The Experts below are selected from a list of 1581 Experts worldwide ranked by ideXlab platform

Paul Christakopoulos - One of the best experts on this subject based on the ideXlab platform.

  • the crystal structure of a fusarium oxysporum Feruloyl Esterase that belongs to the tannase family
    FEBS Letters, 2020
    Co-Authors: Maria Dimarogona, Evangelos Topakas, Paul Christakopoulos, E D Chrysina
    Abstract:

    : Feruloyl Esterases are enzymes of industrial interest that catalyse the hydrolysis of the ester bond between hydroxycinnamic acids such as ferulic acid and sugars present in the plant cell wall. Although there are several structures of biochemically characterized Feruloyl Esterases available, the structural determinants of their substrate specificity are not yet fully understood. Here, we present the crystal structure of a Feruloyl Esterase from Fusarium oxysporum (FoFaeC) at 2.3 A resolution. Similar to the two other tannase-like Feruloyl Esterases, FoFaeC features a large lid domain covering the active site with potential regulatory role and a disulphide bond that brings together the serine and histidine of the catalytic triad. Differences are mainly observed in the metal coordination site and the substrate binding pocket. ENZYMES: E.C.3.1.1.73. DATABASES: The sequence of FoFaeC has been deposited with UniProt with accession code A0A1D3S5H0_FUSOX and the atomic coordinates of the three-dimensional structure with Protein Data Bank, with PDB code: 6FAT.

  • cross linked enzyme aggregates of Feruloyl Esterase preparations from thermothelomyces thermophila and talaromyces wortmannii
    Catalysts, 2018
    Co-Authors: Anastasia Zerva, Io Antonopoulou, Josefine Enman, Laura Iancu, Ulrika Rova, Paul Christakopoulos
    Abstract:

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

  • Feruloyl Esterase catalysed synthesis of glycerol sinapate using ionic liquids mixtures
    Journal of Biotechnology, 2009
    Co-Authors: Christina Vafiadi, Craig B Faulds, Evangelos Topakas, Victoria R Nahmias, Paul Christakopoulos
    Abstract:

    Abstract The ability of a Feruloyl Esterase (AnFaeA), either in free or immobilised (cross-linked enzyme aggregates) form, to catalyse the esterification of glycerol, a major by-product of the biodiesel industry, with sinapic acid was studied in four hexafluorophosphate anion-containing ionic liquids: ([Bmim][PF 6 ], [Omim][PF 6 ], [C 2 OHmim][PF 6 ] and [C 5 O 2 mim][PF 6 ]). Such ionic liquids are considered ‘green’ reaction systems. The synthetic reaction was optimised in [C 2 OHmim][PF 6 ] and the highest conversion yield was 72.5 ± 2.1%, while, at the same reaction conditions in [C 5 O 2 mim] [PF 6 ], a similar conversion yield was obtained (76.7 ± 1.5%). AnFaeA was active in its free and immobilised form, with the latter retaining a part of its synthetic activity after 5 consecutive 24 h-period reaction cycles. Sinapic acid was esterified to one of the primary hydroxyl groups of glycerol and retained, after esterification, 63.1 ± 0.3% and 89.5 ± 1.1% of its antioxidant activity against low-density lipoprotein oxidation, when added at concentrations of 10 and 60 μM, respectively, in the assay mixture.

  • the Feruloyl Esterase system of talaromyces stipitatus determining the hydrolytic and synthetic specificity of tsfaec
    Journal of Biotechnology, 2006
    Co-Authors: Christina Vafiadi, Paul Christakopoulos, Evangelos Topakas, Craig B Faulds
    Abstract:

    The active site of the recombinant Talaromyces stipitatus type-C Feruloyl Esterase (TsFaeC) was probed using a series of C1–C4 alkyl ferulates and methyl esters of phenylalkanoic and cinnamic acids. The enzyme was active on 23 of the 34 substrates tested. Lengthening or shortening the aliphatic side chain while maintaining the same aromatic substitutions completely abolished the enzyme activity. Maintaining the phenylpropenoate structure but altering the substitutions of the aromatic ring demonstrated the importance of hydroxyl groups on meta and/or para position of the benzoic ring. The highest catalytic efficiency of TsFaeC for methyl cinnamates was shown on methyl 3,4-dihydroxy cinnamate and on its hydro form (3,4-dihydroxy-phenyl-propionate). Maintaining the ferulate structure but altering the esterified alkyl group, the comparison of kcat and kcat/Km values showed that the enzyme hydrolysed faster and more efficiently than ethyl ferulate. Alkyl ferulates were applied also for substrate selectivity mapping of Feruloyl Esterase to catalyze Feruloyl group transfer to l-arabinose, using as a reaction system a ternary water–organic mixture consisting of n-hexane, t-butanol and water. The reaction parameters affecting the Feruloylation rate and the conversion of the enzymatic synthesis, such as the composition of the reaction media, temperature, substrate and enzyme concentration have been investigated.

  • sporotrichum thermophile type c Feruloyl Esterase stfaec purification characterization and its use for phenolic acid sugar ester synthesis
    Enzyme and Microbial Technology, 2005
    Co-Authors: Evangelos Topakas, Haralambos Stamatis, Christina Vafiadi, Paul Christakopoulos
    Abstract:

    Abstract A Feruloyl Esterase (StFaeC) produced by Sporotrichum thermophile was purified to homogeneity. The native StFaeC was homodimer with a subunit of M r 23,000 and pI 3.1. The enzyme activity was optimal at pH 6.0 and 55 °C. The Esterase displayed remarkable stability at pH 10.0 and retained 50% of its activity after 133 and 55 min at 55 and 60 °C, respectively. Determination of k cat / K m revealed that the enzyme had a broad spectrum of activity against the (hydroxyl) cinnamate esters indicating a type C Feruloyl Esterase. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose and hydrolysed 4-nitrophenyl-5- O - trans -Feruloyl-α- l -arabinofuranoside three times more efficiently than 4-nitrophenyl-2- O - trans -Feruloyl-α- l -arabinofuranoside. Ferulic acid was efficiently released from wheat bran when the Esterase was incubated together with xylanase from S. thermophile (a maximum of 41% total ferulic acid released after 1 h incubation). StFaeC by itself could release FA but at a level almost 10-fold lower than that obtained in the presence of xylanase. The potential of StFaeC for the synthesis of various phenolic acid esters was examined using as a reaction system a ternary water–organic mixture consisting of n-hexane, 1-butanol and water. Also StFaeC catalyzed the transfer of the Feruloyl group to l -arabinose in a similar system using t -butanol, with about a 40% conversion of l -arabinose to Feruloylated derivative was achieved. This work is the first example of enzymatic Feruloylation of a carbohydrate.

Marcel Asther - One of the best experts on this subject based on the ideXlab platform.

  • Phylogenetic analysis of the Aspergillus niger aggregate in relation to Feruloyl Esterase activity.
    Research in Microbiology, 2007
    Co-Authors: Frédéric Giraud, Marcel Asther, Mireille Haon, Joëlle Dupont, Ourdia Bouzid, Olivier Alibeu, David Navarro, Lucile Sage, Françoise Seigle-murandi, Laurence Lesage-meessen
    Abstract:

    Species of the Aspergillus niger aggregate are known to produce Feruloyl Esterases, enzymes involved in the degradation of cell wall polymers. However, species delineation is difficult in these fungi. We combined AFLP analysis with ITS rDNA and b-tubulin sequencing to characterize the isolates of this aggregate in terms of Feruloyl Esterase production. A preliminary re-examination of isolates based on comparison of ITS rDNA and b-tubulin sequences with those of typical taxa deposited in international collections led us to re-identify the isolates as members of the species A. niger, A. foetidus and A. tubingensis. Molecular clustering based on b-tubulin data and AFLP analysis showed that the strains of A. niger formed a homogenous phylogenetic group distinguished by either zero or type A Feruloyl Esterase activity, while strains A. foetidus and A. tubingensis exhibited type B Feruloyl Esterase activity when grown on sugar beet pulp. 2007 Elsevier Masson SAS. All rights reserved.

  • tracking the connection between evolutionary and functional shifts using the fungal lipase Feruloyl Esterase a family
    BMC Evolutionary Biology, 2006
    Co-Authors: Eric Record, Marcel Asther, Laurence Lesagemeessen, Anthony Levasseur, Philippe Gouret, Pierre Pontarotti
    Abstract:

    Background There have been many claims of adaptive molecular evolution, but what role does positive selection play in functional divergence? The aim of this study was to test the relationship between evolutionary and functional shifts with special emphasis on the role of the environment. For this purpose, we studied the fungal lipase/Feruloyl Esterase A family, whose functional diversification makes it a very promising candidate.

  • respective importance of protein folding and glycosylation in the thermal stability of recombinant Feruloyl Esterase a
    FEBS Letters, 2006
    Co-Authors: Isabelle Benoit, Eric Record, Marcel Asther, G Sulzenbacher, Laurence Marmuse, G Parsiegla, Isabelle Gimbert, Christophe Bignon
    Abstract:

    The thermal stability of four molecular forms (native, refolded, glycosylated, non-glycosylated) of Feruloyl Esterase A (FAEA) was studied. From the most to the least thermo-resistant, the four molecular species ranked as follows: (i) glycosylated form produced native, (ii) non-glycosylated form produced native, (iii) non-glycosylated form produced as inclusion bodies and refolded, and (iv) glycosylated form produced native chemically denatured and then refolded. On the basis of these results and of crystal structure data, we discuss the respective importance of protein folding and glycosylation in the thermal stability of recombinant FAEA.

  • enzymatic saccharification of wheat straw for bioethanol production by a combined cellulase xylanase and Feruloyl Esterase treatment
    Enzyme and Microbial Technology, 2006
    Co-Authors: M G Tabka, Marcel Asther, I Herpoelgimbert, F Monod, Jean-claude Sigoillot
    Abstract:

    The focus of this study was to improve conditions of use of fungal lignocellulolytic enzymes for conversion of lignocellulosic biomass to fermentable sugars for the production of bioethanol. Wheat straw was pre-treated by acid treatment with diluted sulfuric acid followed by steam explosion. Several enzymatic treatments implementing hydrolases (cellulases and xylanases from Trichoderma reesei, recombinant Feruloyl Esterase (FAE) from Aspergillus niger and oxidoreductases (laccases from Pycnoporus cinnabarinus) were investigated to the saccharification of exploded wheat straw. A synergistic effect between cellulases, FAE and xylanase was proven under a critical enzymatic concentration (10 U/g of cellulases, 3 U/g of xylanase and 10 U/g of FAE). The yield of enzymatic hydrolysis was enhanced by increasing the temperature from 37 °C to 50 °C and addition of a non-ionic surfactant, Tween 20. Optimisation of enzymatic hydrolyses allowed the use of lower quantities of enzymes and improved the cost effectiveness of the process.

  • Feruloyl Esterase utilization for simultaneous processing of nonwood plants into phenolic compounds and pulp fibers
    Journal of Agricultural and Food Chemistry, 2006
    Co-Authors: Sandra Tapin, Marcel Asther, Jean-claude Sigoillot, Michel Petitconil
    Abstract:

    Wheat and oilseed flax straws were studied as raw material for papermaking. Two different aspects were investigated to valorize these agricultural byproducts:  the capacity to recover some phenolic compounds and the use of the resulting cellulose fibers in papermaking. Straw phenolic compound composition was analyzed to determine the different accessible molecules and their available quantity. Ferulic acid, coumaric acid, vanillic acid, and vanillin were found in both wheat and oilseed flax straws. To enhance the release of these phenolic compounds, enzymatic treatments with Feruloyl Esterase and xylanase, two enzymes known for their role in lignin destructuration, were tested. These treatments increased the amount of phenolic compounds released, destructured hemicellulose and lignin, and improved the soda cooking conditions of pulps with the reduction of chemical charge need in the papermaking process. Phenolic compound production from this process could enhance the cost-effectiveness of papermaking from...

Evangelos Topakas - One of the best experts on this subject based on the ideXlab platform.

  • the crystal structure of a fusarium oxysporum Feruloyl Esterase that belongs to the tannase family
    FEBS Letters, 2020
    Co-Authors: Maria Dimarogona, Evangelos Topakas, Paul Christakopoulos, E D Chrysina
    Abstract:

    : Feruloyl Esterases are enzymes of industrial interest that catalyse the hydrolysis of the ester bond between hydroxycinnamic acids such as ferulic acid and sugars present in the plant cell wall. Although there are several structures of biochemically characterized Feruloyl Esterases available, the structural determinants of their substrate specificity are not yet fully understood. Here, we present the crystal structure of a Feruloyl Esterase from Fusarium oxysporum (FoFaeC) at 2.3 A resolution. Similar to the two other tannase-like Feruloyl Esterases, FoFaeC features a large lid domain covering the active site with potential regulatory role and a disulphide bond that brings together the serine and histidine of the catalytic triad. Differences are mainly observed in the metal coordination site and the substrate binding pocket. ENZYMES: E.C.3.1.1.73. DATABASES: The sequence of FoFaeC has been deposited with UniProt with accession code A0A1D3S5H0_FUSOX and the atomic coordinates of the three-dimensional structure with Protein Data Bank, with PDB code: 6FAT.

  • Feruloyl Esterase catalysed synthesis of glycerol sinapate using ionic liquids mixtures
    Journal of Biotechnology, 2009
    Co-Authors: Christina Vafiadi, Craig B Faulds, Evangelos Topakas, Victoria R Nahmias, Paul Christakopoulos
    Abstract:

    Abstract The ability of a Feruloyl Esterase (AnFaeA), either in free or immobilised (cross-linked enzyme aggregates) form, to catalyse the esterification of glycerol, a major by-product of the biodiesel industry, with sinapic acid was studied in four hexafluorophosphate anion-containing ionic liquids: ([Bmim][PF 6 ], [Omim][PF 6 ], [C 2 OHmim][PF 6 ] and [C 5 O 2 mim][PF 6 ]). Such ionic liquids are considered ‘green’ reaction systems. The synthetic reaction was optimised in [C 2 OHmim][PF 6 ] and the highest conversion yield was 72.5 ± 2.1%, while, at the same reaction conditions in [C 5 O 2 mim] [PF 6 ], a similar conversion yield was obtained (76.7 ± 1.5%). AnFaeA was active in its free and immobilised form, with the latter retaining a part of its synthetic activity after 5 consecutive 24 h-period reaction cycles. Sinapic acid was esterified to one of the primary hydroxyl groups of glycerol and retained, after esterification, 63.1 ± 0.3% and 89.5 ± 1.1% of its antioxidant activity against low-density lipoprotein oxidation, when added at concentrations of 10 and 60 μM, respectively, in the assay mixture.

  • the Feruloyl Esterase system of talaromyces stipitatus determining the hydrolytic and synthetic specificity of tsfaec
    Journal of Biotechnology, 2006
    Co-Authors: Christina Vafiadi, Paul Christakopoulos, Evangelos Topakas, Craig B Faulds
    Abstract:

    The active site of the recombinant Talaromyces stipitatus type-C Feruloyl Esterase (TsFaeC) was probed using a series of C1–C4 alkyl ferulates and methyl esters of phenylalkanoic and cinnamic acids. The enzyme was active on 23 of the 34 substrates tested. Lengthening or shortening the aliphatic side chain while maintaining the same aromatic substitutions completely abolished the enzyme activity. Maintaining the phenylpropenoate structure but altering the substitutions of the aromatic ring demonstrated the importance of hydroxyl groups on meta and/or para position of the benzoic ring. The highest catalytic efficiency of TsFaeC for methyl cinnamates was shown on methyl 3,4-dihydroxy cinnamate and on its hydro form (3,4-dihydroxy-phenyl-propionate). Maintaining the ferulate structure but altering the esterified alkyl group, the comparison of kcat and kcat/Km values showed that the enzyme hydrolysed faster and more efficiently than ethyl ferulate. Alkyl ferulates were applied also for substrate selectivity mapping of Feruloyl Esterase to catalyze Feruloyl group transfer to l-arabinose, using as a reaction system a ternary water–organic mixture consisting of n-hexane, t-butanol and water. The reaction parameters affecting the Feruloylation rate and the conversion of the enzymatic synthesis, such as the composition of the reaction media, temperature, substrate and enzyme concentration have been investigated.

  • sporotrichum thermophile type c Feruloyl Esterase stfaec purification characterization and its use for phenolic acid sugar ester synthesis
    Enzyme and Microbial Technology, 2005
    Co-Authors: Evangelos Topakas, Haralambos Stamatis, Christina Vafiadi, Paul Christakopoulos
    Abstract:

    Abstract A Feruloyl Esterase (StFaeC) produced by Sporotrichum thermophile was purified to homogeneity. The native StFaeC was homodimer with a subunit of M r 23,000 and pI 3.1. The enzyme activity was optimal at pH 6.0 and 55 °C. The Esterase displayed remarkable stability at pH 10.0 and retained 50% of its activity after 133 and 55 min at 55 and 60 °C, respectively. Determination of k cat / K m revealed that the enzyme had a broad spectrum of activity against the (hydroxyl) cinnamate esters indicating a type C Feruloyl Esterase. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2 linkages of arabinofuranose and hydrolysed 4-nitrophenyl-5- O - trans -Feruloyl-α- l -arabinofuranoside three times more efficiently than 4-nitrophenyl-2- O - trans -Feruloyl-α- l -arabinofuranoside. Ferulic acid was efficiently released from wheat bran when the Esterase was incubated together with xylanase from S. thermophile (a maximum of 41% total ferulic acid released after 1 h incubation). StFaeC by itself could release FA but at a level almost 10-fold lower than that obtained in the presence of xylanase. The potential of StFaeC for the synthesis of various phenolic acid esters was examined using as a reaction system a ternary water–organic mixture consisting of n-hexane, 1-butanol and water. Also StFaeC catalyzed the transfer of the Feruloyl group to l -arabinose in a similar system using t -butanol, with about a 40% conversion of l -arabinose to Feruloylated derivative was achieved. This work is the first example of enzymatic Feruloylation of a carbohydrate.

  • mapping the hydrolytic and synthetic selectivity of a type c Feruloyl Esterase stfaec from sporotrichum thermophile using alkyl ferulates
    Tetrahedron-asymmetry, 2005
    Co-Authors: Christina Vafiadi, Evangelos Topakas, Ken K Y Wong, Ian D Suckling, Paul Christakopoulos
    Abstract:

    Abstract The active site of Sporotrichum thermophile type C Feruloyl Esterase (StFaeC) was probed using a series of C1–C4 alkyl ferulates. The affinities for straight and branched alkyl ferulates were demonstrated by the Km values of 1.64–0.51 and 0.19–0.1, respectively. Comparison of kcat and kcat/Km values shows that the enzyme hydrolyzed n-propyl ferulate faster and iso-propyl ferulate more efficiently. Alkyl ferulates were applied also for substrate selectivity mapping of Feruloyl Esterase to catalyze Feruloyl group transfer to l -arabinose, using as a reaction system a ternary water–organic mixture consisting of n-hexane, t-butanol and water. Lengthening the aliphatic side chain was the most significant factor causing lower synthetic activity of the enzyme. The reaction parameters affecting the Feruloylation rate and the conversion of the enzymatic process, such as the temperature and substrate concentration have been investigated. Under identical reaction conditions, the enzyme Feruloylated other monosaccharides such as d -arabinose, d -glucose, d -xylose, d -mannose, d -fructose, d -galactose, d -ribose and model substrates such as 4-nitrophenyl α- l -arabinofuranoside and 4-nitrophenyl α- l -arabinopyranoside.

Lisbeth Olsson - One of the best experts on this subject based on the ideXlab platform.

  • immobilization of bacterial Feruloyl Esterase on mesoporous silica particles and enhancement of synthetic activity by hydrophobic modified surface
    Bioresource Technology, 2019
    Co-Authors: Sunli Chong, Carlos M G A Fontes, Vânia Cardoso, Joana L A Bras, Milene Zezzi Do Valle Gomes, Lisbeth Olsson
    Abstract:

    Here, we demonstrated the immobilization of bacterial Feruloyl Esterase (FAE) from Butyrivibrio sp. XPD2006, Lactobacillus crispatus, Butyrivibrio sp. AE2015, Ruminococcus albus, Cellulosilyticum ruminicola and Clostridium cellulovorans on SBA-15 and their ability to synthesize butyl ferulate (BFA). The BFae2 from Butyrivibrio sp. XPD2006 showed the best catalytic efficiency. High BFA yield was produced when the immobilization of BFae2 took place with a high protein loading and narrow pore sized SBA-15, suggesting alteration of enzyme behavior due to the crowding environment in SBA-15. Grafting of SBA-15 with octyl moieties led to shrinking pore size and resulted in 2.5-fold increment of BFA activity compared to the free enzyme and 70%mol BFA was achieved. The BFae2 encapsulated in hydrophobic-modified SBA-15 endured up to seven reaction cycles while the BFA activity remained above 60%. This is the first report showing the superior performance of hydrophobic-modified surface to entrap FAE to produce fatty phenolic esters.

  • glycosylation influences activity stability and immobilization of the Feruloyl Esterase 1a from myceliophthora thermophila
    AMB Express, 2019
    Co-Authors: Cyrielle Bonzom, Sunli Chong, Silvia Huttner, Ekaterina Mirgorodskaya, Stefan Uthoff, Alexander Steinbuchel, Raymond M D Verhaert, Lisbeth Olsson
    Abstract:

    Heterologous protein production is widely used in industrial biotechnology. However, using non-native production hosts can lead to enzymes with altered post-translational modifications, such as glycosylation. We have investigated how production in a non-native host affects the physicochemical properties and enzymatic activity of a Feruloyl Esterase from Myceliophthora thermophila, MtFae1a. The enzyme was produced in two microorganisms that introduce glycosylation (M. thermophila and Pichia pastoris) and in Escherichia coli (non-glycosylated). Mass spectrometric analysis confirmed the presence of glycosylation and revealed differences in the lengths of glycan chains between the enzymes produced in M. thermophila and P. pastoris. The melting temperature and the optimal temperature for activity of the non-glycosylated enzyme were considerably lower than those of the glycosylated enzymes. The three MtFae1a versions also exhibited differences in specific activity and specificity. The catalytic efficiency of the glycosylated enzymes were more than 10 times higher than that of the non-glycosylated one. In biotechnology, immobilization is often used to allow reusing enzyme and was investigated on mesoporous silica particles. We found the binding kinetics and immobilization yield differed between the enzyme versions. The largest differences were observed when comparing enzymes with and without glycosylation, but significant variations were also observed between the two differently glycosylated enzymes. We conclude that the biotechnological value of an enzyme can be optimized for a specific application by carefully selecting the production host.

  • Feruloyl Esterase immobilization in mesoporous silica particles and characterization in hydrolysis and transesterification
    BMC Biochemistry, 2018
    Co-Authors: Cyrielle Bonzom, Laura Schild, Hanna Gustafsson, Lisbeth Olsson
    Abstract:

    Enzymes display high reactivity and selectivity under natural conditions, but may suffer from decreased efficiency in industrial applications. A strategy to address this limitation is to immobilize the enzyme. Mesoporous silica materials offer unique properties as an immobilization support, such as high surface area and tunable pore size. The performance of a commercially available Feruloyl Esterase, E-FAERU, immobilized on mesoporous silica by physical adsorption was evaluated for its transesterification ability. We optimized the immobilization conditions by varying the support pore size, the immobilization buffer and its pH. Maximum loading and maximum activity were achieved at different pHs (4.0 and 6.0 respectively). Selectivity, shown by the transesterification/hydrolysis products molar ratio, varied more than 3-fold depending on the reaction buffer used and its pH. Under all conditions studied, hydrolysis was the dominant activity of the enzyme. pH and water content had the greatest influence on the enzyme selectivity and activity. Determined kinetic parameters of the enzyme were obtained and showed that Km was not affected by the immobilization but kcat was reduced 10-fold when comparing the free and immobilized enzymes. Thermal and pH stabilities as well as the reusability were investigated. The immobilized biocatalyst retained more than 20% of its activity after ten cycles of transesterification reaction. These results indicate that this enzyme is more suited for hydrolysis reactions than transesterification despite good reusability. Furthermore, it was found that the immobilization conditions are crucial for optimal enzyme activity as they can alter the enzyme performance.

  • Feruloyl Esterase immobilization in mesoporous silica particles and characterization in hydrolysis and transesterification
    BMC Biochemistry, 2018
    Co-Authors: Cyrielle Bonzom, Laura Schild, Hanna Gustafsson, Lisbeth Olsson
    Abstract:

    Background Enzymes display high reactivity and selectivity under natural conditions, but may suffer from decreased efficiency in industrial applications. A strategy to address this limitation is to immobilize the enzyme. Mesoporous silica materials offer unique properties as an immobilization support, such as high surface area and tunable pore size. Results The performance of a commercially available Feruloyl Esterase, E-FAERU, immobilized on mesoporous silica by physical adsorption was evaluated for its transesterification ability. We optimized the immobilization conditions by varying the support pore size, the immobilization buffer and its pH. Maximum loading and maximum activity were achieved at different pHs (4.0 and 6.0 respectively). Selectivity, shown by the transesterification/hydrolysis products molar ratio, varied more than 3-fold depending on the reaction buffer used and its pH. Under all conditions studied, hydrolysis was the dominant activity of the enzyme. pH and water content had the greatest influence on the enzyme selectivity and activity. Determined kinetic parameters of the enzyme were obtained and showed that K_m was not affected by the immobilization but k_cat was reduced 10-fold when comparing the free and immobilized enzymes. Thermal and pH stabilities as well as the reusability were investigated. The immobilized biocatalyst retained more than 20% of its activity after ten cycles of transesterification reaction. Conclusions These results indicate that this enzyme is more suited for hydrolysis reactions than transesterification despite good reusability. Furthermore, it was found that the immobilization conditions are crucial for optimal enzyme activity as they can alter the enzyme performance.

Craig B Faulds - One of the best experts on this subject based on the ideXlab platform.

  • Feruloyl Esterase catalysed synthesis of glycerol sinapate using ionic liquids mixtures
    Journal of Biotechnology, 2009
    Co-Authors: Christina Vafiadi, Craig B Faulds, Evangelos Topakas, Victoria R Nahmias, Paul Christakopoulos
    Abstract:

    Abstract The ability of a Feruloyl Esterase (AnFaeA), either in free or immobilised (cross-linked enzyme aggregates) form, to catalyse the esterification of glycerol, a major by-product of the biodiesel industry, with sinapic acid was studied in four hexafluorophosphate anion-containing ionic liquids: ([Bmim][PF 6 ], [Omim][PF 6 ], [C 2 OHmim][PF 6 ] and [C 5 O 2 mim][PF 6 ]). Such ionic liquids are considered ‘green’ reaction systems. The synthetic reaction was optimised in [C 2 OHmim][PF 6 ] and the highest conversion yield was 72.5 ± 2.1%, while, at the same reaction conditions in [C 5 O 2 mim] [PF 6 ], a similar conversion yield was obtained (76.7 ± 1.5%). AnFaeA was active in its free and immobilised form, with the latter retaining a part of its synthetic activity after 5 consecutive 24 h-period reaction cycles. Sinapic acid was esterified to one of the primary hydroxyl groups of glycerol and retained, after esterification, 63.1 ± 0.3% and 89.5 ± 1.1% of its antioxidant activity against low-density lipoprotein oxidation, when added at concentrations of 10 and 60 μM, respectively, in the assay mixture.

  • the Feruloyl Esterase system of talaromyces stipitatus determining the hydrolytic and synthetic specificity of tsfaec
    Journal of Biotechnology, 2006
    Co-Authors: Christina Vafiadi, Paul Christakopoulos, Evangelos Topakas, Craig B Faulds
    Abstract:

    The active site of the recombinant Talaromyces stipitatus type-C Feruloyl Esterase (TsFaeC) was probed using a series of C1–C4 alkyl ferulates and methyl esters of phenylalkanoic and cinnamic acids. The enzyme was active on 23 of the 34 substrates tested. Lengthening or shortening the aliphatic side chain while maintaining the same aromatic substitutions completely abolished the enzyme activity. Maintaining the phenylpropenoate structure but altering the substitutions of the aromatic ring demonstrated the importance of hydroxyl groups on meta and/or para position of the benzoic ring. The highest catalytic efficiency of TsFaeC for methyl cinnamates was shown on methyl 3,4-dihydroxy cinnamate and on its hydro form (3,4-dihydroxy-phenyl-propionate). Maintaining the ferulate structure but altering the esterified alkyl group, the comparison of kcat and kcat/Km values showed that the enzyme hydrolysed faster and more efficiently than ethyl ferulate. Alkyl ferulates were applied also for substrate selectivity mapping of Feruloyl Esterase to catalyze Feruloyl group transfer to l-arabinose, using as a reaction system a ternary water–organic mixture consisting of n-hexane, t-butanol and water. The reaction parameters affecting the Feruloylation rate and the conversion of the enzymatic synthesis, such as the composition of the reaction media, temperature, substrate and enzyme concentration have been investigated.

  • barley contains two cationic acetylxylan Esterases and one anionic Feruloyl Esterase
    Cereal Chemistry, 2005
    Co-Authors: Craig B Faulds, Charles W Bamforth
    Abstract:

    ABSTRACT Various carbohydrate-active Esterases are detected in extracts of malted barley when analyzed by polyacrylamide gel electophoresis. The slowest migrating and most heat-resistant of these are relatively cationic acetylxylan Esterases. Two such activities, one with a high affinity for Esterase substrates including acetylated xylan, and one with a low affinity, are indicated. These enzymes did not hydrolyze methyl ferulate. A relatively heat-labile anionic Feruloyl Esterase has also been purified. It has some, albeit low, ability to act on acetylated xylan. The Feruloyl Esterase effects extensive release of ferulate from endosperm cell walls isolated from barley, whereas the acetylxylan Esterases are only capable of very limited release of acetate.

  • probing the determinants of substrate specificity of a Feruloyl Esterase anfaea from aspergillus niger
    FEBS Journal, 2005
    Co-Authors: Craig B Faulds, J Sanzaparicio, Rafael Molina, Nathalie Juge, Ramon Gonzalez, Fiona Husband, Juan A Hermoso
    Abstract:

    Feruloyl Esterases hydrolyse phenolic groups involved in the cross-linking of arabinoxylan to other polymeric structures. This is important for opening the cell wall structure making material more accessible to glycoside hydrolases. Here we describe the crystal structure of inactive S133A mutant of type-A Feruloyl Esterase from Aspergillus niger (AnFaeA) in complex with a Feruloylated trisaccharide substrate. Only the ferulic acid moiety of the substrate is visible in the electron density map, showing interactions through its OH and OCH3 groups with the hydroxyl groups of Tyr80. The importance of aromatic and polar residues in the activity of AnFaeA was also evaluated using site-directed mutagenesis. Four mutant proteins were heterologously expressed in Pichia pastoris, and their kinetic properties determined against methyl esters of ferulic, sinapic, caffeic and p-coumaric acid. The kcat of Y80S, Y80V, W260S and W260V was drastically reduced compared to that of the wild-type enzyme. However, the replacement of Tyr80 and Trp260 with smaller residues broadened the substrate specificity of the enzyme, allowing the hydrolysis of methyl caffeate. The role of Tyr80 and Trp260 in AnFaeA are discussed in light of the three-dimensional structure.

  • the crystal structure of Feruloyl Esterase a from aspergillus niger suggests evolutive functional convergence in Feruloyl Esterase family
    Journal of Molecular Biology, 2004
    Co-Authors: Juan A Hermoso, J Sanzaparicio, Rafael Molina, Nathalie Juge, Ramon Gonzalez, Craig B Faulds
    Abstract:

    Abstract As a component of the array of enzymes produced by micro-organisms to deconstruct plant cell walls, Feruloyl Esterases hydrolyze phenolic groups involved in the cross-linking of arabinoxylan to other polymeric structures. This is important for opening the cell wall structure, making material more accessible to glycosyl hydrolases. Here, we describe the first crystal structure of the non-modular type-A Feruloyl Esterase from Aspergillus niger (AnFaeA) solved at 2.5 A resolution. AnFaeA displays an α/β hydrolase fold similar to that found in fungal lipases and different from that reported for other Feruloyl Esterases. Crystallographic and site-directed mutagenesis studies allow us to identify the catalytic triad (Ser133-His247-Asp194) that forms the catalytic machinery of this enzyme. The active-site cavity is confined by a lid (residues 68–80), on the analogy of lipases, and by a loop (residues 226–244) that confers plasticity to the substrate-binding site. The lid presents a high ratio of polar residues, which in addition to a unique N -glycosylation site stabilises the lid in an open conformation, conferring the Esterase character to this enzyme. A putative model for bound 5,5′-diferulic acid-linked arabinoxylan has been built, pointing to the more relevant residues involved in substrate recognition. Comparison with structurally related lipases reveals that subtle amino acid and conformational changes within a highly conserved protein fold may produce protein variants endowed with new enzymatic properties, while comparison with functionally related proteins points to a functional convergence after evolutionary divergence within the Feruloyl Esterases family.