Driving enzymatic carboxylation using supercritical CO2

While enzymes have evolved to work in aqueous environment, or with biological membranes, industrial applications have often sought to make use of nonaqueous solvents. Recently, the use of supercritical CO2 (scCO2) has been explored by various groups, due the "green" credentials of this solvent. In fact, CO2 is inexpensive, abundant, nontoxic, non-flammable and chemically inert. Furthermore, products are easily recovered upon depressurisation as scCO2 becomes gaseous. Furthermore, unfavourable thermodynamic equilibria in aqueous solvents are suitably altered as in the case of carboxylation by (de)carboxylase enzymes. However, enzyme inactivation can occur, due to pressure, carbamoylation or pH effects. A limited set of examples is available from the literature where natural enzymes are shown to be robust catalysist under scCO2 or CO2/aqueous systems. We wish to explore the suitability of the UbiD family of (de)carboxylases to act as catalysts under such conditions. The mechanism of action of this particular family was discovered in our group, with support from our industrial partner, and the wide spread nature of these enzymes offers a rich biological resource in terms of varied substrate specificity and temperature/pressure/pH stability. In fact, we have shown in preliminary data these enzymes readily act as carboxylases at elevated [CO2]. Hence, we are ideally positioned to explore to what extend UbiD enzymes can catalyse carboxylation in scCO2 or CO2/aqueous systems. We initially will explore the reaction of the thermophylic HmfF enzyme as a model system of biotechnological interest. This enzyme catalyses the carboxylation of furoic acid to furan dicarboxylic acid (FDCA), an alternative green polymer precursor. We now wish to explore the yield and scalability under scCO2 conditions. Furthermore, we plan to explore whether enzyme stability/catalytic properties for scCO2 based catalysis can be improved using (semi)rational mutagenesis. Removal of lysine residues that are subject to carbamoylation will be explored, as well as determining the structural effects of pressure/CO2 on enzyme structure using simulation. The long term goal is to bring these UbiD systems to a sufficient level of productivity to support industrial take up and application.