Single-Molecule Study of Thermomyces lanuginosus Lipase in a Detergency Application System Reveals Diffusion Pattern Remodeling by Surfactants and Calcium.

Lipases comprise one of the major enzyme classes in biotechnology with applications within, e.g., baking, brewing, biocatalysis, and the detergent industry. Understanding the mechanisms of lipase function and regulation is therefore important to facilitate the optimization of their function by protein engineering. Advances in single-molecule studies in model systems have provided deep mechanistic insights on lipase function, such as the existence of functional states, their dependence on regulatory cues, and their correlation to activity. However, it is unclear how these observations translate to enzyme behavior in applied settings. Here, single-molecule tracking of individual Thermomyces lanuginosus lipase (TLL) enzymes in a detergency application system allowed real-time direct observation of spatiotemporal localization, and thus diffusional behavior, of TLL enzymes on a lard substrate. Parallelized imaging of thousands of individual enzymes allowed us to observe directly the existence and quantify the abundance and interconversion kinetics between three diffusional states that we recently provided evidence to correlate with function. We observe redistribution of the enzyme's diffusional pattern at the lipid-water interface as well as variations in binding efficiency in response to surfactants and calcium, demonstrating that detergency effectors can drive the sampling of lipase functional states. Our single-molecule results combined with ensemble activity assays and enzyme surface binding efficiency readouts allowed us to deconvolute how application conditions can significantly alter protein functional dynamics and/or surface binding, both of which underpin enzyme performance. We anticipate that our results will inspire further efforts to decipher and integrate the dynamic nature of lipases, and other enzymes, in the design of new biotechnological solutions.

Direct observation of Thermomyces lanuginosus lipase diffusional states by Single Particle Tracking and their remodeling by mutations and inhibition.

Lipases are interfacially activated enzymes that catalyze the hydrolysis of ester bonds and constitute prime candidates for industrial and biotechnological applications ranging from detergent industry, to chiral organic synthesis. As a result, there is an incentive to understand the mechanisms underlying lipase activity at the molecular level, so as to be able to design new lipase variants with tailor-made functionalities. Our understanding of lipase function primarily relies on bulk assay averaging the behavior of a high number of enzymes masking structural dynamics and functional heterogeneities. Recent advances in single molecule techniques based on fluorogenic substrate analogues revealed the existence of lipase functional states, and furthermore so how they are remodeled by regulatory cues. Single particle studies of lipases on the other hand directly observed diffusional heterogeneities and suggested lipases to operate in two different modes. Here to decipher how mutations in the lid region controls Thermomyces lanuginosus lipase (TLL) diffusion and function we employed a Single Particle Tracking (SPT) assay to directly observe the spatiotemporal localization of TLL and rationally designed mutants on native substrate surfaces. Parallel imaging of thousands of individual TLL enzymes and HMM analysis allowed us to observe and quantify the diffusion, abundance and microscopic transition rates between three linearly interconverting diffusional states for each lipase. We proposed a model that correlate diffusion with function that allowed us to predict that lipase regulation, via mutations in lid region or product inhibition, primarily operates via biasing transitions to the active states.

Using Polarized Spectroscopy to Investigate Order in Thin-Films of Ionic Self-Assembled Materials Based on Azo-Dyes.

Three series of ionic self-assembled materials based on anionic azo-dyes and cationic benzalkonium surfactants were synthesized and thin films were prepared by spin-casting. These thin films appear isotropic when investigated with polarized optical microscopy, although they are highly anisotropic. Here, three series of homologous materials were studied to rationalize this observation. Investigating thin films of ordered molecular materials relies to a large extent on advanced experimental methods and large research infrastructure. A statement that in particular is true for thin films with nanoscopic order, where X-ray reflectometry, X-ray and neutron scattering, electron microscopy and atom force microscopy (AFM) has to be used to elucidate film morphology and the underlying molecular structure. Here, the thin films were investigated using AFM, optical microscopy and polarized absorption spectroscopy. It was shown that by using numerical method for treating the polarized absorption spectroscopy data, the molecular structure can be elucidated. Further, it was shown that polarized optical spectroscopy is a general tool that allows determination of the molecular order in thin films. Finally, it was found that full control of thermal history and rigorous control of the ionic self-assembly conditions are required to reproducibly make these materials of high nanoscopic order. Similarly, the conditions for spin-casting are shown to be determining for the overall thin film morphology, while molecular order is maintained.