Enzymatic synthesis of vanillin and related catalytic mechanism

There has been an increasing demand for bio vanillin for a variety of applications. Although vanillin production using biotechnological methods based on microbial fermentation has been extensively explored, the use of isolated enzymes for vanillin synthesis has not yet attracted much attention. Lipoxygenase (LOX) is an iron-containing dioxygenase well known for catalysing the hydroperoxidation of polyunsaturated fatty acids and esters. Its newly discovered ability to catalyse the oxidative cleavage of isoeugenol to vanillin offers a promising solution for synthesizing bio vanillin. In this study, soybean LOX was used as the catalyst for vanillin synthesis. The reaction was optimised by examining the effects of pH, temperature, substrate concentration, and enzyme dosage. By investigating the impact of various additives, including two denaturants, seven chelators, six surfactants, 10 organic solvents, eight deep eutectic solvents, and 13 natural deep eutectic solvents, it was found that denaturants (urea and guanidine) and some chelators (e.g. ethylenediaminetetraacetic acid, EDTA) are effective in improving synthetic yields, by up to 133 and 406%, respectively, relative to the additive-free reaction system. Two free-radical catalytic mechanisms were proposed to explain the new activity of the enzyme for vanillin synthesis.