Engineering of Thermovibrio ammonificans carbonic anhydrase mutants with increased thermostability

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 ?C 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 ?C. A significantly 50-fold increased ester hydrolysis activity was also observed with the most thermostable variant at 95 ?C compared to 25 ?C, suggesting an increased flexibility of the active site at high temperatures