Directed evolution of an ultrastable carbonic anhydrase for highly efficient carbon capture from flue gas

Abstract

Carbonic anhydrase (CA) is one of nature's fastest enzymes and can dramatically improve the economics of carbon capture under demanding environments such as coal-fired power plants. The use of CA to accelerate carbon capture is limited by the enzyme's sensitivity to the harsh process conditions. Using directed evolution, the properties of a β-class CA from Desulfovibrio vulgaris were dramatically enhanced. Iterative rounds of library design, library generation, and high-throughput screening identified highly stable CA variants that tolerate temperatures of up to 107 °C in the presence of 4.2 M alkaline amine solvent at pH >10.0. This increase in thermostability and alkali tolerance translates to a 4,000,000-fold improvement over the natural enzyme. At pilot scale, the evolved catalyst enhanced the rate of CO2 absorption 25-fold compared with the noncatalyzed reaction.

Keywords: carbon capture; carbonic anhydrase; directed evolution.

Fig. 1.

Flue gas from a coal-fired power plant is piped into an absorber column (blue) where CO₂ chemisorbs into an amine solvent, catalyzed by CA, and is hydrated to a proton and a bicarbonate ion. The CO₂-depleted flue gas is released into the atmosphere and the HCO₃⁻-loaded amine solvent and CA is transferred to a second column where CO₂ is stripped at elevated temperatures (>87 °C), resulting in solvent regeneration. The pure CO₂ stream can be compressed and stored in depositories or used in industrial processes. The regenerated solvent is returned to the absorber column to repeat the process.

Fig. 2.

A56S and A84Q mutations are shown in yellow and the hydrogen bonds as red dashed lines. Based on its closest crystallized homolog (14) the quaternary structure of DvCA is predicted to be a dimer of dimers. (A) The upper inset shows the A56S mutation hydrogen bonding to the backbone of the opposing monomer presumably stabilizing the dimer interface. The hydrogen bonds are thought to form at opposite ends of the antiparallel β-strand interaction at the core of the folded protein. (B) The lower inset depicts the hypothesized interactions between the A84Q mutations across the dimer–dimer interface. Position 84 is at the center of the tetramer interaction. It is the only amino acid that interacts with the corresponding amino acids in all four monomers.

Fig. 3.

(A) Compounded fold improvement after each round of evolution. The relative fold improvement for each round is shown in red, whereas the temperature at which the half-life was determined is shown in black above each bar. (B) The round 8 parent (DvCA8.0) was exposed to 4.2 M MDEA at 50 and 60 °C for 14 wk. At regular intervals a sample was assayed for activity at 25 °C. The half-life is estimated to be 14 and 6 wk at 50 and 60 °C, respectively. This compares to a half-life of 15 min at 50 °C for the wild-type enzyme DvCA and translates to a 10,000-fold improvement in half-life of the DvCA8.0 variant. At 60 °C, the wild-type enzyme has no detectable activity.

Fig. 4.

(A) Mutations accumulated in nine rounds of evolution of DvCA are shown in orange with the corresponding residues of the wild-type enzyme shown above. The pI_calc of the enzyme increases by more than two pH units. The best round 9 variant (DvCA10) accumulated 39 mutations and shares less than 85% sequence identity with the natural sequence. (B) A surface view of one of the monomers is shown in blue with the mutations accumulated in each of the parents highlighted in red. The wild-type (DvCA) rendition also contains the ribbon diagram of the three additional monomers in red, yellow, and green. (C) Same image as in B but rotated by 180° to show the mutations at the tetramer interface.

Fig. 5.

(A) Mass transfer coefficient (K_G∙a) for the carbon capture process was determined at various CA and MDEA concentrations in the test rig. The fold improvement over the no-enzyme control is shown above each data point in red. The circles indicate the results obtained at 2.1 M MDEA where the mass transfer of CO₂ from the gas to the liquid phase is 25-fold greater than in the absence of enzyme. The squares show the performance of the test unit using 4.2 M MDEA. At high enzyme loadings and MDEA concentrations the absorption of CO₂ becomes mass transfer limited. This is highlighted by the nonlinear correlation between K_G∙a and enzyme loading at 2.1 M MDEA and by the drop in performance at 4.2 M MDEA as the solution becomes more viscous. (B) The long-term stability demonstration test was run for 60 h with DvCA8.0 in 25% MDEA solution. The enzyme was exposed to temperatures between 20 and 87 °C as it was cycled between the absorber and the desorber. The top blue trace shows the percent CO₂ in the flue gas entering the absorber column, whereas the bottom blue trace shows it exiting the column. The red trace depicts the percent CO₂ captured. It is clear that the CA remained active throughout the run and that there was no measurable decrease in percent carbon capture after 60 h of testing. The 60-h test spanned 5 d, operating for about 12 h per day.