Ferulic acid is esterified to glucuronoarabinoxylans in pineapple cell walls

The ester-linkage of ferulic acid (mainly E) to polysaccharides in primary cell walls of pineapple fruit (Ananas comosus) (Bromeliaceae) was investigated by treating a cell-wall preparation with 'Driselase' which contains a mixture of endo- and exo-glycanases, but no hydroxycinnamoyl esterase activity. The most abundant feruloyl oligosaccharide released was O-[5-O-(E-feruloyl)-alpha-L-arabinofuranosyl](1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (FAXX). This indicated that the ferulic acid is ester-linked to glucuronoarabinoxylans in the same way as in the primary walls of grasses and cereals (Poaceae). Glucuronoarabinoxylans are the major non-cellulosic polysaccharides in the pineapple cell walls.     

Purification and partial characterization of two feruloyl esterases from the anaerobic fungus Neocallimastix strain MC-2.

Two extracellular feruloyl esterases (FAE-I and FAE-II) produced by the anaerobic fungus Neocallimastix strain MC-2 which cleave ferulic acid from O-(5-O-[(E)-feruloyl]-alpha-L- arabinofuranosyl)-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (FAXX) were purified. The molecular masses of FAE-I and FAE-II were 69 and 24 kDa, respectively, under both denaturing and nondenaturing conditions. Apparent Km and maximum rate of hydrolysis with FAXX were 31.9 microM and 2.9 mumol min-1 mg-1 for FAE-I and 9.6 microM and 11.4 mumol min-1 mg-1 for FAE-II. FAE-II was specific for FAXX, but FAE-I hydrolyzed FAXX and PAXX, the equivalent p-coumaroyl ester, at a maximum rate of metabolism ratio of 3:1.     

Purification and characterization of a feruloyl esterase from the intestinal bacterium Lactobacillus acidophilus

Dietary ferulic acid (FA), a significant antioxidant substance, is currently the subject of extensive research. FA in cereals exists mainly as feruloylated sugar ester. To release FA from food matrices, it is necessary to cleave ester cross-linking by feruloyl esterase (FAE) (hydroxycinnamoyl esterase; EC 3.1.1.73). In the present study, the FAE from a human typical intestinal bacterium, Lactobacillus acidophilus, was isolated, purified, and characterized for the first time. The enzyme was purified in successive steps including hydrophobic interaction chromatography and anion-exchange chromatography. The purified FAE appeared as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular mass of 36 kDa. It has optimum pH and temperature characteristics (5.6 and 37 degrees C, respectively). The metal ions Cu(2+) and Fe(3+) (at a concentration of 5 mmol liter(-1)) inhibited FAE activity by 97.25 and 94.80%, respectively. Under optimum pH and temperature with 5-O-feruloyl-L-arabinofuranose (FAA) as a substrate, the enzyme exhibited a K(m) of 0.0953 mmol liter(-1) and a V(max) of 86.27 mmol liter(-1) min(-1) mg(-1) of protein. Furthermore, the N-terminal amino acid sequence of the purified FAE was found to be A R V E K P R K V I L V G D G A V G S T. The FAE released FA from O-(5-O-feruloyl-alpha-L-arabinofuranosyl)-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (FAXX) and FAA obtained from refined corn bran. Moreover, it released two times more FA from FAXX in the presence of added xylanase.     

Do domain interactions of glycosyl hydrolases from Clostridium thermocellum contribute to protein thermostability?

Cellulolytic and hemicellulolytic enzymes usually have a domain composition. The mutual influence of a cellulose-binding domain and a catalytic domain was investigated with cellobiohydrolase CelK and xylanase XynZ from Clostridium thermocellum. CelK is composed of an N-terminal family IV cellulose-binding domain (CBDIV(CelK)), a family 9 glycosyl hydrolase domain (Gh9(CelK)) and a dockerin domain (DD). CelK without the DD, (CBDIV-Gh9)(CelK) and CBDIV(CelK) bound cellulose. The thermostability of (CBDIV-Gh9)(CelK) was significantly higher than that of CBDIV(CelK) and Gh9(CelK). The temperature optima of (CBDIV-Gh9)(CelK) and Gh9(CelK) were 65 and 45 degrees C, respectively. XynZ consists of an N-terminal feruloyl esterase domain (FAE(XynZ)), a linker (L), a family VI CBD (CBDVI(XynZ)), a DD and a xylanase domain. FAE(XynZ) and (FAE-L-CBDVI)(XynZ), used in the present study did not bind cellulose, but both were highly thermostable. Replacement of CBDVI(XynZ) with CBDIV(CelK) resulted in chimeras with feruloyl esterase activity and the ability to bind cellulose. CBDIV(CelK)-FAE(XynZ) bound cellulose with parameters similar to that of (CBDIV-Gh9)(CelK). (FAE-L)(XynZ)-CBDIV(CelK) and FAE(XynZ)-CBDIV(CelK) had lower relative affinities and binding capacities than those of (CBDIV-Gh9)(CelK). The three chimeras were much less thermostable than FAE(XynZ) and (FAE-L-CBDVI)(XynZ). The results indicate that domains of glycosyl hydrolases are not randomly combined and that domain interactions affect properties of these domain-structured enzymes.     

Purification and partial characterization of two feruloyl esterases from the anaerobic fungus Neocallimastix strain MC-2.

Two extracellular feruloyl esterases (FAE-I and FAE-II) produced by the anaerobic fungus Neocallimastix strain MC-2 which cleave ferulic acid from O-(5-O-[(E)-feruloyl]-alpha-L- arabinofuranosyl)-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (FAXX) were purified. The molecular masses of FAE-I and FAE-II were 69 and 24 kDa, respectively, under both denaturing and nondenaturing conditions. Apparent Km and maximum rate of hydrolysis with FAXX were 31.9 microM and 2.9 mumol min-1 mg-1 for FAE-I and 9.6 microM and 11.4 mumol min-1 mg-1 for FAE-II. FAE-II was specific for FAXX, but FAE-I hydrolyzed FAXX and PAXX, the equivalent p-coumaroyl ester, at a maximum rate of metabolism ratio of 3:1.     

嗜热细菌Clostridium thermocellum阿魏酸酯酶基因的克隆、异源表达及酶学特性分析

【目的】阐明嗜热细菌Clostridium thermocellum Xyn Z蛋白的阿魏酸酯酶催化域的酶学特性,为其在生物质能源及其它发酵工业中的应用奠定基础。【方法】分别构建了C.thermocellum Xyn Z的阿魏酸酯酶催化域(FAE)及该阿魏酸酯酶催化域和碳水化合物结合域(FAE-CBM6)编码基因的原核表达载体,并在大肠杆菌菌株BL21(DE3)中异源表达,在此基础上分析比较了温度、pH、底物、金属离子及CBM6结合域对阿魏酸酯酶活性的影响。【结果】重组FAE酶及FAE-CBM6酶发挥催化活性的适宜pH值为5.0-9.0,适宜温度为50-70°C,它们对不同金属离子的响应有差异。【结论】在同一反应条件下,FAE-CBM6酶的酶活均比FAE高,说明CBM6结合域的存在对于阿魏酸酯酶活性有促进作用。     

Structural basis for the substrate specificity of the feruloyl esterase domain of the cellulosomal xylanase Z from Clostridium thermocellum

Feruloyl esterases function in the cleavage of ferulic acid's bonds to arabinoxylan and pectin where the ferulic acid moieties cross-link the layers of polysaccharide chains within hemicellulose. This work presents the crystal structure of FAE_XynZ, the domain of Clostridium thermocellum's cellulosomal xylanase Z that displays feruloyl esterase activity. The structure was obtained via multiple isomorphous replacement with anomalous scattering (MIRAS) using three heavy atom derivatives and refined against X-ray diffraction data of up to 1.75 A resolution. The R-value of the final model was 0.187 (R(free) = 0.21). FAE_XynZ displays an eight-stranded alpha/beta-fold with the characteristic "catalytic triad" at the heart of the active site. To define the substrate specificity determinants of the enzyme, the crystal structures of FAE_XynZ and the inactive FAE_XynZ(S172A) mutant were determined in complexes with the feruloyl-arabinoxylans FAXX and FAX(3), respectively. In the complex crystals, the ferulic acid moieties are clearly recognizable and allowed identification of the hydrophobic binding pocket. The carbohydrate part of both substrates is not visible in either structure. The location of the putative carbohydrate binding-pocket was inferred based on the location and orientation of the adjacent ferulic acid molecule. Five of the six residues lining the pocket were found to be conserved in FAE A from Orpinomyces sp., which further supports the proposed role of these amino acids.     

The structure of the feruloyl esterase module of xylanase 10B from Clostridium thermocellum provides insights into substrate recognition.

BACKGROUND: Degradation of the plant cell wall requires the synergistic action of a consortium of predominantly modular enzymes. In Clostridiae, these biocatalysts are organized into a supramolecular assembly termed a "cellulosome." This multienzyme complex possesses, in addition to its well-described cellulolytic activity, an apparatus specific for xylan degradation. Cinnamic acid esterases hydrolyze the ferulate groups involved in the crosslinking of arabinoxylans to lignin and thus play a key role in the degradation of the plant cell wall in addition to having promising industrial and medical applications. RESULTS: We have cloned and overexpressed the feruloyl esterase module from a 5 domain xylanase, Xyn10B from Clostridium thermocellum. The native structure at 1.6 A resolution has been solved with selenomethionine multiple wavelength anomalous dispersion and refined to a final R(free) of 17.8%. The structure of a hydrolytically inactive mutant, S954A, in complex with the reaction product ferulic acid has been refined at a resolution of 1.4 A with an R(free) of 16.0%. CONCLUSIONS: The C. thermocellum Xyn10B ferulic acid esterase displays the alpha/beta-hydrolase fold and possesses a classical Ser-His-Asp catalytic triad. Ferulate esterases are characterized by their specificity, and the active center reveals the binding site for ferulic acid and related compounds. Ferulate binds in a small surface depression that possesses specificity determinants for both the methoxy and hydroxyl ring substituents of the substrate. There appears to be a lack of specificity for the xylan backbone, which may reflect the intrinsic chemical heterogeneity of the natural substrate.     

Cloning of a gene encoding cinnamoyl ester hydrolase from the ruminai bacterium Butyrivibrio fibrisolvens E14 by a novel method

Abstract A gene ( cin I) encoding a cinnamoyl ester hydrolase (CEH) has been isolated from the ruminai bacterium, Butyrivibrio fibrisohens E14, using a model substrate, MUTMAC [4-methylumbelliferoyl ( p -trimethylammonium cinnamate chloride)]. CinI has significant amino-acid similarities with members of a large and diverse family of hydrolases with a serine/aspartic acid/ histidine catalytic triad. Our analyses identified two previously unclassified amino acid sequences, the amino-terminal domain of unknown function in XynZ from Clostridium thermocellum and XynC, an acetylxylan esterase from Caldicellulosiruptor saccharolyticus , as members of the same family of hydrolases. A previously described esterase with CEH activity, XylD from Pseudomonas fluorescens ssp. cellulosa , is not similar to CinI. CinI was expressed at high levels in the periplasmic fraction of E. coli TOPP2 and released ferulic acid from Fara [5- O -( trans -feruloyl)-arabinofuranose] prepared from wheat bran.     

Acetylation at O-2 of arabinofuranose residues in feruloylated arabinoxylan from bamboo shoot cell-walls.

Hydrolysis of bamboo shoot cell-walls with Driselase (a fungal enzyme preparation) gave an arabinoxylan trisaccharide with ferulic and acetic acids as ester groups. The structure of this oligosaccharide was determined to be O-[2-O-acetyl-5-O-[E)-feruloyl)-alpha-L-arabinofuranosyl]-(1----3) -O-beta- D-xylopyranosyl-(1----4)-D-xylopyranose, on the basis of spectroscopy and methylation analysis.     

Purification and characterization of a feruloyl esterase from the fungus Penicilliumexpansum

An extracellular phenolic acid esterase produced by the fungus Penicillium expansum in solid state culture released ferulic and ρ-coumaric acid from methyl esters of theacids, and from the phenolic-carbohydrate esters O-[5-O-(trans-feruloyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose (FAXX) and O-[5-O-((E)-ρ-coumaroyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose(PAXX). The esterase was purified 360-fold in successive stepsinvolving ultrafiltration and column chromatography by gel filtration, anion exchange andhydrophobic interaction. These chromatographic methods separated the phenolic acid esterasefrom α- l -arabinofuranosidase, pectate and pectin lyase, polygalacturonase,xylanase and β- d -xylosidase activities. The phenolic acid esterase had an apparentmass of 65 kDa under non-denaturing conditions and a mass of 57·5 kDa underdenaturing conditions. Optimal pH and temperature were 5·6 and 37 °C,respectively and the metal ions Cu2+ and Fe³+ atconcentrations of 5 mmol l⁻¹ inhibited feruloyl esterase activity by 95% and44%, respectively, at the optimum pH and temperature. The apparent K_m and V_max of the purified feruloyl esterase for methyl ferulate at pH 5·6 and 37 °Cwere 2·6 mmol l⁻¹ and 27·1 μmol min⁻¹ mg⁻¹. The corresponding constants of ρ-coumaroylesterase for methyl coumarate were 2·9 mmol l⁻¹ and 18·6μmol min−1 mg⁻¹.     

Isolation and characterization of p-coumaroyl esterase from the anaerobic fungus Neocallimastix strain MC-2.

An extracellular p-coumaroyl esterase produced by the anaerobic fungus Neocallimastix strain MC-2 released p-coumaroyl groups from 0-[5-0-((E)-p-coumaroyl)-alpha-L-arabinofuranosyl]-(1----3)-0-beta -D-xylopyranosyl-(1----4)-D-xylopyranose (PAXX). The esterase was purified 121-fold from culture medium in successive steps involving ultrafiltration column chromatography on S-sepharose and hydroxylapatite, isoelectric focusing, and gel filtration. The native enzyme had an apparent mass of 11 kDa under nondenaturing conditions and a mass of 5.8 kDa under denaturing conditions, suggesting that the enzyme may exist as a dimer. The isoelectric point was 4.7, and the pH optimum was 7.2. The purified esterase had 100 times more activity towards PAXX than towards the analogous feruloyl ester (FAXX). The apparent Km and Vmax of the purified p-coumaroyl esterase for PAXX at pH 7.2 and 40 degrees C were 19.4 microM and 5.1 microM min(-1), respectively. p-Coumaroyl tetrasaccharides isolated from plant cell walls were hydrolyzed at rates similar to that for PAXX, whereas a dimer of PAXX was hydrolyzed at a rate 20-fold lower, yielding 4,4'-dihydroxy-alpha-truxillic acid as an end product. Ethyl and methyl p-coumarates were hydrolyzed at very slow rates, if at all. The purified esterase released p-coumaroyl groups from finely, but not coarsely, ground plant cell walls, and this activity was enhanced by the addition of xylanase and other cell wall-degrading enzymes.     

Determination of immunological homology between cellulosome subunits and cloned endoglucanases and xylanase of Clostridium thermocellum

Immuno-cross reactivity between the subunits of Clostridium thermocellum cellulosome and cloned endogucanases and xylanase from the same bacterium was studied using the polyclonal antibodies raised against cloned enzymes. Dot blot analysis showed that the cellulosome, S8 and S11 subunits cross-reacted strongly with the antibodies of all cloned enzymes tested except that raised against CelC. Western blot analysis revealed that S8 and S11 subunits cross-reacted with the antibodies of CelA, CelB, CelD, CelG, CelH and XynZ, but the antibodies of CelB and CelG were highly specific for S8 and S11 subunits, respectively. Similar analysis using dissociated cellulosome showed that the antibodies of all cloned enzymes tested cross-reacted with more than one subunit of the cellulosome. Antibodies of CelC showed a very low cross-reactivity against all subunits of the cellulosome. The results indicate that immunological cross-reactivity studies could be useful, not only for demonstrating the similarities between native and cloned enzymes, but also for identifying native enzymes using antibodies of cloned enzymes.     

Purification and properties of a feruloyl esterase involved in lignocellulose degradation by Aureobasidium pullulans

The lignocellulolytic fungus Aureobasidium pullulans NRRL Y 2311-1 produces feruloyl esterase activity when grown on birchwood xylan. Feruloyl esterase was purified from culture supernatant by ultrafiltration and anion-exchange, hydrophobic interaction, and gel filtration chromatography. The pure enzyme is a monomer with an estimated molecular mass of 210 kDa in both native and denatured forms and has an apparent degree of glycosylation of 48%. The enzyme has a pI of 6.5, and maximum activity is observed at pH 6.7 and 60 degrees C. Specific activities for methyl ferulate, methyl p-coumarate, methyl sinapate, and methyl caffeate are 21.6, 35.3, 12.9, and 30.4 micro mol/min/mg, respectively. The pure feruloyl esterase transforms both 2-O and 5-O arabinofuranosidase-linked ferulate equally well and also shows high activity on the substrates 4-O-trans-feruloyl-xylopyranoside, O-[5-O-[(E)-feruloyl]-alpha-L-arabinofuranosyl]-(1,3)-O-beta-D-xylopyranosyl-(1,4)-D-xylopyranose, and p-nitrophenyl-acetate but reveals only low activity on p-nitrophenyl-butyrate. The catalytic efficiency (k(cat)/K(m)) of the enzyme was highest on methyl p-coumarate of all the substrates tested. Sequencing revealed the following eight N-terminal amino acids: AVYTLDGD.     

A halotolerant type A feruloyl esterase from Pleurotus eryngii

An extracellular feruloyl esterase (PeFaeA) from the culture supernatant of Pleurotus eryngii was purified to homogeneity using cation exchange, hydrophobic interaction, and size exclusion chromatography. The length of the complete coding sequence of PeFaeA was determined to 1668 bp corresponding to a protein of 555 amino acids. The catalytic triad of Ser-Glu-His demonstrated the uniqueness of the enzyme compared to previously published FAEs. The purified PeFaeA was a monomer with an estimated molecular mass of 67 kDa. Maximum feruloyl esterase (FAE) activity was observed at pH 5.0 and 50 °C, respectively. Metal ions (5 mM), except Hg²⁺, had no significant influence on the enzyme activity. Substrate specificity profiling characterized the enzyme as a type A FAE preferring bulky natural substrates, such as feruloylated saccharides, rather than small synthetic ones. K_m and k_cat of the purified enzyme for methyl ferulate were 0.15 mM and 0.85 s⁻¹. In the presence of 3 M NaCl activity of the enzyme increased by 28 %. PeFaeA alone released only little ferulic acid from destarched wheat bran (DSWB), whereas after addition of Trichoderma viride xylanase the concentration increased more than 20 fold.     

Penicillium brasilianum as an enzyme factory; the essential role of feruloyl esterases for the hydrolysis of the plant cell wall

The production of arabinoxylan-degrading enzymes by the fungus Penicillium brasilianum, grown on different carbon and nitrogen sources as well as different environmental conditions was investigated. Highest feruloyl esterase (225 mU/ml) and alpha-L-arabinofuranosidase (211 mU/ml) activities were obtained when P. brasilianum was grown on sugar beet pulp, whereas maximum xylanase (17 U/ml) activity was found during growth on oat spelt xylan. Yeast extract was the preferable nitrogen source for the production of all the three enzymes. Further optimization of the production of the crude enzyme mixture was examined by experimental design using a D-optimal quadratic model. Investigation of the microbial regulation of enzyme production showed that the presence of free ferulic acid further stimulated the production and pointing to that the fungal regulatory mechanism involved a coordinated production and secretion of feruloyl esterase, xylanase and alpha-L-arabinofuranosidase. Since agroindustrial by-products are a potential source of phenolic acids, crude enzyme mixtures of P. brasilianum were tested for their hydrolysis abilities against eight complex or model substrates. While total release of phenolic acids and pentoses was not observed, the synergistic enhancement of hydrolysis in the presence of feruloyl esterase was clearly demonstrated.     

Sporotrichum thermophile type C feruloyl esterase (StFaeC): purification,characterization, and its use for phenolic acid (sugar) ester synthesis

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.     

Feruloyl esterase production by Aspergillus terreus CECT 2808 and subsequent application to enzymatic hydrolysis

Ferulic acid esterases (FAE) were produced by Aspergillus terreus CECT 2808 from vine trimming shoots (VTS) and corn cob. Later, the fungal extracts thus obtained were used to enzymatically release ferulic acid (FA) from both substrates. Our findings showed a higher FAE activity in the enzymatic extracts produced on corn cob (0.070 ± 0.004 U/mL). Nevertheless, the enzymatic extracts produced on VTS demonstrated a better performance for FA release from both corn cob (2.05 ± 0.01 mg/g) and VTS (0.19 ± 0.003 mg/g). This result was probably because of the higher xylanase/FAE ratio determined in VTS extract. Therefore, an additional assay was carried out by supplementing corn cob extract with a commercial xylanase to test the influence of FAE/xylanase ratio in FA release. The results revealed the relevance of the FAE/xylanase ratio for an optimal FA release.     

Influence of ferulic acid on the production of feruloyl esterases by Aspergillus niger

Extracellular feruloyl esterases from the filamentous fungus Aspergillus niger are induced by growth on oat spelt xylan (OSX), which contains no detectable esterified ferulic acid. FAE-III accounted for most of the feruloyl esterase activity. Addition of free ferulic acid to OSX at the start of the culture induced FAE-III secretion a further 2.3-fold, and also induced other feruloyl esterases which could not be ascribed to FAE-III. Wheat bran- (WB)-grown cultures, containing 1% (m/v) ester-linked ferulic acid, gave almost identical FAE-III and total feruloyl esterase activities as the cultures grown on OSX plus ferulic acid. De-esterification of WB yielded less total feruloyl esterase, and 2.4-fold less FAE-III, compared to untreated WB. A slightly modified form of FAE-III was produced on de-esterified WB. These results show that production of FAE-III does not absolutely require ferulic acid. However, production is stimulated by the presence of free ferulic acid through increased expression, and is reduced by the removal of esterified ferulic acid from the growth substrate.     

Expression, characterization and structural modelling of a feruloyl esterase from the thermophilic fungus Myceliophthora thermophila

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.