Cloning, expression and purification of the α-carbonic anhydrase from the mantle of the Mediterranean mussel, Mytilus galloprovincialis

Abstract

We cloned, expressed, purified, and determined the kinetic constants of the recombinant α-carbonic anhydrase (rec-MgaCA) identified in the mantle tissue of the bivalve Mediterranean mussel, Mytilus galloprovincialis. In metazoans, the α-CA family is largely represented and plays a pivotal role in the deposition of calcium carbonate biominerals. Our results demonstrated that rec-MgaCA was a monomer with an apparent molecular weight of about 32 kDa. Moreover, the determined kinetic parameters for the CO₂ hydration reaction were k_cat =  4.2 × 10⁵ s⁻¹ and k_cat/K_m of 3.5 × 10⁷ M⁻¹ ×s⁻¹. Curiously, the rec-MgaCA showed a very similar kinetic and acetazolamide inhibition features when compared to those of the native enzyme (MgaCA), which has a molecular weight of 50 kDa. Analysing the SDS-PAGE, the protonography, and the kinetic analysis performed on the native and recombinant enzyme, we hypothesised that probably the native MgaCA is a multidomain protein with a single CA domain at the N-terminus of the protein. This hypothesis is corroborated by the existence in mollusks of multidomain proteins with a hydratase activity. Among these proteins, nacrein is an example of α-CA multidomain proteins characterised by a single CA domain at the N-terminus part of the entire protein.

Keywords:

• Carbonic anhydrase
• metalloenzymes
• α-class enzyme
• hydratase activity
• mussel
• multidomain protein
• protonography
• bivalve

Acknowledgements

This 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”. We also thank the Distinguished Scientist Fellowship Program (DSFP) of King Saud University, Riyadh, Saudi Arabia.

Disclosure statement

The authors report no conflicts of interest.

Additional information

Funding

This 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”. We also thank the Distinguished Scientist Fellowship Program (DSFP) of King Saud University, Riyadh, Saudi Arabia.