Alpha Carbonic Anhydrase from Nitratiruptor tergarcus Engineered for Increased Activity and Thermostability

The development of carbon capture and storage technologies has resulted in a rising interest in the use of carbonic anhydrases (CAs) for CO₂ fixation at elevated temperatures. In this study, we chose to rationally engineer the α-CA (NtCA) from the thermophilic bacterium Nitratiruptor tergarcus, which has been previously suggested to be thermostable by in silico studies. Using a combination of analyses with the DEEPDDG software and available structural knowledge, we selected residues in three regions, namely, the catalytic pocket, the dimeric interface and the surface, in order to increase thermostability and CO₂ hydration activity. A total of 13 specific mutations, affecting seven amino acids, were assessed. Single, double and quadruple mutants were produced in Escherichia coli and analyzed. The best-performing mutations that led to improvements in both activity and stability were D168K, a surface mutation, and R210L, a mutation in the dimeric interface. Apart from these, most mutants showed improved thermostability, with mutants R210K and N88K_R210L showing substantial improvements in activity, up to 11-fold. Molecular dynamics simulations, focusing particularly on residue fluctuations, conformational changes and hydrogen bond analysis, elucidated the structural changes imposed by the mutations. Successful engineering of NtCA provided valuable lessons for further engineering of α-CAs.