Cloning, expression and characterization of β- and γ‑carbonic anhydrase from Bacillus sp. SS105 for biomimetic sequestration of CO2

Abstract

Bacterium Bacillus sp. SS105, isolated from Free Air CO₂ Enriched (FACE) soil was previously screened for carbonic anhydrase activity and CO₂ sequestration. In this study, strain was selected to amplify carbonic anhydrase encoding genes. The CA genes from Bacillus sp. SS105 were found to be homologous with beta‑carbonic anhydrase (β-CA) and gamma‑carbonic anhydrase (γ-CA). Both types of CA genes was cloned in pET30b (+) and expressed in E coliBL21 (DE3) with His-tag at the N-terminus. The recombinant proteins were purified by Ni-NTA affinity chromatography. The molecular size of β-CA and γ-CA were approximately 27 kDa and 25 kDa respectively. The optimum pH and temperature were found to be 8.0 and 37 °C respectively. The Zn⁺ was enhancing the CAs enzyme activity. Anions and modulators showed inhibitory effect on CAs at specific concentration. Functional domain analysis of both CA proteins showed conserved region of respective proteins. Recombinant enzymes were used for bio-mineralization based conversion of atmospheric CO₂ into valuable calcite. Calcite formation was evaluated with or without use of enzymes and confirmed by SEM and XRD analysis. SEM result confirmed the conversion of flower-shaped unstable form of vaterite to hexagonal cubic stable form of calcite in presence of enzymes.

Keywords: Bacillus sp. SS105; CO(2) sequestration; Carbonic anhydrase.