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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 CO 2 absorption 25-fold compared with the noncatalyzed reaction.carbonic anhydrase | directed evolution | carbon capture R eleasing over 9 billion metric tons of CO 2 worldwide each year, coal-fired power plants are the leading anthropogenic source of CO 2 emission. A potential solution to reduce the release of CO 2 into the atmosphere is the implementation of carbon capture and sequestration (CCS) technology on coal-and natural-gas-fired power plants. Currently, one of the most viable postcombustion CCS technology options uses an amine solvent to remove CO 2 from the flue gas (1). Unfortunately, the large amount of energy required to release the CO 2 and regenerate the solvent would significantly increase the cost of electricity generated (2). The ideal solvent capture process uses a solvent such as an aqueous amine with fast CO 2 absorption kinetics and a low heat of desorption to minimize the solvent regeneration energy. However, a high rate of CO 2 absorption kinetics for an amine solvent correlates with a higher temperature of desorption, whereas solvents with low heat of desorption tend to have much slower adsorption kinetics (3, 4).With reaction rates approaching the limits of diffusion, carbonic anhydrase (CA) is one of the fastest enzymes known. The active site of CAs can turnover CO 2 and water to bicarbonate and a proton up to a million times per second, and are used by almost every living organism to maintain pH balance and transport carbon dioxide. It has been shown that CAs can be used to accelerate the capture of CO 2 (5) by serving as a catalyst in alkaline capture solvents with slow absorption kinetics (6, 7). The potential improvements of CAs on CCS process economics stems from several considerations. The faster absorption and desorption kinetics allows for smaller processing equipment with reduced capital and operating costs (6). The improved kinetics also allows the use of lower temperature capture and solvent regeneration process conditions, and decreases energy losses in the capture process. Another benefit is the improved range of solvent candidates with attractive thermochemical stability (e.g., low vapor pressure, low formation of heat stable salts, etc.), but otherwise unacceptable unca...

Dissolved salts are known to affect properties of proteins in solution including solubility and melting temperature, and the effects of dissolved salts can be ranked qualitatively by the Hofmeister series. We seek a quantitative model to predict the effects of salts in the Hofmeister series on the deactivation kinetics of enzymes. Such a model would allow for a better prediction of useful biocatalyst lifetimes or an improved estimation of protein-based pharmaceutical shelf life. Here we consider a number of salt properties that are proposed indicators of Hofmeister effects in the literature as a means for predicting salt effects on the deactivation of horse liver alcohol dehydrogenase (HL-ADH), alpha-chymotrypsin, and monomeric red fluorescent protein (mRFP). We find that surface tension increments are not accurate predictors of salt effects but find a common trend between observed deactivation constants and B-viscosity coefficients of the Jones-Dole equation, which are indicative of ion hydration. This trend suggests that deactivation constants (log k(d,obs)) vary linearly with chaotropic B-viscosity coefficients but are relatively unchanged in kosmotropic solutions. The invariance with kosmotropic B-viscosity coefficients suggests the existence of a minimum deactivation constant for proteins. Differential scanning calorimetry is used to measure protein melting temperatures and thermodynamic parameters, which are used to calculate the intrinsic irreversible deactivation constant. We find that either the protein unfolding rate or the rate of intrinsic irreversible deactivation can control the observed deactivation rates.

A variety of proteins are capable of converting from their soluble forms into highly ordered fibrous cross-b aggregates (amyloids). This conversion is associated with certain pathological conditions in mammals, such as Alzheimer disease, and provides a basis for the infectious or hereditary protein isoforms (prions), causing neurodegenerative disorders in mammals and controlling heritable phenotypes in yeast. The N-proximal region of the yeast prion protein Sup35 (Sup35NM) is frequently used as a model system for amyloid conversion studies in vitro. Traditionally, amyloids are recognized by their ability to bind Congo Red dye specific to b-sheet rich structures. However, methods for quantifying amyloid fibril formation thus far were based on measurements linking Congo Red absorbance to concentration of insulin fibrils and may not be directly applicable to other amyloid-forming proteins. Here, we present a corrected formula for measuring amyloid formation of Sup35NM by Congo Red assay. By utilizing this corrected procedure, we explore the effect of different sodium salts on the lag time and maximum rate of amyloid formation by Sup35NM. We find that increased kosmotropicity promotes amyloid polymerization in accordance with the Hofmeister series. In contrast, chaotropes inhibit polymerization, with the strength of inhibition correlating with the B-viscosity coefficient of the Jones-Dole equation, an increasingly accepted measure for the quantification of the Hofmeister series.

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Enzyme instability is a major factor preventing widespread adoption of enzymes for catalysis. Stability at high temperatures and in the presence of high salt concentrations and organic solvents would allow enzymes to be employed for transformations of compounds not readily soluble in low temperature or in purely aqueous systems. Furthermore, many redox enzymes require costly cofactors for function and consequently a robust cofactor regeneration system. In this work, we demonstrate how thermostable variants developed via an amino acid sequence-based consensus method also showed improved stability in solutions with high concentrations of kosmotropic and chaotropic salts and water-miscible organic solvents. This is invaluable to protein engineers since deactivation in salt solutions and organic solvents is not well understood, rendering a priori design of enzyme stability in these media difficult. Variants of glucose 1-dehydrogenase (GDH) were studied in solutions of different salts along the Hofmeister series and in the presence of varying amounts of miscible organic solvent. Only the most stable variants showed little deactivation dependence on salt-type and salt concentration. Kinetic stability, expressed by the deactivation rate constant k(d,obs), did not always correlate with thermodynamic stability of variants, as measured by melting temperature T(m). However, a strong correlation (R(2) > 0.95) between temperature stability and organic solvent stability was found when plotting T(50)(60) versus C(50)(60) values. All GDH variants retained stability in homogeneous aqueous-organic solvents with >80% v/v of organic solvent.

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Enzymes display high substrate specificity and can catalyze reactions that are not possible in a single step through traditional synthesis. [1] In addition, biocatalysts usually function at relatively mild aqueous conditions with moderate temperature, pressure, and pH, and thus can allow for process routes that can potentially replace less environmentally friendly steps in chemical synthesis. The ability, or in many cases, the need to function in relatively mild reaction media can also limit the utility of biocatalysts. Many interesting, often prochiral, compounds are water insoluble and thus unavailable to biocatalytic conversion. Numerous schemes have been developed to use biocatalysts to transform waterinsoluble substrates.[2] These schemes employ soluble and immobilized enzymes in simple one-and two-phase organicaqueous mixtures or more-complex mixtures by using reversed micelles, [3] supercritical fluids, and ionic liquids.[4]Two-phase approaches, either liquid-liquid or solid-liquid (as in the case of immobilized enzymes), can suffer from reduced reaction rates owing to interphase mass-transfer limitations. Furthermore, immobilized enzymes are susceptible to activity loss owing to the immobilization process and leaching of the enzyme from the solid support. Monophasic systems can avoid these limitations; however, recovery and reuse of the biocatalyst, which is imperative for large-scale processes or the isolation of pharmaceutical products, is more challenging. Herein, we demonstrate an approach to take advantage of the higher reaction rates of homogeneous biocatalysis while providing a simple method for biocatalyst recycling by using organic-aqueous tunable solvent (OATS) systems. OATS mixtures are engineered to couple a reaction and separation as shown in Figure 1. As in other latent biphasic systems, [5] OATS mixtures allow homogeneous reactions between hydrophobic and hydrophilic components, therefore eliminating mass-transfer limitations. CO 2 can be added to split the reaction mixture into a gas-expanded liquid organic phase containing hydrophobic components and an aqueous phase containing the hydrophilic catalyst [6,7] The CO 2 -induced separation allows for a one-pot reaction and separation scheme.The successful application of CO 2 as a reversible switch to modulate miscibility of aqueous and organic phases and the phase-separation behavior for a number of OATS systems has previously been studied with solvents such as acetonitrile, THF, and dioxane. [6,7] Recent investigation of this miscibility switch as a vehicle for catalyst recovery was tested on the hydrophobic substrate 1-octene with a water-soluble Rhtriphenylphosphine tris-sulfonated salt (TPPTS) complex as the catalyst. The use of an OATS system increased the