Identification of the carbonic anhydrases from the unicellular green alga Dunaliella salina strain CCAP 19/18

Dunaliella salina is a unicellular halophilic green alga, which can survive even in saturated brine solutions (up to 5.0 M NaCl). In D. salina, the carbonic anhydrase (CA, EC 4.2.1.1) is essential for cells to acquire carbon and to cope with the low CO2 environment under high salt conditions. For the first time, the present study describes the existence of eight genes coding for different types of D. salina CAs: five alpha-type (DsCAs) and three gamma-type (DsgCAs). Beta-type CAs appear to be lacking in D. salina, as they could not be found. Under either high salt or limited CO2 condition, the CAs from D. salina showed different expression patterns, with phylogenetically close CAs exhibiting similar gene expression patterns. For the biological characterization of DsCA2b, which is a newly identified α-type CA, the enzyme was successfully produced as a soluble protein by truncating the membrane spanning regions at both ends (trDsCA2b). It was demonstrated that this truncated trDsCA2b version of the CA enzyme was active. Purified trDsCA2b clearly showed increased CA activity at increased NaCl concentrations of up to 3.0 M. Based on in silico analysis and our predicted 3D structure of DsCA2b, we propose that this enzyme is localized in the plasma membrane of cells and active on the extracellular side. Our results are in agreement with the hypothesis that the DsCAs are essential enzymes for carbon acquisition mechanism under salt stress and CO2 stress in D. salina.