Carbonic anhydrase can be used as an additive to improve the efficiency of carbon capture and utilisation processes, due to its ability to increase the rate of CO₂ absorption into solvents. Successful industrial application requires robust carbonic anhydrases, able to withstand process conditions and to perform consistently over long periods of time. Tolerance of high temperatures, pH and salt concentrations are particularly desirable features. We have previously used molecular dynamics simulations to rationally design four mutants of Thermovibrio ammonificans carbonic anhydrase with increased rigidity, and we hypothesized that this will result in an increased thermostability. Herein, we report on the successful recombinant expression and characterization of these mutants. Four of the TaCA variants showed increased stability at 90 ᵒC during 1 h, compared to wild-type. Two out of the four mutations predicted by the theoretical studies resulted in marked stabilization of the protein, with up to 3-fold higher time of half-life for mutant N140 G compared to the wild-type enzyme at 60 ᵒC. A significantly 50-fold increased ester hydrolysis activity was also observed with the most thermostable variant at 95 ᵒC compared to 25 ᵒC, suggesting an increased flexibility of the active site at high temperatures.

碳酸酐酶由于能够提高吸收到溶剂中的CO ₂的速率，因此可以用作提高碳捕获和利用过程效率的添加剂。成功的工业应用需要坚固的碳酸酐酶，能够承受工艺条件并长期稳定运行。高温，pH和盐浓度的耐受性是特别理想的功能。我们以前曾使用分子动力学模拟来合理设计铵化热弧菌的四个突变体碳酸酐酶具有增加的刚性，我们假设这将导致增加的热稳定性。在本文中，我们报告了这些突变体的成功重组表达和表征。与野生型相比，TaCA的四个变体在1小时内在90°C的条件下显示出更高的稳定性。理论研究预测的四个突变中有两个导致蛋白质显着稳定，与60°C的野生型酶相比，突变型N140 G的半衰期延长了3倍。与25°C相比，在95°C时最热稳定的变体还观察到酯水解活性显着提高了50倍，这表明在高温下活性位点的柔性增加了。