NAD+ regeneration-coupled enzymatic bioconversion of lignin-derived vanillin into vanillic acid: A cleaner production approach

Abstract

Vanillic acid is a well-known phenolic compound widely used in the food, flavour, cosmetic, and pharmaceutical industries and has been shown to exhibit various biological activities. Considering the environmental burden caused by chemical synthesis of vanillic acid, the enzymatic transformation of vanillin derived from lignin offers a greener alternative to the chemical synthesis of vanillic acid offers a greener and alternative avenue. In the present study, vanillin dehydrogenase (VDH) from Bacillus ligniniphilus L1 and NADH oxidase (NOX) from Streptococcus pyogenes were used for biotransformation of vanillin into vanillic acid. The addition of NOX regenerates NAD⁺ from NADH generated by VDH during the biotransformation of vanillin into vanillic acid, which eliminates the need for continuous addition of the expensive coenzyme NAD⁺. To further improve the stability and reusability of VDH and NOX, cross-linked enzyme aggregates (Combi-CLVNAs) were obtained and the results showed better NAD⁺ regeneration and vanillic acid production. Fed-batch addition of vanillin to the reaction resulted in the production of 44.21 mM vanillic acid using 2 mM NAD⁺ in 31 h which was 2.06- and 22.5-fold higher than that of free enzymes and without NOX, respectively. A total of 149.98 mM vanillic acid was synthesized using Combi-CLVNAs at the end of 4 cycles. As a poof of this concept, Combi-CLVNAs were successfully used to produce vanillic acid (38.69 mM) from vanillin-rich extracts derived from lignin depolymerization. Overall, this study presents an eco-friendly method for transforming vanillin into vanillic acid with NAD+ regeneration, without creating unwanted by-products.