A one-step procedure for immobilising the thermostable carbonic anhydrase (SspCA) on the surface membrane of Escherichia coli

Abstract

The carbonic anhydrase superfamily (CA, EC 4.2.1.1) of metalloenzymes is present in all three domains of life (Eubacteria, Archaea, and Eukarya), being an interesting example of convergent/divergent evolution, with its seven families (α-, β-, γ-, δ-, ζ-, η-, and θ-CAs) described so far. CAs catalyse the simple, but physiologically crucial reaction of carbon dioxide hydration to bicarbonate and protons. Recently, our groups characterised the α-CA from the thermophilic bacterium, Sulfurihydrogenibium yellowstonense finding a very high catalytic activity for the CO₂ hydration reaction (k_cat = 9.35 × 10⁵ s⁻¹ and k_cat/K_m = 1.1 × 10⁸ M⁻¹s⁻¹) which was maintained after heating the enzyme at 80 °C for 3 h. This highly thermostable SspCA was covalently immobilised within polyurethane foam and onto the surface of magnetic Fe₃O₄ nanoparticles. Here, we describe a one-step procedure for immobilising the thermostable SspCA directly on the surface membrane of Escherichia coli, using the INPN domain of Pseudomonas syringae. This strategy has clear advantages with respect to other methods, which require as the first step the production and the purification of the biocatalyst, and as the second step the immobilisation of the enzyme onto a specific support. Our results demonstrate that thermostable SspCA fused to the INPN domain of P. syringae ice nucleation protein (INP) was correctly expressed on the outer membrane of engineered E. coli cells, affording for an easy approach to design biotechnological applications for this highly effective thermostable catalyst.

Keywords:

• Carbonic anhydrase
• thermostable enzyme
• ice nucleation protein
• hydratase activity
• protonography
• outer membrane

Acknowledgements

The authors also thank the Distinguished Scientist Fellowship Program (DSFP) of King Saud University, Riyadh, Saudi Arabia.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

SMART GENERATION – Sistemi e tecnologie sostenibili per la generazione di energia10.13039/501100001474PON03PE_00157_1

SMART GENERATION – Sistemi e tecnologie sostenibili per la generazione di energia10.13039/501100001474PON03PE_7_1This research was financed by the grant “SMART GENERATION – Sistemi e tecnologie sostenibili per la generazione di energia – PON03PE_00157_1, OR3 – Bio-sistemi di cattura ed utilizzazione della CO₂.”