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.
Title: Identification of a type-D feruloyl esterase from Neurospora crassa Crepin VF, Faulds CB, Connerton IF Ref: Applied Microbiology & Biotechnology, 63:567, 2004 : PubMed
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.
Several extracellular feruloyl esterases were produced by the mesophilic fungus Talaromyces stipitatus when grown on selective carbon sources in liquid media. Type-A and Type-B feruloyl esterases, as defined by their substrate specificity against methyl hydroxycinnamates, were produced during growth on wheat bran and sugar beet pulp, respectively. In addition, Tal. stipitatus produced a new type of esterase (TsFaeC) during growth on sugar beet pulp with a broader spectrum of activity (Type-C) against the (hydroxy)cinnamate esters than those previously described. All three enzymes were purified and N-terminal amino acid sequences and internal peptide sequences determined. The TsFaeC sequences were used to amplify a gene fragment from Tal. stipitatus genomic DNA. The flanking sequences were identified with the aid of RACE-RTPCR, and a full-length clone constructed. The faeC gene is present as a single copy and contains a single intron. The complete cDNA fragment contains an ORF of 1590bp, faeC, which is predicted to encode a 530 amino acid pre-protein, including a 25-residue signal peptide, and to produce a mature protein of M(R) 55 340Da. There was no evidence for a carbohydrate-binding domain in TsFaeC.
Title: A non-modular type B feruloyl esterase from Neurospora crassa exhibits concentration-dependent substrate inhibition Crepin VF, Faulds CB, Connerton IF Ref: Biochemical Journal, 370:417, 2003 : PubMed
Feruloyl esterases, a subclass of the carboxylic acid esterases (EC 3.1.1.1), are able to hydrolyse the ester bond between the hydroxycinnamic acids and sugars present in the plant cell wall. The enzymes have been classified as type A or type B, based on their substrate specificity for aromatic moieties. We show that Neurospora crassa has the ability to produce multiple ferulic acid esterase activities depending upon the length of fermentation with either sugar beet pulp or wheat bran substrates. A gene identified on the basis of its expression on sugar beet pulp has been cloned and overexpressed in Pichia pastoris. The gene encodes a single-domain ferulic acid esterase, which represents the first report of a non-modular type B enzyme (fae-1 gene; GenBank accession no. AJ293029). The purified recombinant protein has been shown to exhibit concentration-dependent substrate inhibition (K(m) 0.048 mM, K (i) 2.5 mM and V(max) 8.2 units/mg against methyl 3,4-dihydroxycinnamate). The kinetic behaviour of the non-modular enzyme is discussed in terms of the diversity in the roles of the feruloyl esterases in the mobilization of plant cell wall materials and their respective modes of action.
Title: Production and characterization of the Talaromyces stipitatus feruloyl esterase FAEC in Pichia pastoris: identification of the nucleophilic serine Crepin VF, Faulds CB, Connerton IF Ref: Protein Expr Purif, 29:176, 2003 : PubMed
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 cDNA encoding Aspergillus niger cinnamoyl esterase (FAEA) with its native signal sequence was isolated by reverse transcriptase-polymerase chain reaction, sequenced, and expressed in Pichia pastoris. Secretion yields up to 300 mg l(-1) were obtained in buffered medium. The recombinant FAEA was purified to homogeneity using a one-step purification protocol and found to be identical to the native enzyme with respect to size, pI, immunoreactivity and N-terminal sequence. Specific activity, pH and temperature optimum, and kinetic parameters were also found similar to the native esterase. FAEA is thus the first fungal esterase efficiently produced using a heterologous system.
Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.
