim
    University of Ghent.
    Kekos, Dimitris
    National Technical University of Athens.
    Macris, Basil
    National Technical University of Athens.
    Claeyssens, Marc
    University of Ghent.
    The alkaline xylanase III from Fusarium oxysporum F3 belongs to family F/101997In: Carbohydrate Research, ISSN 0008-6215, E-ISSN 1873-426X, Vol. 302, no 3-4, p. 191-195Article in journal (Refereed)
    Abstract [en]

    Xylanase III from Fusarium oxysporum F3 was purified to homogeneity by ion-exchange chromatography and gel filtration. The enzyme has a molecular mass of 38 kDa, an isoelectric point of 9.5, and is maximally active on oat spelt xylan at pH 7 and 45 °C with a Km of 0.8 mg/mL. The xylanase displays remarkable stability at pH 9.0. It is not active on xylotriose but hydrolyzes the 4-methylumbelliferyl glycosides of β-xylobiose and --- , and to a lower extent 4-methylumbelliferyl β-cellobioside. When acted on xylooligosaccharides and xylan, analysis of reaction mixtures by high-pressure liquid chromatography shows preferred internal glycoside cleavage. Thus the purified enzyme appears to be a true endo-β-1,4-xylanase. Partial amino acid analysis of xylanase III shows high sequence homology with xylanases of family F/10.Xylanase III from Fusarium oxysporum F3 was purified to homogeneity by ion-exchange chromatography and gel filtration, and was functionally characterised. The enzyme displays remarkable stability at pH 9.0, appears to be a true endo-β-1,4-xylanase, and shows high sequence homology with xylanases of family F/10.

  • 48.
    Christakopoulos, Paul
    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.
    Project: Organosolv Biomass Pretreatment for Flexible Fuel Production (Swedish Energy Agency)2016Other (Other (popular science, discussion, etc.))
  • 49.
    Christakopoulos, Paul
    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.
    Sjöblom, Magnus
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Topakas, Evangelos
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Project: BIOcatalytic Carbon Capture and Conversion of steel flue gas to liquid hydrocarbons (FORMAS)2016Other (Other (popular science, discussion, etc.))
  • 50.
    Christakopoulos, Paul
    et al.
    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.
    Editorial note: Advances in enzymology and enzyme engineering2012In: Computational and Structural Biotechnology Journal, ISSN 2001-0370, Vol. 2, no 3, article id e201209001Article in journal (Refereed)
123456 1 - 50 of 254
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