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

Abstract

In hydrothermal liquefaction (HTL) of biomass, only the oil and the char phases are consideredimperative while the aqueous phase is discarded. However, the aqueous phase is rich in phen-olic compounds like guaiacol and vanillyl alcohol that can be upgraded to vanillin.Commercially vanillin is synthesized from phenolic compounds derived from fossil fuels usingchemical synthesis methods. In this investigation, the method was developed to use raw materi-als and enzymatic conversion of substrates which is sustainable and renewable. The purpose ofthis investigation is to evaluate the applicability of enzymatic vanillin synthesis within the HTLaqueous 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. Theeffect on the enzyme reaction of each of the major components of the aqueous phase asdetected by HPLC (High Performance Liquid Chromatography) analysis was evaluated. The com-ponents evaluated in this study included different phenolic compounds namely guaiacol, vanil-lic acid, vanillyl alcohol, phenol, catechol and acetic acid. These components were evaluatedindividually as well as in different combinations. Vanillic acid increased the reaction rate whileguaiacol inhibited the reaction rate. Acetic acid exhibited the largest inhibition of the enzymereaction after 15 min. It is known that acids are often used to stop enzyme reactions as it resultsin precipitation of the enzyme. Therefore, to successfully convert vanillin from vanillyl alcoholusing a recombinantly expressed vanillyl alcohol oxidase, the acetic acid would first need to beremoved from the HTL aqueous phase.