Bio-catalytic upgrading of phenolic components present in waste water of a hydrothermal liquefaction plant

Abstract

Vanillyl alcohol oxidase is a flavoenzyme naturally sourced from Penicillium simplicissimum. It is a potential candidate in the commercial production of vanillin, an expensive natural flavourant in the cosmetic, food, beverages and pharmaceutical industries. During hydrothermal liquefaction of biomass, only the oil and the char phases are considered as essential 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. A method channeled towards the production of vanillin from the phenolic components of the aqueous phase using vanillyl alcohol oxidase was investigated. The vanillyl alcohol oxidase enzyme was expressed in Escherichia coli BL21(DE3) bacteria cells and purified in order to characterize the production of vanillin from vanillyl alcohol. The expression and purification of the recombinant expressed vanillyl alcohol oxidase was preliminary detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and a band of 65 kDa was observed. The total yield of purified protein after purification from a 50 ml bacterial culture was 17.6 mg (w/v). The effect of the aqueous phase components of HTL on the enzyme reaction was investigated. High Performance Liquid Chromatography (HPLC) analysis was done to detect the phenolic and sugar composition of the aqueous phase of biomass sourced from municipality sewage waste, then used as a reference in making the synthetic aqueous phase. Vanillic acid increased the reaction rate while guaiacol inhibited the reaction rate at 95% confidence. However, in the presence of acetic acid, there was a noticeable and statistically significant difference from 15 minutes onwards, where acetic acid containing reactions were slower than all the other reactions. Conclusively, active vanillyl alcohol oxidase can be expressed and purified using BL21(DE3) cells. The enzyme reaction is affected by other phenolic compounds in the hydrothermal liquefaction aqueous phase, but a component of the HTL, acetic acid can slow down the rate of this reaction with a greater margin.