Family Report for: FaeC

In this study, we report the identification and characterization of an acetyl xylan esterase, designated as AoAXEC, which was previously annotated as a hypothetical protein encoded by AO090023000158 in the Aspergillus oryzae genomic database. Based on its amino acid sequence, a low sequence identity to known acetyl xylan esterases was observed in the sequence of characterized acetyl xylan esterase. The gene fused with alpha-factor signal sequence of Saccharomyces cerevisiae instead of the native signal sequence was cloned into a vector, pPICZalphaC, and expressed successfully in Pichia pastoris as an active extracellular protein. The purified recombinant protein had pH and temperature optima of 7.0 and 50 degreesC, respectively, and was stable up to 50 degreesC. The optimal substrate for hydrolysis by the purified recombinant AoAXEC, among a panel of alpha-naphthyl esters (C2-C16), was alpha-naphthyl propionate (C3), with an activity of 0.35 +/- 0.006 units/mg protein. No significant difference of the K(m) value was observed between C3 (2.3 +/- 0.7 mM) and C2 (1.9 +/- 0.4 mM). In contrast, k(cat) value for C3 (18 +/- 3.9 s(-1)) was higher compared to C2 (4.5 +/- 0.7 s(-1)). The purified recombinant enzyme displayed a low activity toward acyl chain substrates containing eight or more carbon atoms. Recombinant AoAXEC catalyzed the release of acetic acid from wheat arabinoxylan. However, no activity was detected on methyl esters of ferulic, p-coumaric, caffeic, or sinapic acids. Additionally, the liberation of phenolic acids, such as ferulic acid, from wheat arabinoxylan was not exhibited by the recombinant protein.

Two hypothetical proteins XP_001818628 and XP_001819091 (designated AoFaeB and AoFaeC, respectively), showing sequence identity with known type-C feruloyl esterases, have been found in the genomic sequence of Aspergillus oryzae. We cloned the putative A. oryzae feruloyl esterase-encoding genes and expressed them in Pichia pastoris. Both purified recombinant AoFaeB (rAoFaeB) and AoFaeC (rAoFaeC) had apparent relative molecular masses of 61,000 and 75,000, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After N-deglycosylation, both proteins had a relative molecular mass of 55,000. The optimum pH for rAoFaeB was 6.0, although it was stable at pH values ranging from 3.0 to 9.0; rAoFaeC had an optimum pH of 6.0 and was stable in the pH range of 7.0-10.0. Thermostability of rAoFaeC was greater than that of rAoFaeB. Whereas rAoFaeC displayed hydrolytic activity toward methyl caffeate, methyl p-coumarate, methyl ferulate, and methyl sinapate, rAoFaeB displayed hydrolytic activity toward methyl caffeate, methyl p-coumarate, and methyl ferulate but not toward methyl sinapate. Substrate specificity profiling of rAoFaeB and rAoFaeC revealed type-B and type-C feruloyl esterases, respectively. Ferulic acid was efficiently released from wheat arabinoxylan when both esterases were applied with xylanase from Thermomyces lanuginosus. Both recombinant proteins also exhibited hydrolytic activity toward chlorogenic acid.

To facilitate analysis of plant cell wall polysaccharide structure and composition, we cloned 74 genes encoding polysaccharide-degrading enzymes from Aspergillus nidulans, Aspergillus fumigatus, and Neurospora crassa and expressed the genes as secreted proteins with C-terminal Myc and 6x His tags. Most of the recombinant enzymes were active in enzyme assays, and optima for pH and temperature were established. A subset of the enzymes was used to fragment polysaccharides from the irregular xylem 9 (irx9) mutant of Arabidopsis. The analysis revealed a decrease in the abundance of xylan in the mutant, indicating that the IRX9 gene, which encodes a putative family 43 glycosyltransferase, is required for xylan synthesis.

In this study, we report the identification and characterization of an acetyl xylan esterase, designated as AoAXEC, which was previously annotated as a hypothetical protein encoded by AO090023000158 in the Aspergillus oryzae genomic database. Based on its amino acid sequence, a low sequence identity to known acetyl xylan esterases was observed in the sequence of characterized acetyl xylan esterase. The gene fused with alpha-factor signal sequence of Saccharomyces cerevisiae instead of the native signal sequence was cloned into a vector, pPICZalphaC, and expressed successfully in Pichia pastoris as an active extracellular protein. The purified recombinant protein had pH and temperature optima of 7.0 and 50 degreesC, respectively, and was stable up to 50 degreesC. The optimal substrate for hydrolysis by the purified recombinant AoAXEC, among a panel of alpha-naphthyl esters (C2-C16), was alpha-naphthyl propionate (C3), with an activity of 0.35 +/- 0.006 units/mg protein. No significant difference of the K(m) value was observed between C3 (2.3 +/- 0.7 mM) and C2 (1.9 +/- 0.4 mM). In contrast, k(cat) value for C3 (18 +/- 3.9 s(-1)) was higher compared to C2 (4.5 +/- 0.7 s(-1)). The purified recombinant enzyme displayed a low activity toward acyl chain substrates containing eight or more carbon atoms. Recombinant AoAXEC catalyzed the release of acetic acid from wheat arabinoxylan. However, no activity was detected on methyl esters of ferulic, p-coumaric, caffeic, or sinapic acids. Additionally, the liberation of phenolic acids, such as ferulic acid, from wheat arabinoxylan was not exhibited by the recombinant protein.

Feruloyl esterases (FAEs) are accessory enzymes for plant biomass degradation, which catalyse hydrolysis of carboxylic ester linkages between hydroxycinnamic acids and plant cell-wall carbohydrates. They are a diverse group of enzymes evolved from, e.g. acetyl xylan esterases (AXEs), lipases and tannases, thus complicating their classification and prediction of function by sequence similarity. Recently, an increasing number of fungal FAEs have been biochemically characterized, owing to their potential in various biotechnological applications and multitude of candidate FAEs in fungal genomes. However, only part of the fungal FAEs are included in Carbohydrate Esterase family 1 (CE1) of the carbohydrate-active enzymes (CAZy) database. In this work, we performed a phylogenetic analysis that divided the fungal members of CE1 into five subfamilies of which three contained characterized enzymes with conserved activities. Conservation within one of the subfamilies was confirmed by characterization of an additional CE1 enzyme from Aspergillus terreus. Recombinant A. terreus FaeD (AtFaeD) showed broad specificity towards synthetic methyl and ethyl esters, and released ferulic acid from plant biomass substrates, demonstrating its true FAE activity and interesting features as potential biocatalyst. The subfamily division of the fungal CE1 members enables more efficient selection of candidate enzymes for biotechnological processes.

Two hypothetical proteins XP_001818628 and XP_001819091 (designated AoFaeB and AoFaeC, respectively), showing sequence identity with known type-C feruloyl esterases, have been found in the genomic sequence of Aspergillus oryzae. We cloned the putative A. oryzae feruloyl esterase-encoding genes and expressed them in Pichia pastoris. Both purified recombinant AoFaeB (rAoFaeB) and AoFaeC (rAoFaeC) had apparent relative molecular masses of 61,000 and 75,000, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After N-deglycosylation, both proteins had a relative molecular mass of 55,000. The optimum pH for rAoFaeB was 6.0, although it was stable at pH values ranging from 3.0 to 9.0; rAoFaeC had an optimum pH of 6.0 and was stable in the pH range of 7.0-10.0. Thermostability of rAoFaeC was greater than that of rAoFaeB. Whereas rAoFaeC displayed hydrolytic activity toward methyl caffeate, methyl p-coumarate, methyl ferulate, and methyl sinapate, rAoFaeB displayed hydrolytic activity toward methyl caffeate, methyl p-coumarate, and methyl ferulate but not toward methyl sinapate. Substrate specificity profiling of rAoFaeB and rAoFaeC revealed type-B and type-C feruloyl esterases, respectively. Ferulic acid was efficiently released from wheat arabinoxylan when both esterases were applied with xylanase from Thermomyces lanuginosus. Both recombinant proteins also exhibited hydrolytic activity toward chlorogenic acid.

To facilitate analysis of plant cell wall polysaccharide structure and composition, we cloned 74 genes encoding polysaccharide-degrading enzymes from Aspergillus nidulans, Aspergillus fumigatus, and Neurospora crassa and expressed the genes as secreted proteins with C-terminal Myc and 6x His tags. Most of the recombinant enzymes were active in enzyme assays, and optima for pH and temperature were established. A subset of the enzymes was used to fragment polysaccharides from the irregular xylem 9 (irx9) mutant of Arabidopsis. The analysis revealed a decrease in the abundance of xylan in the mutant, indicating that the IRX9 gene, which encodes a putative family 43 glycosyltransferase, is required for xylan synthesis.

Feruloyl esterases constitute an interesting group of enzymes that have the potential for use over a broad range of applications in the agri-food industries. In order to expand the range of available enzymes, we have examined the presence of feruoyl esterase genes present in the genome sequence of the filamentous fungus Neurospora crassa. We have identified an orphan gene (contig 3.544), the translation of which shows sequence identity with known feruloyl esterases. This gene was cloned and the corresponding recombinant protein expressed in Pichia pastoris to confirm that the enzyme (NcFaeD-3.544) exhibits feruloyl esterase activity. Unusually the enzyme was capable of p-coumaric acid release from untreated crude plant cell wall materials. The substrate utilisation preferences of the recombinant enzyme place it in the recently recognised type-D sub-class of feruloyl esterase.