Characterization of bacterial α‑carbonic anhydrase with high CO2 hydration efficiency from Bacillus yunxiaonensis S3Wahi

Anthropogenic emissions of CO₂ continue to rise, contributing significantly to global warming and climate change. Among the available mitigation strategies, carbonic anhydrase (CA)-mediated CO₂ capture and conversion represents a green and efficient approach for accelerating the hydration of CO2 under mild conditions. α‑carbonic anhydrases (α-CAs) are highly efficient metalloenzymes for CO2 hydration, yet a limited number of bacterial α-CAs combine high catalytic turnover with adequate thermostability. Herein, this study presents a detailed characterization of an α-CA from Bacillus yunxiaonensis S3Wahi (S3WahiCAα). A mature α-CA encoded 657 bp gene shares only 45.5% amino acid sequence identity with the closest characterized bacterial α-CA from Sulfurihydrogenibium azorense. 3D homology model revealed the canonical ten-stranded β-sheet flanked by three α-helices with a Zn2+ ion coordinated by H83, H85, H102. Recombinant S3WahiCAα has a molecular weight of ~29 kDa. The purified S3WahiCAα displayed an optimal activity at 40 °C and pH 7.0. S3WahiCAα retained >50% activity after 300 min of incubation at 60 °C. Zn2+ and Ca2+ stimulated activity (up to 160% and 120% at 1 mM) whereas Cu2+ and Fe3+ caused >50% inhibition. Kinetic analysis for CO2 hydration activity showed a catalytic efficiency (kcat/Km) of 1.4 × 108 M-1 s-1 which is comparable to the most efficient bacterial α-CAs. Furthermore, S3WahiCAα accelerated the onset of CaCO3 precipitation demonstrating its potential to promote CO2 conversion into stable carbonate minerals. These attributes positioned S3WahiCAα as a promising biocatalyst for CO2 mineralization and carbon capture utilization applications.