Using Esterase and Laccase Enzymes to Derivatize Bioactive Plant Phenolics for Altered Chemistry

Plant phenolics have notable antioxidant activity and there is potential to improve their action by chemical modification. Two enzyme classes carry out reactions that can act on the hydroxyl moiety of phenolics. Esterase enzymes can be used in non-aqueous solvents to esterify a long chain acyl group onto the phenolic compound. Laccase enzymes can be used to form phenoxy radicals that can then couple to form larger molecular weight oligomers. Both enzymatic modifications may produce a new antioxidant with altered chemistry. One archaeal esterase (AF1753) from Archaeoglobus fulgidus and one bacterial esterase (PP3645) from Pseudomonas putida were assayed for activity in organic solvents. Both enzymes catalyzed hydrolysis of phenyl acetate and vinyl acetate in 98:2 (v/v) (t-amyl alcohol):buffer; with continued activity up to 96 h of reaction. However, the enzymes were not able to catalyze transesterification of 4’-hydroxyacetophenone with vinyl acetate in 9:1 (v/v) cyclohexane:(t-amyl alcohol), which was not explained by enzyme inactivation during lyophilization. Still, alanine scanning mutagenesis revealed that R37A substitution improved activity of AF1753 on long-chain p-nitrophenyl (pNP) esters. A multicopper oxidase (SCO6712) from Streptomyces coelicolor displayed activity on a variety of phenolics including caffeic acid, ferulic acid, resveratrol, quercetin, morin, kaempferol and myricetin. Among the products formed by action on flavonols were dimers of quercetin, morin, and myricetin. Quercetin and myricetin dimers showed longer retention time on reversed phase chromatography. All three dimers could be detected by 5 min of reaction but depleted by 3 h and 24 h. The TRAP and FRAP antioxidant activity of the whole reaction mixture of modified quercetin, morin, and myricetin decreased, as starting phenolic was depleted over 24 h. Accordingly, mass spectrometry was used to shed light on the molecular structure of the dimers produced from quercetin and myricetin. In both cases, mass spectrometric analyses ruled out dimer formation through the A ring of each monomer. For myricetin, the most likely linkage structure was determined to be between either two B rings or a B ring with a C ring. These predicted linkage positions are in agreement to those observed for quercetin dimers previously extracted from natural plant sources.Ph.D