Development of a Glucuronoyl Esterase Assay -biochemical characterization of three putative Glucuronoyl Esterases

Abstract

Glucuronoyl Esterases (GEs) have been proven to hydrolyze the ester bond between lignin and glucuronic acid units on the xylan chain. Separation of lignin and hemicellulose is vital for complete utilization of plant biomass for production of ethanol, chemicals and materials. Direct detection of enzymatic cleavage of the lignin carbohydrate ester bond is, however, difficult due the low concentration of bonds and the complex structure of the native material. Therefore, there is a need for a well-working GE assay with a variety of model substrates that imitate the natural substrate, for investigation of GE kinetics and substrate specificity. In this study, the already established assay developed and validated by Sunner for commercially available substrates and the newly published Fraňová assay using a synthesized model substrate were used for characterization of three putative GEs. The Fraňová assay was for this work established and evaluated in terms of GE activity and pH working range. The assay was assessed in both a stopped and continuous mode. Due to a substrate containing p-nitrophenol, the assay can be used in a stopped mode from pH 5-9, however, not continuously at a pH below 6. The kinetic parameters of the three GEs from the bacterium Solibacter usitatus differed significantly on various substrates. For all GEs, the highest specific activity was reached with the synthesized compound, methyl ester D-glucuronic acid 4-nitrophenol, using the Fraňová assay. The Fraňová assay is thus a suitable assay for activity measurements of GE activity. Moreover, kinetic parameters were determined for both methyl ester D-glucuronic acid (using the Sunner assay) and methyl ester D-glucuronic acid 4-nitrophenol (using the Fraňová assay) for one of the GE candidates (SuC). The results showed a higher catalytic efficiency for the p-nitrophenol substrate compared to the substrate lacking p-nitrophenol. In conclusion, the Fraňová assay was further developed into a continuous assay and validated. The assay is useful for GE research, since high specific GE activity was observed for the enzymes used in this study. Moreover, a range of model LC ester substrates can potentially, be synthesized.