Biotransformation of Natural Compounds by Free and Immobilized Fungal Laccase using Mediator Combinations in the LMS Reaction Model

Abstract

The ternary systems constructed on the basis of isotropic properties of various compositions of a hydrocarbon and a short chain alcohol in a low water content were used to carry out the oxidation of Mastic oil, d-limonene and α-Pinene at 30 °C using fungal laccase from Trametes versicolor in the presence of different concentrations and combinations of three mediators ,2,2'-azino-bis 3-methylbenzothiazoline-6-sulfonic acid(ABTS), 1-hydroxybenzotriazole (HBT) and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) in Laccase Mediator System (LMS) model . The biotransformation was conducted with the use of either free or immobilized laccase on organogels prepared by introducing appropriate amounts of Dioctyl Sulfosuccinate (AOT) microemulsions containing laccase to a second solution of polymer Hydroxy Propyl Methyl Cellulose (HPMC) . The main oxidation product of α-pinene was verbenone and verbenol, but low amounts of α-pinene oxide was also detected. Similarly, d-limonene was oxidized to limonene epoxide, while mastic oil was oxidized to α-pinene oxide, limonene epoxide, verbenol, verbenone . In all cases, some of the reaction products remained unidentified. The amounts of unidentified products were rather high especially in the case of d-limonene and crude mastic oil. Mediator Combinations with HBT in different ratio were used for further transformation optimization of the three natural compounds in the ternary systems. Ascorbic and Gallic acids in 40mM concentration also were used in combination with HBT to transform the natural compounds. All combinations revealed different level of oxidation and only one combination of Gallic acid and HBT was unable to oxidize any of three compounds. The natural compounds oxidation rate correlated with the mediator concentration in the reaction mixture, and clear synergetic effect were found when two mediators were present, 10mM ABTS and 80mM HBT , while the best triple combination was 10mM ABTS: 40mM HBT: 40mM TEMPO .