Understanding the activation mechanism of Thermomyces lanuginosus lipase using rational design and tryptophan‐induced fluorescence quenching

Abstract

The attractiveness of lipases as industrial biocatalysts underlines the importance of understanding their molecular action in detail, helping future efforts to improve performance and applicability. Lipases represent a special class of carboxyl ester hydrolases, which act on long‐chained water insoluble acyl‐glycerols at the water‐lipid interface. This heterogeneous catalysis makes it very difficult to monitor the structural changes taking place in the enzyme during activation in its preferred milieu. In this review, we cover recent developments toward a deeper understanding of the interfacial activation phenomenon of lipases and discuss a selection of biophysical methods suitable for studying lipase activation. For many lipases, such as that from Thermomyces lanuginosus, a structural domain called the “lid” is directly involved in the activation process. This has been shown through alteration of the lid‐residue composition using rational design. The resulting lid‐mutants have highlighted the lid's role in governing enzymatic activity and interfacial activation. Moreover, a recently developed fluorescence‐based technique called the Tryptophan Induced Quenching method has proved to enable the direct measurement of lipase activation in water‐ethanol mixtures, and thus represents an intriguing method to gain further insight into the structural movements taking place at the water‐lipid interface. Practical applications: Lipases are important industrial biocatalysts widely used in industrial applications including detergent, leather, paper, fuel, fine chemicals, baking, cosmetics, and food. Knowledge of structural changes of lipase upon activation and identification of the amino acids involved in lipase activation is crucial for design of new variants with improved or new properties. Rational design of the lid‐region in lipases has an interesting potential for optimization of lipase function and adapting them to different environments. In order to leverage on the design of new lipase variants and their altered function, methods that enable direct measurement of lipase action directly in solution, in real time, are desired. In this context, fluorescence based methods have shown great potential and provides interesting perspectives for probing the delicate movements occurring in lipases upon activation. Interfacial activation of lipases and the lid movements studied by rational design, fluorescence quenching methods, and solvent effect – polarity driven activation.

Effect of Protein Flexibility from Coarse-Grained Elastic Network Parameterizations on the Calculation of Free Energy Profiles of Ligand Binding

“…Then, at distances very close to the active site, the free energy rises again. The test case used in this work, namely, the TOG molecule, belongs to a set of enzyme/substrate systems whose reaction occurrence is well known. ,,, Therefore, the energy increase when the TOG molecule approaches the center of the TLL active site must be mainly due to a binding resistance imposed by the eventual lack of flexibility of the protein. Moreover, the free energy starts to increase at the same region of the reaction coordinate for both TOG and TAG solutes.…”

A Temporal Evolution Perspective of Lipase Production by Yarrowia lipolytica in Solid-State Fermentation

“…The interfacial activation, commonly observed in lipases, is the result of combined effects of the amino acid composition of the lid (and therefore the structure resulting from interactions with the environment) and the properties of the interface (such as the dielectric constant and ionic strength) [4]. The enzyme undergoes rearrangements at the water-oil interface upon exposure to a less polar solvent, making the catalytic site available for substrate access [32]. The exception is the lipase B from Candida antartica.…”

“…Esterases show activity before the critical micellar concentration (CMC) and may be produced concurrently with lipases. After interaction with an interface, the latter acts majorly, needing a structural rearrangement to reach an active conformation [32]. Therefore the reaction media may be formulated differently to favor either type of enzymatic activity.…”

“…In addition, anything that alters the properties, or the size of the surface, may change the reaction rates. For instance, smaller droplets are beneficial for higher reaction rates [34], and calcium ions tend to neutralize negative charges maintaining a low dielectric constant at the interface [32].…”

Advances in Recombinant Lipases: Production, Engineering, Immobilization and Application in the Pharmaceutical Industry

“…Although lipases have tremendous potential industrial use, they often need to have their properties improved due to the biocatalytic bioprocess requirements. Therefore, using techniques, such as site directed mutagenesis based on rational design, different mutants can be constructed showing improved thermal stability, tolerance against organic solvents and optimized selectivity [36,37]. This is due to the necessity to achieve optimal performance in different industrial fields, and to effectively catalyze synthetic reactions in micro-aqueous conditions where organic solvents are required.…”