Biocatalytic oxidative conversion of valencene to nootkatone mediated by lipoxygenase and cytochrome P450

Abstract

Nootkatone (NK) is an oxygen-containing sesquiterpene with a significant grapefruit aroma and plays an important role in the flavour and fragrance industry. The natural production of NK through extraction produces trace amounts and is therefore not a viable option to meet industrial needs. The chemical synthesis often utilises reagents harmful to the environment. The purpose of this research was to explore the use of crude lipoxygenase (LOX) enzyme extracted from soya beans, commercial mutants of CYP450 as well as laccase enzymes for the conversion of valencene (VL), the aroma components of citrus fruits to NK. For the LOX reactions, a conversion of 28.79% (mol/mol) was obtained when the reactions were performed with LOX only. The best conversion of 74.46 % was realised when FeSO4.7H2O and MnSO4 were added to the reaction. In the temperature studies, 70 ˚C was shown to be the optimal temperature for the conversion. In addition, we observed that vegetable oils provided sufficient unsaturated fatty acids to facilitate the conversion of VL to NK with sunflower oil being the best. In exploring the potential of LOX to oxidise other organic molecules, caryophyllene was oxidised to novel caryophyllene oxide, and styrene was oxidised to benzoic acid, 1-phenylethane-1,2-diol, and 2-hydroxyl-2-phenylethyl benzoate. This is the first time that such oxidations are reported, and this underlines the potential of LOX in biotransformation and organic synthesis. For CYP450 reactions, the best conversion of 16.70 % was obtained using a variant sourced from Prozomix. Evaluation of parameters such as temperature, pH (7.0 to 7.5), using buffer solutions should be explored to optimise the activity of the enzyme. Laccase from Novoprime Base 268 showed no activity for the conversion of VL to NK despite making use of mediators. Therefore, we should explore changing the reaction conditions, varying the pH of the reaction, buffer strength or mediator and laccases from other sources such as Cerrena unicolor and Trametes versicolor, as well as a fresh batch of laccase from Trichoderma (Merck) should be assessed in the conversion of VL to NK.