Comparative Characterization of α-Carbonic Anhydrases from Bacteria Aeromonas hydrophila, Halomonas binhaiensis, and Roseibium salinum: Insights into Halotolerance and Thermostability

Abstract

Halotolerant enzymes are promising biocatalysts for industrial applications due to their ability to function under high salinity and temperature conditions that typically inactivate conventional enzymes. This is particularly relevant for biomineralization-based carbon capture, where carbonic anhydrases (CAs) catalyze the reversible hydration of CO2 but often lack stability in harsh environments such as brine. Here, α-CA genes from three halotolerant bacteria, Aeromonas hydrophila (AhCA), Halomonas binhaiensis (HbCA), and Roseibium salinum (RsCA), were cloned and expressed, and the corresponding enzymes were purified and characterized. Enzymatic activity was evaluated following incubation under different conditions: NaCl concentrations (0–4 M), temperatures (30–80 °C), and divalent metal ions. AhCA and RsCA retained high activity under elevated salinity, with RsCA exhibiting pronounced salt activation after incubation at 4 M NaCl, whereas HbCA displayed comparatively lower salt tolerance but greater thermostability, maintaining activity after incubation at 80 °C for 1 h. Co2+ and Mg2+ produced the strongest activation for AhCA and RsCA, respectively, with metal-dependent effects varying considerably among the three enzymes. Molecular dynamics simulations performed at different NaCl concentrations revealed enhanced structural stability of RsCA under saline conditions, consistent with its superior catalytic performance. A surface physicochemical analysis identified an extensive negatively charged surface characteristic of salt-adapted proteins. Together, these findings identify RsCA as a candidate for further evaluation and provide structural insight into the molecular basis of halotolerance in α-CAs.