(17e) Periplasmic Expression of Carbonic Anhydrase to Mediate Carbon Mineralization As a Permanent Carbon Capture and Storage Solution

Uncatalyzed, the hydration rate of carbon dioxide to bicarbonate ions is very low.  Carbonic anhydrases (CA) are Zn-binding metalloenzymes that are capable of catalyzing this reversible hydration.  Ubiquitous in nature, almost all living organisms express at least one form of this enzyme.  In plants, CA is used to help the conversion of CO₂ into glucose during photosynthesis. Mammals need these enzymes to maintain the acid-base levels in their blood, and to remove CO₂ from their tissues.  However, regardless of the native organism, all carbonic anhydrases can convert carbon dioxide to bicarbonate ions (HCO₃^-), which spontaneously shed their remaining proton, resulting in a carbonate ion (CO₃^-2).  When these ions are combined with divalent cations such as Ca⁺² or Mg⁺², carbonate salts are formed and precipitate in aqueous solution.  This process is known as carbon mineralization and can potentially be used as a large-scale combined carbon capture and sequestration technology.  By bubbling the flue gas from a coal-fired power plant through a reactor containing carbonic anhydrase, CO₂ could be quickly converted to CO₃^-2 and then precipitated as a thermodynamically stable salt.  Since it is highly unlikely that carbon dioxide would spontaneously leach out of these solid crystals, this is a much more permanent solution to traditional sequestration methods in which purified and compressed carbon dioxide gas is geologically sequestered.  In such a process, while dissolving a large amount of purified carbonic anhydrase would work well, it would be very difficult to recycle the catalyst back into the process.  As such, immobilization of the protein on a carrier particle would ease recyclability and reduce the cost of operation.  Protein immobilization is a well-studied method by which a protein’s recyclability, and in some cases stability, can be increased.  Traditionally, proteins are attached to solid surfaces or membranes by covalent linkage or physisorption.  In both of these methods, a large quantity of enzyme would need to be purified before attachment in order to obtain a sufficient amount of immobilized activity.  In this study, carbonic anhydrases from two different archeabacteria were recombinantly expressed in the periplasm of E. coli to be used as whole-cell biocatalysts.  While this method does introduce an additional transport barrier that will slow the reaction, the elimination of purification costs and enhanced recyclability should account for this loss in activity.  The whole-cell biocatalysts were analyzed using quantitative western blots of fractionation experiments and stopped-flow kinetics.  Using these methods, it was determined that the recombinant carbonic anhydrase was almost entirely located in the periplasm, with the possibility of a small fraction in the cytoplasm.  Also, it was found that each cell contains approximately 350 fg of enzyme.  Using kinetic measurements, the transport-limited reaction was found to reduce the activity of purified enzyme by up to ~75%.  However, given that carbonic anhydrases are among the fastest known enzymes (10⁴-10⁶ sec^-1), even these diffusion-limited reactions exhibited turnover numbers on the order of 10³ sec^-1.  The whole-cell biocatalysts were also analyzed for their stability and lifetime over various temperatures, pressures, and timescales.  Finally, using a bubble column reactor, the overall conversion rate of CO2 to precipitated carbonate salt was determined.