methyl-ferulate and methyl-caffeate

The filamentous fungus Trametes versicolor is a rich source of laccase (Tvlac). Laccases catalyze reactions that convert substituted phenol substrates into diverse derivatives through aromatic oxidation. We investigated methyl p-coumarate, methyl ferulate, and methyl caffeate biotransformation by Trametes versicolor ATCC 200801. Despite substrate similarity, the biotransformation reactions varied widely. Only methyl p-coumarate was converted into three derivatives. We isolated and identified the chemical structures of such derivatives by NMR and IR analysis. Hydroxylation, methylation, and hydrolysis were the main reactions resulting from the studied biotransformation. We also analyzed the interactions between Tvlac (PDB ID: 1GYC) and the three phenolic substrates by molecular docking simulations. The substituents in the phenol ring influenced substrate conformation and orientation in the Tvlac site. The biotransformation reaction selectivity correlated with the different binding energies to the Tvlac site. Our results demonstrated that docking studies successfully predict the biotransformation of cinnamic acid analogs by T. versicolor.

Topics: Biotransformation; Caffeic Acids; Catalysis; Cinnamates; Environmental Restoration and Remediation; Hydrolysis; Hydroxylation; Industrial Microbiology; Laccase; Magnetic Resonance Spectroscopy; Molecular Conformation; Molecular Docking Simulation; Oxygen; Phenols; Polyporaceae; Solvents; Spectrophotometry, Infrared

Lactobacillus plantarum is frequently found in the fermentation of plant-derived food products, where hydroxycinnamoyl esters are abundant. L. plantarum WCFS1 cultures were unable to hydrolyze hydroxycinnamoyl esters; however, cell extracts from the strain partially hydrolyze methyl ferulate and methyl p-coumarate. In order to discover whether the protein Lp_0796 is the enzyme responsible for this hydrolytic activity, it was recombinantly overproduced and enzymatically characterized. Lp_0796 is an esterase that, among other substrates, is able to efficiently hydrolyze the four model substrates for feruloyl esterases (methyl ferulate, methyl caffeate, methyl p-coumarate, and methyl sinapinate). A screening test for the detection of the gene encoding feruloyl esterase Lp_0796 revealed that it is generally present among L. plantarum strains. The present study constitutes the description of feruloyl esterase activity in L. plantarum and provides new insights into the metabolism of hydroxycinnamic compounds in this bacterial species.

Topics: Caffeic Acids; Carboxylic Ester Hydrolases; Cinnamates; Gene Expression; Lactobacillus plantarum; Recombinant Proteins

Feruloyl esterases are key enzymes involved in the complete hydrolysis of hemicellulose. In the present study, the encoding sequence of putative feruloyl esterase A (AfFaeA) was cloned from genomic DNA from Aspergillus flavus and expressed in Pichia pastoris. The purified recombinant AfFaeA had apparent relative molecular mass of about 40,000 and had an optimum pH of 6.0, although it was stable at pH values ranging from 4.5 to 8.0. The optimum temperature for AfFaeA was 58°C. AfFaeA had hydrolytic activity toward methyl caffeate, methyl p-coumarate, methyl ferulate and methyl sinapate. Substrate specificity profiling of AfFaeA demostrated it is a type-A feruloyl esterase. The good performance of AfFaeA to release ferulic acid from steam exploded corn stalk in concert with Geobacillus stearothermophilus xylanase mutant indicated it is a promising biocatalyst for biomass degradation.

Topics: Aspergillus flavus; Caffeic Acids; Carboxylic Ester Hydrolases; Cloning, Molecular; Coumaric Acids; Pichia; Recombinant Proteins; Substrate Specificity