In laundry formulations, synergies between amphiphiles and other additives such as enzymes increase sustainability through a large decrease in energy consumption. However, traditional surfactants are derived from petroleum, requiring chemical modifications (sulfonation, ethoxylation, or esterification) and generating environmental pollution through toxicity and low degradability. Use of biosurfactants removes these issues. To provide a firmer basis for the use of biosurfactants, we report on the interactions between the industrial lipase LIPEX® and three common biosurfactants, rhamnolipids, sophorolipids, and surfactin. The model surfactant sodium dodecyl sulfate (SDS) is included in the study for comparison. A thorough characterization by Small-angle X-ray scattering (SAXS) provides valuable information on the enzyme's oligomerization and the surfactant micelles' ellipsoidal morphology. Additionally, the enzymatic activity and complex formation in different surfactant mixtures are studied using isothermal titration calorimetry, activity assays, and SAXS. SDS activates the enzyme while promoting a controlled association of monomers while the biosurfactants inhibit the enzyme, independent of their effects on its quaternary structure. Rhamnolipids and surfactin promote lipase dimerization while sophorolipids have no significant effect on lipase quaternary structure. Based on these data, we propose a partial replacement that allows the enzyme to retain enzymatic activity while improving the environmental footprint of the formulation.

An essential structural component of bacterial biofilms is functional amyloid (FuA), which also has great potential as an engineerable nano‐biomaterial. However, experimentally based high resolution structures of FuA that resolve individual residues are lacking. A fully experimentally based 3.2 Å resolution cryo‐electron microscopy density map of the FuA protein FapC from Pseudomonas sp. UK4 is presented, which reveals a Greek key‐shaped protofilament. The structure supports bioinformatic identification of conserved motifs and is broadly consistent with the AlphaFold prediction but with important modifications. Each FapC monomer consists of three imperfect repeats (IRs), with each repeat forming one cross‐β layer. An array of highly conserved Asn and Gln residues with an extensive H‐bonding network underpins this conserved Greek key‐shape and reveals the role of heterogeneous cross‐β stacking in amyloid cross‐seeding. The covariation of residues in the hydrophobic core among different IRs suggests a cooperative monomer folding process during fibril elongation, while heterogeneous stacking of IRs reduces charge repulsion between layers to stabilize the monomer fold. The FapC fibrils show intrinsic catalytic activity and strain‐dependent nanomechanical properties. Combined with mutagenesis data, the structure provides mechanistic insights into formation of FapC FuA from disordered monomers and a structural foundation for the design of novel biomaterials.

Cold-active enzymes hold promise for energy-efficient processes. Amylases are widely used in household and industrial applications, but only a few are cold-active. Here we describe three novel secreted amylases, Rho13, Ika2 and I3C6, all from bacteria growing in the cold and alkaline ikaite columns in Greenland. They all hydrolyzed starch to smaller malto-oligomers, but only Rho13 and Ika2 hydrolyzed cyclodextrins, and only Ika2 displayed transglycosylation activity. Ika2 forms a stable dimer, while both Rho13 and I3C6 are mainly monomeric. They all have optimal active temperatures around 30–35 °C and significant enzymatic activity below 20 °C, but Rho13 and I3C6 had an alkaline optimal pH, while Ika2 was markedly acidophilic. They showed complex dependence on Ca²⁺ concentration, with the activity of Rho13 and I3C6 following a bell-shaped curve and Ika2 being unaffected; however, removal of Ca²⁺ reduced the stability of all three enzymes. Loss of structure occurred well above the temperature of optimal activity, showing the characteristic psychrophilic divorce between activity and stability. MD simulations showed that Ika2 did not have a well-defined Ca²⁺ binding site, while Rho13 and I3C6 both maintained one stably bound Ca²⁺ ion. We identified psychrophilic features as higher levels of backbone fluctuations compared to mesophilic counterparts, based on a lower number of internal hydrogen bonds and salt bridges. This increased fluctuation was also found in regions outside the active site and may provide easier substrate access and accommodation, as well as faster barrier transitions. Our work sheds further light on the many ways in which psychrophilic enzymes adapt to increased catalysis at lower temperatures.

Amyloids, once viewed solely as pathological hallmarks, are now recognized as widespread and versatile functional protein assemblies. Bacterial functional amyloids (FuBAs), particularly curli (CsgA) from Escherichia coli and FapC from Pseudomonas , have emerged as paradigms for understanding amyloid structure, assembly, and function. The recent cryo‐EM‐based structure of FapC, together with others’ combined cryo‐EM and integrative computational studies on CsgA, reveal a β‐solenoid fold stabilized by imperfect repeats, producing fibrils of exceptional stability and low polymorphism, whose biogenesis is tightly controlled through dedicated accessory factors, ensuring precise secretion and nucleation. FuBAs not only scaffold biofilms but also display intrinsic catalytic activity, expanding the biochemical repertoire of extracellular matrices. They also exhibit hierarchical mechanical properties ranging from GPa stiffness at the fibril core to kPa elasticity in hydrated biofilms. FuBA operons are phylogenetically widespread, with repeat variation contributing to sequence diversity and functional adaptability. FuBAs might be seen as evolutionary intermediates between disordered peptides with significant self‐interaction tendencies and highly structured globular proteins. Their simple structures make them robust platforms for biomaterial engineering. Understanding the interplay between sequence repeats, fibril architecture, and emergent functions opens avenues for harnessing amyloids as programmable nanomaterials with applications in catalysis, synthetic biology, and biofilm control.

Protein stability in surfactant environments is crucial for various applications including industrial enzyme formulations. The interaction between Ubiquitin (UBI) and Sodium Dodecyl Sulfate (SDS) is investigated to understand the effect of pH (2 - 9.5) on the unfolding of UBI in SDS. We carried out kinetic studies in which tyrosine fluorescence (Yfl), circular dichroism (CD) and Small-angle X-ray scattering (SAXS) monitored changes in UBI’s tertiary and secondary structure and structural dynamics of the UBI-SDS system from initial interactions to endpoint complex formation. All approaches concurred in demonstrating that the unfolding rate of UBI in SDS increased significantly with decreasing pH, with an inflexion point around UBI’s isoelectric point due to changes in its net charge. The study demonstrates that the highly stable UBI unfolds by a similar mechanism across the studied pH range, through an asymmetric core-shell structure that leads to protein decorated micelles with an increase in alpha helical structure. However, the kinetics of the process and the UBI-SDS aggregation states are highly dependent on electrostatic interactions between UBI and SDS.