(515ah) A Novel Fusion Carbonic Anhydrase/cellulose Binding Protein For Capturing CO2

The enzyme carbonic anhydrase (CA) catalyzes the reaction: CO2 + H2O = HCO3-+ H+ CA is a robust enzyme that maintains its activity over a broad range of temperature (20 - 65 °C) and pH 4-9. We have explored the possibility of crating hybrid enzyme comprised of a suitable Cellulose-Binding Domain (CBD), with the carbonic anhydrase (CA) for use in CO2 capture processes. A number of enzymes that catalyze cellulose degradation contain domains, referred to as cellulose binding domain or CBD, that allow the protein to bind tightly to the cellulose fiber during the catalytic hydrolysis of cellulose. The use of a CBD-CA hybrid versus the native CA, provide important advantages. It eliminates the need for enzyme purification as the cell extract containing CBD-CA can be directly loaded onto cellulose. Since non-specific binding of cellulose to proteins is minimal, the CBD-CA will constitute the majority of bound proteins. Moreover, the strong binging of CBD-CA could remove the need for any immobilization chemistry to fix the enzyme.

The carbonic anhydrase from Neisseria gonorrhoeae was selected for our initial experiments. It hydrates CO2 at rates as high as kcat = 1.1*106 s-1 which translates to about 5 million kilograms/hour using just one kilogram of enzyme under ideal conditions. Full length CA gene was amplified from N. gonorrhoeae genomic DNA using PCR with appropriate primers and cloned into the vector plasmid pUC19. Plasmid DNA from a selected cone was isolated and sequenced. The sequence confirmed the presence of CA gene.

Our early attempts focused on fusion of CBD of Clostridium cellulovorans to the full length carbonic anhydrase of N. gonorrhoeae but the fusion product did not yield active protein. Next a CBD-CA fusion lacking 26 amino acids of signal peptide of CA was constructed and shown to produce active protein. Several versions of CBD-CA fusions were made. Cellulose binding domains (CBD) from Bacillus licheniformis, Clostridium cellulovorans and C. thermocellum were fused to CA. Production of these CBD-CAs were examined using different expression vectors and cultivation conditions. In majority of cases CBD-CA was produced as inclusion bodies and the yield of active protein after denaturing and refolding of the inclusion bodies was very low. One of the construct comprised of the C. thermocellum CBD fused to CA in pREST B expression vector produced high levels of soluble CBD-CA, in E.coli BL21 (DE3)pLysS as host strain, reaching 8% of the total cell.

The CBD-CA from this construct was purified and tested for its enzymatic activity and affinity for phosphoric acid-swollen cellulose (PASC) and CM-cellulose(CMC). Analysis of enzyme kinetics data showed that the CBD-CA maintains high levels of enzymatic activity. Binding isotherms reveled high level of affinity of CBD-CA to phosphoric acid-swollen cellulose (PASC) and CM-cellulose(CMC) in a wide range of pH. In summary, our results may enable new approaches for CO2 capture with the potential to contribute significantly to meeting targeted reductions in greenhouse gas emissions.