Rational engineering of a highly active and resilient α-carbonic anhydrase from the hydrothermal vent species Persephonella hydrogeniphila.

Carbonic anhydrases (CAs) are ideal catalysts for carbon dioxide sequestration in efforts to alleviate climate change. Here, we report the characterisation of three α-CAs that originate from the thermophilic bacteria Persephonella hydrogeniphila (PhyCA), Persephonella atlantica (PaCA), and Persephonella sp. KM09-Lau-8 (PlauCA) isolated from hydrothermal vents. The three α-Cas, showing high sequence similarities, were produced in Escherichia coli, purified and characterised. Surprisingly, they revealed very different behaviours with regards to their thermostability profiles. PhyCA presented a more stable thermostability profile amongst the three, thus we chose it for rational engineering to improve it further. PhyCA's residue K88, a proton transfer residue in α-CAs, was mutated to His, Ala, Gln and Tyr. A 4-fold activity improvement was noted for variants K88H and K88Q at 30 °C, owing to the higher proton transfer efficiency of the replacement proton transfer residues. K88Q also proved more stable than PhyCA. K88Y did not increase activity, but notably increased thermal stability, with this enzyme variant retaining 50% of its initial activity after incubation for 1 h at 90 °C. Removal of the two main proton shuttles (variant H85A_K88A) resulted in diminished activity of the enzyme. Molecular dynamics simulations performed for PhyCA and all its variants revealed differences in residue fluctuations, with K88A resulting in a general reduction in root mean square fluctuation (RMSF) of active site residues as well as most of the CA's residues. Its specific activity and stability in turn increased compared to the wild type.