Driving enzymatic carboxylation using supercritical CO2

虽然酶已经进化到可以在水环境中工作，或者与生物膜一起工作，但工业应用经常寻求利用非水溶剂。最近，由于超临界二氧化碳（scCO2）溶剂的“绿色”证书，各种团体都在探索使用超临界二氧化碳。事实上，二氧化碳价格低廉、储量丰富、无毒、不易燃且具有化学惰性。此外，当scCO2变为气态时，减压后产品很容易回收。此外，在水溶液中不利的热力学平衡被适当地改变，如在羧化酶（去）羧化酶的情况下。然而，由于压力、氨基甲酰化或pH值的影响，酶可能会失活。从文献中可以得到有限的一组例子，其中天然酶在scCO2或CO2/水系统下被证明是强大的催化剂。我们希望探索UbiD家族（脱）羧化酶在这种条件下作为催化剂的适用性。这个特殊家族的作用机制是在我们的工业合作伙伴的支持下由我们的团队发现的，这些酶的广泛分布性质在不同的底物特异性和温度/压力/pH稳定性方面提供了丰富的生物资源。事实上，我们已经在初步数据中表明，这些酶在升高的[CO2]中很容易起羧化酶的作用。因此，我们理想地定位于探索UbiD酶在scCO2或CO2/水系统中催化羧化的程度。我们最初将探索热源性HmfF酶的反应作为生物技术兴趣的模型系统。该酶催化呋喃二羧酸的羧基化生成呋喃二羧酸（FDCA），一种可选的绿色聚合物前体。我们现在希望探索在scCO2条件下的产量和可扩展性。此外，我们计划探索是否可以通过（半）合理诱变来改善scCO2基催化的酶稳定性/催化性能。将探索氨基甲酸基化的赖氨酸残基的去除，以及通过模拟确定压力/CO2对酶结构的结构影响。长期目标是使这些UbiD系统达到足够的生产力水平，以支持工业采用和应用。

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.