Abstract
Biocatalytic fixation of CO2 into carboxylic acids offers a sustainable route to carbon capture and utilization. Microbial cofactor-free non-oxidative decarboxylases catalyze the reversible ortho-carboxylation of phenolic substrates under mild conditions in the presence of adequate concentrations of bicarbonate ions. The development of industrial biocatalysts based on immobilized DHDBs asks for a rapid method for the screening of immobilization techniques and optimization of carboxylation operating conditions. UV spectroscopy methods applied to DHDBs activity assay are scarce and not standardized, differing in solvent composition, wavelengths, and data analysis, while HPLC methods are more popular, as they are useful for substrate screening over a variety of phenols. The present contribution reports the development of a UV spectroscopic method for the analysis of both the carboxylation and decarboxylation reactions catalyzed by decarboxylases with 2,3-dihydroxybenzoic acid (2,3-DHBA) and catechol as substrates/products. Absorption spectra show which wavelengths provided the most significant differences among catechol and 2,3-DHBA, and the optimal ones were set to follow catechol and 2,3-DHBA concentration dynamics in the reaction mixture. The activity assay of 2,3-DHBD from Aspergillus oryzae was defined to the developed method with 0.05 g/L enzyme and 10 mM 2,3-DHBA. Further carboxylation tests of catechol proved the effectiveness of the method at relevant conditions for future optimization of 2,3-DHBD immobilization and continuous flow monitoring of phenols conversion by enzymatic carboxylation.
