Ancestral carbonic anhydrase with significantly enhanced stability and activity for CO₂ capture and utilization

Carbonic anhydrases (CAs) has garnered increasing attention in carbon capture, utilization and storage (CCUS) due to their ecological friendliness. However, most of them suffer susceptibility to deactivation in harsh conditions. Herein, a reliable dataset was adopted for creating ancestral CAs through an optimized ancestral sequence reconstruction (ASR) method. After prescreening, the ancestor AncCA19 was obtained and successfully expressed. The hydration activity of AncCA19 was as high as 58,859 WAU/mg, with the optimum temperature and pH obtained by esterase assay at 100 ℃ and 9, respectively. AncCA19 had the longest half-life (1.7 h) at 95 ℃ compared with existing CAs. After 2 weeks' incubation in artificial seawater at 30 ℃ or 25.0 % N-methyldiethanolamine (MDEA) at 60 ℃, the activities remained above 47,370 WAU/mg and 6,596 WAU/mg, respectively. Thus, AncCA19, as a novel benchmark of CAs, exhibits exceptional stability in a variety CCUS applications, establishing a versatile candidate for effective CO₂ capture.