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  • 1.
    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)
  • 2.
    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.

  • 3.
    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

  • 4. 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.

  • 5. 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.

  • 6. Christakopoulos, Paul
    et al.
    Tzia, Constantina
    National Technical University of Athens.
    Kekos, Dimitris
    National Technical University of Athens.
    Macris, Basil J.
    National Technical University of Athens.
    Production and characterization of extracellular lipase from Calvatia gigantea1992In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 38, no 2, p. 194-197Article in journal (Refereed)
    Abstract [en]

    A number of factors affecting production of extracellular lipase by the edible fungus Calvatia gigantea were investigated. Consecutive optimization of carbon and nitrogen sources, initial pH of culture medium and growth temperature resulted in an increase in lipase activity of 87%. Under optimum conditions, activities as high as 22.4 units ml−1 of culture medium were obtained, competing favourably with most activities reported for other lipase hyperproducing microorganisms. The enzyme was optimally active at pH 7.0 and 30°C and had, at optimum pH, half-lives of 75.7 and 22.9 min at 45 and 55°C. Both high activity and kinetic characteristics of the enzyme make this process worthy of further investigation.

  • 7.
    Dimarogona, Maria
    et al.
    Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Topakas, Evangelos
    Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Sustainable Process Engineering.
    Recalcitrant polysaccharide degradation by novel oxidative biocatalysts2013In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 97, no 19, p. 8455-8465Article in journal (Refereed)
    Abstract [en]

    The classical hydrolytic mechanism for the degradation of plant polysaccharides by saprophytic microorganisms has been reconsidered after the recent landmark discovery of a new class of oxidases termed lytic polysaccharide monooxygenases (LPMOs). LPMOs are of increased biotechnological interest due to their implication in lignocellulosic biomass decomposition for the production of biofuels and high-value chemicals. They act on recalcitrant polysaccharides by a combination of hydrolytic and oxidative function, generating oxidized and non-oxidized chain ends. They are copper-dependent and require molecular oxygen and an external electron donor for their proper function. In this review, we present the recent findings concerning the mechanism of action of these oxidative enzymes and identify issues and questions to be addressed in the future

  • 8.
    Faulds, C.B.
    et al.
    Institute of Food Research, Norwich.
    Mandalari, G.
    Institute of Food Research, Norwich.
    Curto, R.B. Lo
    University of Messina.
    Bisignano, G.
    University of Messina.
    Christakopoulos, Paul
    Waldron, K.W.
    Institute of Food Research, Norwich.
    Synergy between xylanases from glycoside hydrolase family 10 and family 11 and feruloyl esterase in the release of phenolic acids from cereal arabinoxylan2006In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 71, no 5, p. 622-629Article in journal (Refereed)
    Abstract [en]

    The bioconversion of waste residues (by-products) from cereal processing industries requires the cooperation of enzymes able to degrade xylanolytic and cellulosic material. The type A feruloyl esterase from Aspergillus niger, AnFaeA, works synergistically with (1→4)-β-d-xylopyranosidases (xylanases) to release monomeric and dimeric ferulic acid (FA) from cereal cell wall-derived material. The esterase was more effective with a family 11 xylanase from Trichoderma viride in releasing FA and with a family 10 xylanase from Thermoascus aurantiacus in releasing the 5,5′ form of diferulic acid from arabinoxylan (AX) derived from brewers’ spent grain. The converse was found for the release of the phenolic acids from wheat bran-derived AXs. This may be indicative of compositional differences in AXs in cereals.

  • 9.
    Karnaouri, Anthi C
    et al.
    Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Evangelos, Topakas
    Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Cloning, expression, and characterization of a thermostable GH7 endoglucanase from Myceliophthora thermophila capable of high-consistency enzymatic liquefaction2014In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 98, no 1, p. 231-242Article in journal (Refereed)
    Abstract [en]

    An endoglucanase gene from the thermophilic fungus Myceliophthora thermophila, belonging to the glycoside hydrolase family 7, was functionally expressed in methylotrophic yeast Pichia pastoris. The putative endoglucanase from the genomic DNA was successfully cloned in P. pastoris X-33 and the recombinant enzyme was purified to its homogeneity (65 kDa) and subsequently characterized. Substrate specificity analysis revealed that the enzyme exhibits high activity on substrates containing β-1,4-glycosidic bonds such as carboxymethyl cellulose, barley β-glucan, and cello-oligosaccharides, as well as activity on xylan-containing substrates, including arabinoxylan and oat spelt xylan. MtEG7a was proved to liquefy rapidly and efficiently pretreated wheat straw, indicating its key role to the initial step of hydrolysis of high-solids lignocellulose substrates. High thermostability of the endoglucanase reflects potential commercial significance of the enzyme

  • 10. Katapodis, P.
    et al.
    Kalogeris, E.
    Kekos, D.
    Macris, J.
    Christakopoulos, Paul
    Biosynthesis of fructo-oligosaccharides by Sporotrichum thermophile during submerged batch cultivation in high sucrose media2004In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 63, no 4, p. 378-382Article in journal (Refereed)
    Abstract [en]

    A feruloyl esterase (StFAE-A) produced by Sporotrichum thermophile was purified to homogeneity. The purified homogeneous preparation of native StFAE-A exhibited a molecular mass of 57.0±1.5 kDa, with a mass of 33±1 kDa on SDS-PAGE. The pI of the enzyme was estimated by cation-exchange chromatofocusing to be at pH 3.1. The enzyme activity was optimal at pH 6.0 and 55–60 °C. The purified esterase was stable at the pH range 5.0–7.0. The enzyme retained 70% of activity after 7 h at 50 °C and lost 50% of its activity after 45 min at 55 °C and after 12 min at 60 °C. Determination of k cat/K m revealed that the enzyme hydrolyzed methyl p-coumarate 2.5- and 12-fold more efficiently than methyl caffeate and methyl ferulate, respectively. No activity on methyl sinapinate was detected. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose and it hydrolyzed 4-nitrophenyl 5-O-trans-feruloyl-α-l-arabinofuranoside (NPh-5-Fe-Araf) 2-fold more efficiently than NPh-2-Fe-Araf. Ferulic acid (FA) was efficiently released from destarched wheat bran when the esterase was incubated together with xylanase from S. thermophile (a maximum of 34% total ferulic acid released after 1 h incubation). StFAE-A by itself could release FA, but at a level almost 47-fold lower than that obtained in the presence of xylanase. The potential of StFAE-A for the synthesis of various phenolic acid esters was tested using a ternary water-organic mixture consisting of n-hexane, 1-butanol and water as a reaction system.

  • 11.
    Katsimpouras, Constantinos
    et al.
    Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Bénarouche, Anaïs
    INRA, Aix Marseille Université, UMR1163, Polytech Marseille, 13288, Marseille.
    Navarro, David
    INRA, Aix Marseille Université, UMR1163, Polytech Marseille, 13288, Marseille.
    Karpusas, Michael
    Physics Laboratory, Department of Biotechnology, Agricultural University of Athens.
    Dimarogona, Maria
    Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Berrin, Jean-Guy
    INRA, Aix Marseille Université, UMR1163, Polytech Marseille, 13288, Marseille.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Topakas, Evangelos
    Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Enzymatic synthesis of model substrates recognized by glucuronoyl esterases from Podospora anserina and Myceliophthora thermophila2014In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 98, no 12, p. 5507-5516, article id 10Article in journal (Refereed)
    Abstract [en]

    Glucuronoyl esterases (GEs) are recently discovered enzymes that are suggested to cleave the ester bond between lignin alcohols and xylan-bound 4-O-methyl-d-glucuronic acid. Although their potential use for enhanced enzymatic biomass degradation and synthesis of valuable chemicals renders them attractive research targets for biotechnological applications, the difficulty to purify natural fractions of lignin-carbohydrate complexes hampers the characterization of fungal GEs. In this work, we report the synthesis of three aryl alkyl or alkenyl d-glucuronate esters using lipase B from Candida antarctica (CALB) and their use to determine the kinetic parameters of two GEs, StGE2 from the thermophilic fungus Myceliophthora thermophila (syn. Sporotrichum thermophile) and PaGE1 from the coprophilous fungus Podospora anserina. PaGE1 was functionally expressed in the methylotrophic yeast Pichia pastoris under the transcriptional control of the alcohol oxidase (AOX1) promoter and purified to its homogeneity (63 kDa). The three d-glucuronate esters contain an aromatic UV-absorbing phenol group that facilitates the quantification of their enzymatic hydrolysis by HPLC. Both enzymes were able to hydrolyze the synthetic esters with a pronounced preference towards the cinnamyl-d-glucuronate ester. The experimental results were corroborated by computational docking of the synthesized substrate analogues. We show that the nature of the alcohol portion of the hydrolyzed ester influences the catalytic efficiency of the two GEs.

  • 12.
    Katsimpouras, Constantinos
    et al.
    Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Dimarogona, Maria
    National Technical University of Athens, Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Petropoulos, Pericles
    Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Topakas, Evangelos
    National Technical University of Athens, School of Chemical Engineering, National Technical University of Athens, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens.
    A thermostable GH26 endo-β-mannanase from Myceliophthora thermophila capable of enhancing lignocellulose degradation2016In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 100, no 19, p. 8385-8397Article in journal (Refereed)
    Abstract [en]

    The endomannanase gene em26a from the thermophilic fungus Myceliophthora thermophila, belonging to the glycoside hydrolase family 26, was functionally expressed in the methylotrophic yeast Pichia pastoris. The putative endomannanase, dubbed MtMan26A, was purified to homogeneity (60 kDa) and subsequently characterized. The optimum pH and temperature for the enzymatic activity of MtMan26A were 6.0 and 60 °C, respectively. MtMan26A showed high specific activity against konjac glucomannan and carob galactomannan, while it also exhibited high thermal stability with a half-life of 14.4 h at 60 °C. Thermostability is of great importance, especially in industrial processes where harsh conditions are employed. With the aim of better understanding its structure–function relationships, a homology model of MtMan26A was constructed, based on the crystallographic structure of a close homologue. Finally, the addition of MtMan26A as a supplement to the commercial enzyme mixture Celluclast® 1.5 L and Novozyme® 188 resulted in enhanced enzymatic hydrolysis of pretreated beechwood sawdust, improving the release of total reducing sugars and glucose by 13 and 12 %, respectively.

  • 13.
    Lezinou, V,
    et al.
    National Technical University of Athens.
    Christakopoulos, Paul
    Li, L.W.
    National Technical University of Athens.
    Kekos, D.
    National Technical University of Athens.
    Macris, B.J.
    National Technical University of Athens.
    Study of a single and mixed culture for the direct bio-conversion of sorghum carbohydrates to ethanol1995In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 43, no 3, p. 412-415Article in journal (Refereed)
    Abstract [en]

    Fusaium oxysporum F3 alone or in mixed culture with Saccharomyces cerevisiae 2541 fermented soluble and insoluble carbohydrates of sweet sorghum stalk directly to ethanol. Both microorganisms were first grown aerobically and fermented sorghum stalk to ethanol thereafter. During fermentation, insoluble carbohydrates were hydrolysed to soluble sugars by the celluloytic system of F. oxysporum. Ethanol yields as high as 24.4 and 33.5 g/100 g dry stalks were obtained by F. oxysporum and the mixed culture respectively, representing a theoretical yield enhancement of 11.6% and 53.6% respectively. The corresponding ethanol concentrations in the fermentation medium were 4.6% and 6.4% (w/v). These results clearly demonstrated that a large portion of insoluble carbohydrate from sorghum was converted by simultaneous saccharification and fermentation to ethanol, making the process promising for bioethanol production.

  • 14.
    Moukouli, Maria
    et al.
    National Technical University of Athens.
    Topakas, Evangelos
    National Technical University of Athens.
    Christakopoulos, Paul
    Cloning, characterization and functional expression of an alkalitolerant type C feruloyl esterase from Fusarium oxysporum2008In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 79, no 2, p. 245-254Article in journal (Refereed)
    Abstract [en]

    A hypothetical protein FoFaeC-12213 of Fusarium oxysporum was found to have high amino acid sequence identity with known type C feruloyl esterases (FAEs) containing a 13-amino acid conserved region flanking the characteristic G-X-S-X-G motif of a serine esterase. The putative FAE from the genomic DNA was successfully cloned in frame with the Saccharomyces cerevisiae α-factor secretion signal under the transcriptional control of the alcohol oxidase (AOX1) promoter and integrated in Pichia pastoris X-33 to confirm that the enzyme exhibits FAE activity. The molecular weight (62 kDa) and pI (6.8) were in agreement with the theoretical calculated values indicating the correct processing of the secretion signal in P. pastoris. The recombinant FAE was purified to its homogeneity and subsequently characterized using a series of model substrates including methyl esters of hydroxycinnamates, alkyl ferulates and monoferuloylated 4-nitrophenyl glycosides. The substrate specificity profiling reveals that the enzyme is a type C FAE showing broad hydrolytic activity against the four methyl esters of hydroxycinnamic acids and strong preference for the hydrolysis of n-propyl ferulate. Ferulic acid (FA) was efficiently released from destarched wheat bran when the esterase was incubated together with xylanase from Trichoderma longibrachiatum (a maximum of 67% total FA released after 1-h incubation). The esterase showed broad pH stability making it an important candidate for alkaline applications such as pulp treatment in the paper industry.

  • 15.
    Topakas, E.
    et al.
    National Technical University of Athens.
    Stamatis, H.
    University of Ioannina.
    Biely, P.
    Slovak Academy of Sciences, Bratislava.
    Christakopoulos, Paul
    Purification and characterization of a type B feruloyl esterase (StFAE-A) from the thermophilic fungus Sporotrichum thermophile2004In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 63, no 6, p. 686-690Article in journal (Refereed)
    Abstract [en]

    A feruloyl esterase (StFAE-A) produced by Sporotrichum thermophile was purified to homogeneity. The purified homogeneous preparation of native StFAE-A exhibited a molecular mass of 57.0±1.5 kDa, with a mass of 33±1 kDa on SDS-PAGE. The pI of the enzyme was estimated by cation-exchange chromatofocusing to be at pH 3.1. The enzyme activity was optimal at pH 6.0 and 55–60 °C. The purified esterase was stable at the pH range 5.0–7.0. The enzyme retained 70% of activity after 7 h at 50 °C and lost 50% of its activity after 45 min at 55 °C and after 12 min at 60 °C. Determination of k cat/K m revealed that the enzyme hydrolyzed methyl p-coumarate 2.5- and 12-fold more efficiently than methyl caffeate and methyl ferulate, respectively. No activity on methyl sinapinate was detected. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose and it hydrolyzed 4-nitrophenyl 5-O-trans-feruloyl-α-l-arabinofuranoside (NPh-5-Fe-Araf) 2-fold more efficiently than NPh-2-Fe-Araf. Ferulic acid (FA) was efficiently released from destarched wheat bran when the esterase was incubated together with xylanase from S. thermophile (a maximum of 34% total ferulic acid released after 1 h incubation). StFAE-A by itself could release FA, but at a level almost 47-fold lower than that obtained in the presence of xylanase. The potential of StFAE-A for the synthesis of various phenolic acid esters was tested using a ternary water-organic mixture consisting of n-hexane, 1-butanol and water as a reaction system

  • 16.
    Topakas, Evangelos
    et al.
    School of Chemical Engineering, National Technical University of Athens.
    Moukouli, Maria
    School of Chemical Engineering, National Technical University of Athens.
    Dimarogona, Maria
    School of Chemical Engineering, National Technical University of Athens.
    Christakopoulos, Paul
    Expression, characterization and structural modelling of a feruloyl esterase from the thermophilic fungus Myceliophthora thermophila2012In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 94, no 2, p. 399-411Article in journal (Refereed)
    Abstract [en]

    A ferulic acid esterase (FAE) from the thermophilic fungus Myceliophthora thermophila (synonym Sporotrichum thermophile), belonging to the carbohydrate esterase family 1 (CE-1), was functionally expressed in methylotrophic yeast Pichia pastoris. The putative FAE from the genomic DNA was successfully cloned in P. pastoris X-33 to confirm that the enzyme exhibits FAE activity. The recombinant FAE was purified to its homogeneity (39 kDa) and subsequently characterized using a series of model substrates including methyl esters of hydroxycinnamates, alkyl ferulates and monoferuloylated 4-nitrophenyl glycosides. The substrate specificity profiling reveals that the enzyme shows a preference for the hydrolysis of methyl caffeate and p-coumarate and a strong preference for the hydrolysis of n-butyl and iso-butyl ferulate. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose, whilst it was found capable of de-esterifying acetylated glucuronoxylans. Ferulic acid (FA) was efficiently released from destarched wheat bran when the esterase was incubated together with an M3 xylanase from Trichoderma longibrachiatum (a maximum of 41% total FA released after 1 h incubation). Prediction of the secondary structure of MtFae1a was performed in the PSIPRED server whilst modelling the 3D structure was accomplished by the use of the HH 3D structure prediction server.

  • 17.
    Topakas, Evangelos
    et al.
    National Technical University of Athens.
    Moukouli, Maria
    National Technical University of Athens.
    Dimarogona, Maria
    National Technical University of Athens.
    Vafiadi, Christina
    National Technical University of Athens.
    Christakopoulos, Paul
    Functional expression of a thermophilic glucuronoyl esterase from Sporotrichum thermophile: identification of the nucleophilic serine2010In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 87, no 5, p. 1765-1772Article in journal (Refereed)
    Abstract [en]

    A glucuronoyl esterase (GE) from the thermophilic fungus Sporotrichum thermophile, belonging to the carbohydrate esterase family 15 (CE-15), was functionally expressed in the methylotrophic yeast Pichia pastoris. The putative GE gene ge2 from the genomic DNA was successfully cloned in frame with the sequence for the Saccharomyces cerevisiae α-factor secretion signal under the transcriptional control of the alcohol oxidase (AOX1) promoter and integrated in P. pastoris X-33 to confirm that the encoded enzyme StGE2 exhibits esterase activity. The enzyme was active on substrates containing glucuronic acid methyl ester, showing optimal activity at pH 7.0 and 55°C. The esterase displayed broad pH range stability between 4–10 and temperature stability up to 50°C, rendering StGE2 a strong candidate for future biotechnological applications that require robust biocatalysts. ClustalW alignment of StGE2 with characterized GEs and selected homologous sequences, members of CE-15 family, revealed a novel consensus sequence G-C-S-R-X-G that features the characteristic serine residue involved in the generally conserved catalytic mechanism of the esterase family. The putative serine has been mutated, and the corresponding enzyme has been expressed in P. pastoris to prove that the candidate nucleophilic residue is responsible for catalyzing the enzymatic reaction.

  • 18.
    Varriale, Simona
    et al.
    Department of Chemical Sciences, Complesso Universitario Monte S. Angelo, University of Naples Federico II, Naples, Italy.
    Cerullo, Gabriella
    Department of Chemical Sciences, Complesso Universitario Monte S. Angelo, University of Naples Federico II, Naples, Italy.
    Antonopoulou, Io
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Christakopoulos, Paul
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Rova, Ulrika
    Luleå University of Technology, Department of Civil, Environmental and Natural Resources Engineering, Chemical Engineering.
    Tron, Thierry
    Centrale Marseille, CNRS, Aix Marseille Université, Marseille, France.
    Fauré, Régis
    LISBP, CNRS, INRA, INSA, Université de Toulouse, Toulouse, France.
    Jütten, Peter
    Taros Chemicals GmbH & Co. KG, Dortmund, Germany.
    Piechot, Alexander
    Taros Chemicals GmbH & Co. KG, Dortmund, Germany.
    Brás, Joana L. A.
    NzyTech LDA, Estrada Do Paco Do Lumiar Campus Do Lumiar Ed., Portugal.
    Fontes, Carlos M. G. A.
    NzyTech LDA, Estrada Do Paco Do Lumiar Campus Do Lumiar Ed., Portugal.
    Faraco, Vincenza
    Department of Chemical Sciences, Complesso Universitario Monte S. Angelo, University of Naples Federico II, Naples, Italy .
    Evolution of the feruloyl esterase MtFae1a from Myceliophthora thermophila towards improved catalysts for antioxidants synthesis2018In: Applied Microbiology and Biotechnology, ISSN 0175-7598, E-ISSN 1432-0614, Vol. 102, no 12, p. 5185-5196Article in journal (Refereed)
    Abstract [en]

    The chemical syntheses currently employed for industrial purposes, including in the manufacture of cosmetics, present limitations such as unwanted side reactions and the need for harsh chemical reaction conditions. In order to overcome these drawbacks, novel enzymes are developed to catalyze the targeted bioconversions. In the present study, a methodology for the construction and the automated screening of evolved variants library of a Type B feruloyl esterase from Myceliophthora thermophila (MtFae1a) was developed and applied to generation of 30,000 mutants and their screening for selecting the variants with higher activity than the wild-type enzyme. The library was generated by error-prone PCR of mtfae1a cDNA and expressed in Saccharomyces cerevisiae. Screening for extracellular enzymatic activity towards 4-nitrocatechol-1-yl ferulate, a new substrate developed ad hoc for high-throughput assays of feruloyl esterases, led to the selection of 30 improved enzyme variants. The best four variants and the wild-type MtFae1a were investigated in docking experiments with hydroxycinnamic acid esters using a model of 3D structure of MtFae1a. These variants were also used as biocatalysts in transesterification reactions leading to different target products in detergentless microemulsions and showed enhanced synthetic activities, although the screening strategy had been based on improved hydrolytic activity.

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