Directed evolution of an ultrastable carbonic anhydrase from towards enhanced tolerance to flue gas impurities.

Carbonic anhydrases (CAs) have been proved as a highly efficient and selective promoter for conventional Carbon Capture Utilization and Storage (CCUS) industrial processes. The aim of this work was to demonstrate a high-throughput screening system for detecting engineered CAs with resistance to common inhibitors (SO , SO , NO , NO ) present as major impurities in post-combustion flue gases, maintaining their initial thermostability. We established a screening protocol on solid and liquid assays for selecting mutants generated with error-prone PCR (epCA8.0) maintaining the thermostability of the parent DvCA8.0 but having improved resistance to flue gas inhibitors. A library of around 1000 mutants was created. The mutant E12 (G7D) showed 50 % increased stability for a mix of inhibitors corresponding to total concentration of 300-600 mM and 65 % increased stability to 150 mM, compared to the parent DvCA8.0. To our knowledge, this is the first time that a CA was evolved by protein engineering methods to increase its stability to common flue gas inhibitors. Additionally, we have established a premise for screening and characterization of CA libraries, which has not been clearly addressed previously, as presence of ionic inhibitors significantly change the pH of enzyme assays, while the nature of such screening assays is pH dependent. We envision that this study will open the pathway for the development of highly resistant CAs in the near future, overcoming stability and cost issues that are associated with their limited application in CCUS technologies.