Visualizing Cooperative Adsorption of an Enzyme Mixture at an Air-Liquid Interface

Understanding how multiple enzymes adsorb at gas-liquid interfaces is essential to stabilize cascade reactions under process conditions. Here, we provide the first direct, multicomponent visualization of enzyme adsorption at a gas-liquid interface, using confocal laser-scanning microscopy (CLSM) applied to a model CO₂-to-methanol cascade system. CLSM enabled quantitative analysis of interfacial distribution across the enzyme mixture by tracking spectrally labeled components. This revealed how enzyme identity and relative abundance influence surface partitioning in multienzyme systems. Cooperative adsorption increased absolute surface concentration compared to individual enzymes, but this effect was suppressed when a highly surface-active enzyme dominated the interface, reducing overall protein loading. The addition of low concentrations of biobased ionic liquids significantly decreased interfacial protein concentration. This reduction likely involves a combination of enhanced solubility, electrostatic screening, and modified hydration at the interface. The response was enzyme-specific: [Choline][prolinate] most strongly reduced surface activity of formate dehydrogenase and alcohol dehydrogenase, while [tetramethyl guanidinium] [phenolate] primarily affected formaldehyde dehydrogenase. When combined, the ionic liquids exhibited additive suppression, yielding the lowest absolute surface concentration across the cascade.