BACKGROUND AND OBJECTIVESCarbon dioxide (CO۲) capture and storage (CCS) processes play a crucial role in mitigating climate change by reducing CO۲ emissions. Thermostable carbonic anhydrases (CAs) have shown great potential in enhancing the rate of CO۲ absorption into solvents, offering a promising avenue for robust and cost-effective industrial applications. This study focuses on rational engineering of a thermostable CA (SspCA) derived from the thermophilic bacterium Sulfurihydrogenibium yellowstonense, aiming to improve its thermostability while maintaining catalytic efficiency under high-temperature conditions.MATERIALS AND METHODSIn this investigation, in silico tools were employed to rationally design a mutant form of the SspCA enzyme. The joint candidate from the three tools, RosettaDesign, PoPMuSiC and FoldX, with the best score was selected for construction using the site-directed mutagenesis method. The mutant, was analyzed to assess its impact on the thermostability and catalytic activity of the CA. Molecular dynamics (MD) simulations were utilized to gain insight into the structural changes caused by the mutation and understand its effects on local flexibility and stability.RESULTS AND DISCUSSIONThe rational design of the K۱۰۰G mutation resulted in improved thermostability of the CA. Although the general folding and catalytic efficiency of the enzyme were preserved, the melting temperature of the K۱۰۰G mutant increased by ۳°C. Notably, the half-life of CO۲ hydration activity at ۸۵°C was extended twofold, making it more suitable for industrial processes carried out under elevated temperatures. Molecular dynamics simulations revealed that the K۱۰۰G mutation induced a reduction in local flexibility of the protein. This effect was primarily achieved by the rearrangement of salt bridges and hydrogen interaction networks, leading to structural rigidification in the neighboring regions.CONCLUSIONThis study demonstrates the potential of rational engineering in enhancing the thermostability of enzymes like carbonic anhydrases, essential for efficient CO۲ capture and storage processes in high-temperature industrial settings. The K۱۰۰G mutation in the SspCA enzyme indirectly resulted in increased thermostability by confining the flexible parts and promoting local structural rigidity. These findings highlight the importance of targeted mutations to optimize the performance of biocatalysts for industrial applications, ultimately contributing to the advancement of carbon capture technologies and sustainable environmental practices.