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

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 (k_cat/K_m) 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.