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

ABSTRACT: We cloned, expressed, purified, and determined the kinetic constants of the recombinant ?-carbonic anhydrase (rec-MgaCA) identified in the mantle tissue of the bivalve Mediterranean mussel, Mytilus galloprovincialis. In metazoans, the ?-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 kcat?=??4.2?×?105?s-1 and kcat/Km 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 ?-CA multidomain proteins characterised by a single CA domain at the N-terminus part of the entire protein.

SUBMITTER: Perfetto R 

PROVIDER: S-EPMC6010101 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature