Analyses of the efficiency of a recombinant vanillyl alcohol oxidase in producing vanillin in the hydrothermal liquefaction aqueous phase

In hydrothermal liquefaction (HTL) of biomass, only the oil and the char phases are considered imperative while the aqueous phase is discarded. However, the aqueous phase is rich in phenolic compounds like guaiacol and vanillyl alcohol that can be upgraded to vanillin. Commercially vanillin is synthesized from phenolic compounds derived from fossil fuels using chemical synthesis methods. In this investigation, the method was developed to use raw materials and enzymatic conversion of substrates which is sustainable and renewable. The purpose of this investigation is to evaluate the applicability of enzymatic vanillin synthesis within the HTL aqueous phase major components. Vanillyl alcohol oxidase was expressed in E. coli BL21 (DE3) bacteria cells and purified to characterize the production of vanillin from vanillyl alcohol. The effect on the enzyme reaction of each of the major components of the aqueous phase as detected by HPLC (High Performance Liquid Chromatography) analysis was evaluated. The components evaluated in this study included different phenolic compounds namely guaiacol, vanillic acid, vanillyl alcohol, phenol, catechol and acetic acid. These components were evaluated individually as well as in different combinations. Vanillic acid increased the reaction rate while guaiacol inhibited the reaction rate. Acetic acid exhibited the largest inhibition of the enzyme reaction after 15 min. It is known that acids are often used to stop enzyme reactions as it results in precipitation of the enzyme. Therefore, to successfully convert vanillin from vanillyl alcohol using a recombinantly expressed vanillyl alcohol oxidase, the acetic acid would first need to be removed from the HTL aqueous phase.