Cloning, expression and characterization of the δ-carbonic anhydrase of Thalassiosira weissflogii (Bacillariophyceae)
Lee RBY., Smith JAC., Rickaby REM.
Carbonic anhydrase (CA) is a ubiquitous metalloenzyme responsible for accelerating the interconversion of CO 2 and bicarbonate. Although CAs are involved in a broad range of biochemical processes involving carboxylation or decarboxylation reactions, they are of special interest due to their role in photosynthetic CO 2 assimilation in marine phytoplankton, especially under low-CO 2 conditions. Several phylogenetically independent classes of CAs have been identified in a variety of marine phytoplankton. TWCA1, first discovered in Thalassiosira weissflogii (Grunow) G. Fryxell & Hasle, is the founding member of the δ-class of CAs; these appear to be extracellular enzymes, but are still relatively poorly characterized. To date, it has remained uncertain whether TWCA1 possesses true CA activity due to the difficulty in producing a functional protein in a heterologous expression system. Herein we describe the fusion of a full-length open reading frame of TWCA1 to the coding sequence of a self-splicing intein in a pTWIN2 expression vector that has allowed successful production of a functional enzyme in Escherichia coli. Assay of the recombinant protein shows that TWCA1 is a catalytically active δ-CA possessing both CO 2 hydration and esterase activity. © 2012 Phycological Society of America.