Silke Flindt Badino

The enzyme carbonic anhydrase (CA) can accelerate gas-liquid mass transfer of CO2, and extensive research is exploring how to leverage this in industrial carbon capture. At the molecular level, CA promotes capture by catalyzing the conversion of CO2 to bicarbonate near the gas-liquid interface. This mechanism implies that only a minor fraction of the enzyme at the interface actively contributes to capture, and as a result, efficient capture requires high enzyme dosages. Here, we designed and synthesized CA variants with hydrophobic modifications that render them interfacially active. The variants showed dramatic improvements in performance, and in some cases, the specific activity for carbon capture increased by more than two orders of magnitude relative to the unmodified, wild-type enzyme. Conversely, the thermostability and intrinsic enzyme activity in the aqueous bulk were only marginally affected by the modifications. These results implied that the enhanced efficacy of the modified enzymes reflected a general surface-partitioning mechanism. This interpretation provided fundamental insights into the interfacial enzyme process and highlighted a novel, broad avenue for designing catalysts for CO2 capture. Thus, this mechanism suggested that a range of hydrophobic modifications could enhance the activity of any catalyst (enzyme or non-enzyme) during carbon capture in aqueous sorbents.

Enzyme-assisted carbon capture is attracting massive interest, and absorbents composed of aqueous carbonate supplemented with carbonic anhydrase have proven particularly promising. Here, we study basic capture mechanisms using a novel approach grounded in comparative enzymology. We determined initial, steady-state capture rates in potassium carbonate under a range of conditions and observed a characteristic saturation behavior at high concentrations of either enzyme or CO₂. These results could be rationalized by a modified Michaelis–Menten framework applied to a “reaction zone” near the liquid surface. Capture rates corresponded directly to enzyme reaction rates in the reaction zone as determined by K_M and k_cat, and this explained the observed saturation behavior. The kinetic data suggested a depth of the reaction zone of about 20 µm. This meant that equilibrium between CO₂ and HCO₃⁻ was obtained within this shallow film and that enzymes deeper in the liquid had little or no influence on capture rates. This approach also allowed us to rationalize the effect of pH on enzyme-assisted capture rates. Overall, steady-state kinetics can be used in comparative and mechanistic analyses of enzyme-accelerated carbon capture. The approach is theoretically simple, requires limited experimental input, and offers key molecular insights.

Direct removal of carbon dioxide from atmospheric air has been widely advocated as a key tool in the strive towards zero emissions. One class of technology for direct air capture (DAC) uses aqueous sorbents in air-liquid contactors to extract the CO2. Here we present the first systematic data on enzyme assisted DAC and show that even very low (sub µM) concentrations of carbonic anhydrase (CA) exert a strong acceleration on CO2 absorption. We also found that CA promoted the ability to retain a high capture efficiency as the flow rate was raised and we rationalized these observations based on molecular mechanism of enzyme assisted capture. We interpret that acceleration of carbon capture with CA is more efficient under DAC conditions (short contact times and diluted sources) than during capture from CO2 sources. These attributes may eventually pave the way for DAC in sorbents with low regeneration energies such as carbonate.

Kinetic theory of enzymes that modify insoluble substrates is still underdeveloped, despite the prevalence of this type of reaction both in vivo and industrial applications. Here, we present a steady-state kinetic approach to investigate inhibition occurring at the solid-liquid interface. We propose to conduct experiments under enzyme excess (E₀>>S₀), i.e. the opposite limit compared to the conventional Michaelis-Menten framework. This inverse condition is practical for insoluble substrates and elucidates how the inhibitor reduces enzyme activity through binding to the substrate. We claim that this type of inhibition is common for interfacial enzyme reactions because substrate accessibility is low, and we show that it can be analyzed by experiments and rate equations that are analogous to the conventional approach, except that the roles of enzyme and substrate have been swapped. To illustrate the approach, we investigated the major cellulases from Trichoderma reesei (Cel6A and Cel7A) acting on insoluble cellulose. As model inhibitors, we used catalytically inactive variants of Cel6A and Cel7A. We made so-called inverse Michaelis-Menten curves at different concentrations of inhibitors and found that a new rate equation accounted well for the data. In most cases, we found a mixed type of surface-site inhibition mechanism, and this probably reflected that the inhibitor both competed with the enzyme for the productive binding sites (competitive inhibition) and hampered the processive movement on the surface (uncompetitive inhibition). These results give new insights into the complex interplay of Cel7A and Cel6A on cellulose and the approach may be applicable to other heterogeneous enzyme reactions.

Enzyme reactions, both in Nature and technical applications, commonly occur at the interface of immiscible phases. Nevertheless, stringent descriptions of interfacial enzyme catalysis remain sparse, and this is partly due to a shortage of coherent experimental data to guide and assess such work. We have produced and kinetically characterized 83 cellulases, which revealed a conspicuous linear free energy relationship (LFER) between the strength of substrate binding and the activation barrier. This common scaling occurred despite the investigated enzymes were structurally and mechanistically diverse. We suggest that the scaling reflects basic physical restrictions of the hydrolytic process and that evolutionary selection have condensed cellulase phenotypes near the line. One consequence of the LFER is that the activity of a cellulase can be estimated from substrate binding strength, irrespectively of structural and mechanistic details, and this appears promising for in silico selection and design within this industrially important group of enzymes. On a more general note, the LFER may identify a link to inorganic heterogeneous catalysis, and hence open for the implementation of approaches from this field within interfacial enzymology.

The cellobiohydrolase (CBH) Cel6A is an important component of enzyme cocktails for industrial degradation of lignocellulosic biomass. However, the kinetics of this enzyme acting on its natural, insoluble substrate remains sparsely investigated. Here, we studied Cel6A from Trichoderma reesei with respect to adsorption, processivity, and kinetics both in the steady‐state and pre‐steady‐state regimes, on microcrystalline and amorphous cellulose. We found that slow dissociation (k_off) was limiting the overall reaction rate, and we suggest that this leads to an accumulation of catalytically inactive complexes in front of obstacles and irregularities on the cellulose surface. The processivity number of Cel6A was low on both investigated substrates (5–10), and this suggested a rugged surface with short obstacle‐free path lengths. The turnover of the inner catalytic cycle (the reactions of catalysis in one processive step) was too fast to be fully resolved, but a minimum value of about 20 s⁻¹ could be established. This is among the highest values reported hitherto for a cellulase, and it underscores the catalytic efficiency of Cel6A. Conversely, we found that Cel6A had a poor ability to recognize attack sites on the cellulose surface. On amorphous cellulose, for example, Cel6A was only able to initiate hydrolysis on about 4% of the sites to which it could adsorb. This probably reflects high requirements of Cel6A to the architecture of the site. We conclude that compared to the other CBH, Cel7A, secreted by T. reesei, Cel6A is catalytically more efficient but less capable of attacking a broad range of structurally distinct sites on the cellulose surface.Enzymes: TrCel6A, nonreducing end‐acting cellobiohydrolase (EC3.2.1.91) from Trichoderma reesei; TrCel7A, reducing end‐acting cellobiohydrolase (EC3.2.1.176) from T. reesei.

The ascomycete fungus Trichoderma reesei is the predominant source of enzymes for industrial conversion of lignocellulose. Its glycoside hydrolase family 7 cellobiohydrolase (GH7 CBH) TreCel7A constitutes nearly half of the enzyme cocktail by weight and is the major workhorse in the cellulose hydrolysis process. The orthologs from Trichoderma atroviride (TatCel7A) and Trichoderma harzianum (ThaCel7A) show high sequence identity with TreCel7A, ~ 80%, and represent naturally evolved combinations of cellulose-binding tunnel-enclosing loop motifs, which have been suggested to influence intrinsic cellobiohydrolase properties, such as endo-initiation, processivity, and off-rate. The TatCel7A, ThaCel7A, and TreCel7A enzymes were characterized for comparison of function. The catalytic domain of TatCel7A was crystallized, and two structures were determined: without ligand and with thio-cellotriose in the active site. Initial hydrolysis of bacterial cellulose was faster with TatCel7A than either ThaCel7A or TreCel7A. In synergistic saccharification of pretreated corn stover, both TatCel7A and ThaCel7A were more efficient than TreCel7A, although TatCel7A was more sensitive to thermal inactivation. Structural analyses and molecular dynamics (MD) simulations were performed to elucidate important structure/function correlations. Moreover, reverse conservation analysis (RCA) of sequence diversity revealed divergent regions of interest located outside the cellulose-binding tunnel of Trichoderma spp. GH7 CBHs. We hypothesize that the combination of loop motifs is the main determinant for the observed differences in Cel7A activity on cellulosic substrates. Fine-tuning of the loop flexibility appears to be an important evolutionary target in Trichoderma spp., a conclusion supported by the RCA data. Our results indicate that, for industrial use, it would be beneficial to combine loop motifs from TatCel7A with the thermostability features of TreCel7A. Furthermore, one region implicated in thermal unfolding is suggested as a primary target for protein engineering.

We have measured activity and substrate affinity of the thermostable cellobiohydrolase, Cel7A, from Rasamsonia emersonii over a broad range of temperatures. For the wild type enzyme, which does not have a Carbohydrate Binding Module (CBM), higher temperature only led to moderately increased activity against cellulose, and we ascribed this to a pronounced, temperature induced desorption of enzyme from the substrate surface. We also tested a “high affinity” variant of R. emersonii Cel7A with a linker and CBM from a related enzyme. At room temperature, the activity of the variant was similar to the wild type, but the variant was more accelerated by temperature and about two‐fold faster around 70 °C. This better thermoactivation of the high‐affinity variant could not be linked to differences in stability or the catalytic process, but coincided with less desorption as temperature increased. Based on these observations and earlier reports on moderate thermoactivation of cellulases, we suggest that better cellulolytic activity at industrially relevant temperatures may be attained by engineering improved substrate affinity into enzymes that already possess good thermostability.