Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533, selected for the biotransformation of ferulic acid to vanillin, are also able to produce cell wall polysaccharide-degrading enzymes and feruloyl esterases

The filamentous fungal strains Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533, previously selected for the bioconversion of ferulic acid to vanillic acid and vanillin respectively, were grown on sugar beet pulp. A large spectrum of polysaccharide-degrading enzymes was produced by A. niger and very few levels of feruloyl esterases were found. In contrast, P. cinnabarinus culture filtrate contained low amount of polysaccharide-degrading enzymes and no feruloyl esterases. In order to enhance feruloyl esterases in A. niger cultures, feruloylated oligosaccharide-rich fractions were prepared from sugar beet pulp or cereal bran and used as carbon sources. Number of polysaccharide-degrading enzymes were induced. Feruloyl esterases were much higher in maize bran-based medium than in sugar beet pulp-based medium, demonstrating the ability of carbon sources originating from maize to induce the synthesis of feruloyl esterases. Thus, A. niger I-1472 could be interesting to release ferulic acid from sugar beet pulp or maize bran.     

Substrate and positional specificity of feruloyl esterases for monoferuloylated and monoacetylated 4-nitrophenyl glycosides

4-Nitrophenyl glycosides of 2-, 3-, and 5-O-(E)-feruloyl- and 2- and 5-O-acetyl-alpha-L-arabinofuranosides and of 2-, 3-, and 4-O-(E)-feruloyl- and 2-, 3- and 4-O-acetyl-beta-D-xylopyranosides, compounds mimicking natural substrates, were used to investigate substrate and positional specificity of type-A, -B, and -C feruloyl esterases. All the feruloyl esterases behave as true feruloyl esterases showing negligible activity on sugar acetates. Type-A enzymes, represented by AnFaeA from Aspergillus niger and FoFaeII from Fusarium oxysporum, are specialized for deferuloylation of primary hydroxyl groups, with a very strong preference for hydrolyzing 5-O-feruloyl-alpha-L-arabinofuranoside. On the contrary, type-B and -C feruloyl esterases, represented by FoFaeI from F. oxysporum and TsFaeC from Talaromyces stipitatus, acted on almost all ferulates with exception of 4- and 3-O-feruloyl-beta-D-xylopyranoside. 5-O-Feruloyl-alpha-L-arabinofuranoside was the best substrate for both TsFaeC and FoFaeI, although catalytic efficiency of the latter enzyme toward 2-O-feruloyl-alpha-L-arabinofuranoside was comparable. In comparison with acetates, the corresponding ferulates served as poor substrates for the carbohydrate esterase family 1 feruloyl esterase from Aspergillus oryzae. The enzyme hydrolyzed all alpha-L-arabinofuranoside and beta-D-xylopyranoside acetates. It behaved as a non-specific acetyl esterase rather than a feruloyl esterase, with a preference for 2-O-acetyl-beta-D-xylopyranoside.     

Exploration of members of Aspergillus sections Nigri, Flavi, and Terrei for feruloyl esterase production

The ability of members of Aspergillus sections Nigri, Flavi, and Terrei to produce feruloyl esterases was studied according to their substrate specificity against synthetic methyl esters of hydroxycinnamic acids. Type A feruloyl esterases (FAEA), induced during growth on cereal-derived products, show a preference for the phenolic moiety of substrates that contain methoxy substitutions, as found in methyl sinapinate, whereas type B feruloyl esterases (FAEB) show a preference for the phenolic moiety of substrates that contain hydroxyl substitutions, as occurs in methyl caffeate. All the strains of Aspergillus section Nigri (e.g., A. niger and A. foetidus) were able to produce feruloyl esterases with activity profiles similar to those reported for FAEA and FAEB of A. niger when grown on oat-spelt xylan and sugar beet pulp, respectively. The two genes encoding these proteins, faeA and faeB, were identified by Southern blot analysis. The strains of Aspergillus sections Flavi (e.g., A. flavus, A. flavo-furcatus, and A. tamarii) and Terrei (e.g., A. terreus) were able to produce type A and type B enzymes. faeA was revealed in genomic DNA of these strains, and FAEA was determined by immunodetection in cultures grown in oat-spelt xylan. In addition, type B enzymes, not related to faeB, were efficiently induced by oat-spelt xylan and exhibited very original activity profiles on sugar beet pulp. This work confirms that the members of the genus Aspergillus are good feruloyl esterase producers.     

Production and characterization of the Talaromyces stipitatus feruloyl esterase FAEC in Pichia pastoris: identification of the nucleophilic serine

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. We report the over-expression and characterization of a novel feruloyl esterase exhibiting broad substrate specificity from Talaromyces stipitatus (FAEC) in Pichia pastoris. Using various gene constructions, we have investigated the use of alternative signal peptides to produce an authentic feruloyl esterase featuring the N-terminal sequence determined for the native enzyme. We demonstrate that additional amino acids at the N-terminus of the FAEC sequence do not influence the catalytic capacity of the enzyme, and that the nature of the signal sequence has a limited effect on the yield of the secreted enzyme, with the T. stipitatus FAEC signal sequence producing 297 mgL(-1), the Neurospora crassa Fae-1 260 mgL(-1), and the Saccharomyces cerevisiae alpha-factor secretion signal 214 mgL(-1). Mature FAEC contains two internal peptide sequences that correspond with the consensus motif G-X-S-X-G that contains the catalytic serine nucleophile, which is conserved in the esterase enzyme superfamily. The serine residues at the center of these peptide motifs have been independently mutated and the corresponding enzymes have been over-expressed in P. pastoris to identify the candidate nucleophilic residue responsible for catalyzing the enzymatic reaction. Purified recombinant FAEC containing S465A retained the esterase activity and appeared unaffected by the amino acid modification. In contrast, FAEC activity containing S166A was below the HPLC detection limit, suggesting that serine 166 constitutes the nucleophile.     

The feruloyl esterase system of Talaromyces stipitatus: determining the hydrolytic and synthetic specificity of TsFaeC

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 k(cat) and k(cat)/K(m) 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.     

Feruloyl esterases as a tool for the release of phenolic compounds from agro-industrial by-products

Agro-industrial by-products are a potential source of added-value phenolic acids with promising applications in the food and pharmaceutical industries. Here two purified feruloyl esterases from Aspergillus niger, FAEA and FAEB were tested for their ability to release phenolic acids such as caffeic acid, p-coumaric acid and ferulic acid from coffee pulp, apple marc and wheat straw. Their hydrolysis activity was evaluated and compared with their action on maize bran and sugar beet pulp. The specificity of both enzymes against natural and synthetic substrates was evaluated; particular attention was paid to quinic esters and lignin monomers. The efficiency of both enzymes on model substrates was studied. We show the ability of these enzymes to hydrolyze quinic esters and ester linkages between phenolic acids and lignin monomer.     

Production of feruloyl/ρ-coumaroyl esterase activity by Penicillium expansum, Penicillium brevicompactum and Aspergillus niger

Extracellular esterase production by Penicillium expansum, Penicillium brevicompactum and Aspergillus niger was determined in both liquid and solid-state culture. Methyl ferulate was used as the main carbon source in liquid culture whereas wheat bran and sugar beet pulp were used in solid-state culture. Extracted enzyme for each fungus showed activity in the presence of ONP butyrate, methyl ferulate, methyl coumarate and two 'natural'feruloylated carbohydrate esters. Higher enzyme recoveries were obtained using wheat bran in solid-state culture. Higher levels of feruloyl esterase activity were recovered from P. expansum on all feruloylated substrates than from P. brevicompactum or A. niger. Using ONP butyrate as substrate the pH and temperature optima for the esterases of both Penicillium spp. were 6.0 and 25–30°C. Aspergillus niger esterase activity showed a broader temperature range with an optimum at 40°C.     

Release of ferulic acid from agroindustrial by-products by the cell wall-degrading enzymes produced by Aspergillus niger I-1472

Aspergillus niger I-1472 was grown on sugar beet pulp to produce cell wall polysaccharide-degrading enzymes, including feruloyl esterases. Compared to enzymatic activities measured in commercially available mixtures previously used for the release of ferulic acid, the A. niger enzymes were more various. These enzymes were tested to release ferulic acid from sugar beet pulp, maize bran, or autoclaved maize bran. They were as efficient as the commercial mixture to release ferulic acid from sugar beet pulp. On the other hand, they were much more efficient to release ferulic acid from maize bran after autoclaving pretreatment, as 95% of ferulic acid ester were solubilized. Thus, A. niger enzymes exhibited a high interest in the release of ferulic acid from various agro-industrial by-products.     

The crystal structure of feruloyl esterase A from Aspergillus niger suggests evolutive functional convergence in feruloyl esterase family

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 2.5A resolution. AnFaeA displays an alpha/beta 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.     

Identification of two feruloyl esterases in Dickeya dadantii 3937 and induction of the major feruloyl esterase and of pectate lyases by ferulic acid

The plant-pathogenic bacterium Dickeya dadantii (formerly Erwinia chrysanthemi) produces a large array of plant cell wall-degrading enzymes. Using an in situ detection test, we showed that it produces two feruloyl esterases, FaeD and FaeT. These enzymes cleave the ester link between ferulate and the pectic or xylan chains. FaeD and FaeT belong to the carbohydrate esterase family CE10, and they are the first two feruloyl esterases to be identified in this family. Cleavage of synthetic substrates revealed strong activation of FaeD and FaeT by ferulic acid. The gene faeT appeared to be weakly expressed, and its product, FaeT, is a cytoplasmic protein. In contrast, the gene faeD is strongly induced in the presence of ferulic acid, and FaeD is an extracellular protein secreted by the Out system, responsible for pectinase secretion. The product of the adjacent gene faeR is involved in the positive control of faeD in response to ferulic acid. Moreover, ferulic acid acts in synergy with polygalacturonate to induce pectate lyases, the main virulence determinant of soft rot disease. Feruloyl esterases dissociate internal cross-links in the polysaccharide network of the plant cell wall, suppress the polysaccharide esterifications, and liberate ferulic acid, which contributes to the induction of pectate lyases. Together, these effects of feruloyl esterases could facilitate soft rot disease caused by pectinolytic bacteria.     

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.     

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.     

Identification of a type-D feruloyl esterase from<Emphasis Type="Italic"> Neurospora crassa</Emphasis>

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.     

Occurrence,properties, and applications of feruloyl esterases

Feruloyl esterases hydrolyze the ester linkages of ferulic and diferulic acids present in plant cell walls. This interesting group of enzymes also has a potentially broad range of applications in the pharmaceutical and agri-food industries. An overview of the current knowledge of fungal feruloyl esterases focusing on the diverse of substrate specificity and potential applications is presented in this review. Furthermore, biological functions of ferulic acid are discussed.     

Biotechnological applications and potential of fungal feruloyl esterases based on prevalence,classification and biochemical diversity

Feruloyl esterases are part of the enzymatic spectrum employed by fungi and other microorganisms to degrade plant polysaccharides. They release ferulic acid and other aromatic acids from these polymeric structures and have received an increasing interest in industrial applications such as in the food, pulp and paper and bio-fuel industries. This review provides an overview of the current knowledge on fungal feruloyl esterases focussing in particular on the differences in substrate specificity, regulation of their production, prevalence of these enzymes in fungal genomes and industrial applications.     

Functional classification of the microbial feruloyl esterases

Feruloyl esterases have potential uses over a broad range of applications in the agri-food industries. In recent years, the number of microbial feruloyl esterase activities reported has increased and, in parallel, even more related protein sequences may be discerned in the growing genome databases. Based on substrate utilisation data and supported by primary sequence identity, four sub-classes have been characterised and termed type-A, B, C and D. The proposed sub-classification scheme is discussed in terms of the evolutionary relationships existing between carbohydrate esterases.     

Purification and characterization of an extracellular feruloyl esterase from the thermophilic anaerobe Clostridium stercorarium

Feruloyl esterases act as accessory enzymes for the complete saccharification of plant cell wall hemicelluloses. Although many fungal feruloyl esterases have been purified and characterized, few bacterial phenolic acid esterases have been characterized. This study shows the extracellular production of a feruloyl esterase by the thermophilic anaerobe Clostridium stercorarium when grown on birchwood xylan. The feruloyl esterase was purified 500-fold in successive steps involving ultrafiltration, preparative isoelectric focusing and column chromatography by anion exchange, gel filtration and hydrophobic interaction. The purified enzyme released ferulic, rho-coumaric, caffeic and sinapinic acid from the respective methyl esters. The purified enzyme also released ferulic acid from a de-starched wheat bran preparation. At pH 8.0 and 65 degrees C, the Km and Vmax values for the hydrolysis of methyl ferulate were 0.04 mmol l-l and 131 micromol min-1 mg-1, respectively; the respective values for methyl coumarate were 0.86 mmol l-l and 18 micromol min-1 mg-1. The purified feruloyl esterase had an apparent mass of 33 kDa under denaturing conditions and showed optimum activity at pH 8.0 and 65 degrees C. At a concentration of 5 mmol l-l, the ions Ca2+, Cu2+, Co2+ and Mn2+ reduced the activity by 70-80%.     

Two glucuronoyl esterases of <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

The white-rot fungus Phanerochaete chrysosporium produces glucuronoyl esterase, a recently discovered carbohydrate esterase, during growth on sugar beet pulp. Two putative genes encoding this enzyme, ge1 and ge2, were isolated and cloned. Heterologous expression in Aspergillus vadensis, Pycnoporus cinnabarinus and Schizophyllum commune resulted in extracellular glucuronoyl esterase activity, demonstrating that these genes encode this enzymatic function. The amino acid sequence of GE1 was used to identify homologous genes in the genomes of twenty-four fungi. Approximately half of the genomes, both from ascomycetes and basidiomycetes, contained putative orthologues, but their presence could not be assigned to any of fungal class or subclass. Comparison of the amino acid sequences of identified and putative glucuronoyl esterases to other types of carbohydrate esterases (CE) confirmed that they form a separate family of CEs. These enzymes are interesting candidates for biotechnological applications such as the separation of lignin and hemicellulose.