Enhancement of stability of a lipase by subjecting to three phase partitioning (TPP): structures of native and TPP-treated lipase from Thermomyces lanuginosa

Abstract

Background:The lipase enzyme converts long chain acyltriglycerides into di-and monoglycerides, glycerol and fatty acids. The catalytic site in lipase is situated deep inside the molecule. It is connected through a tunnel to the surface of the molecule. In the unbound state under aqueous conditions, the tunnel remains closed. The tunnel can be opened when the enzyme is exposed to a lipid bilayer or a detergent or many hydrophobic/hydrophilic surfaces. Results:In the present study, the lipase was subjected to three-phase partitioning (TPP) which consisted of mixing in tert-butanol and ammonium sulphate to the solution of lipase in the aqueous buffer. The enzyme formed an interfacial precipitate between the tert-butanol rich and water rich phases. The stability of the enzyme subjected to TPP was found to be higher (T m of 80 °C) than the untreated enzyme (T m of 77 °C). The activity of the enzyme subjected to TPP (3.3 U/mg) was nearly half of that of the untreated one (5.8 U/mg). However, the activity of the treated enzyme was higher (17.8 U/mg) than the untreated one (8.6 U/mg) when a detergent was incorporated in the assay buffer. Conclusions:The structure determination showed that the substrate binding site in the treated enzyme was more tightly closed than that of the untreated protein.

Modulation of the Catalytic Properties of Immobilized Recombinant Lipase from Thermomyces lanuginosus in the Reaction of Esterification by the Selection of an Adsorbent

“…With an increase in the length of lipase substrate molecules, the efficiency of biocatalysis decreased. The broader specificity of the immobilized lipase towards the alcohol substrate indicated that the S 2 -binding site of the active site of the lipase was wider in size than the S 1 -binding site, which allowed us to confirm the conclusions about the structure of the active site of the T. lanuginosus lipase made in [27,[30][31][32][33]: the active site of the RML type lipase, to which TLL belongs, is located close to the surface of the enzyme globule and has a narrow gap for S 1 (acid), and a wider one for S 2 (alcohol).…”

“…The crystal structure of TLL with a resolution of 2.3 Å was taken from the PDB database (PDB ID: 4ZGB). For simulation, a form of lipase in which the active site was in the closed conformation, was used [27]. CNT structures of various sizes were generated using the Nanotube Modeler program and optimized using Avogadro.…”

Improving the Thermostability of Thermomyces lanuginosus Lipase by Restricting the Flexibility of N-Terminus and C-Terminus Simultaneously via the 25-Loop Substitutions

“…To evaluate the flexibility of the protein, B-factors of the Cα atoms in TLL were extracted from the crystal structure file (PDB ID: 4ZGB and 1DT3) [42,43]. The B-factor values of each atom were normalized to have a distribution of zero mean and unit variance based on the following equation:…”

Rebuilding the lid region from conformational and dynamic features to engineering applications of lipase in foods: Current status and future prospects

“…Early studies on lid motion mainly focused on the fungal lipase family, particularly the triacylglycerol lipase family (called the RmL lipase family), such as Rhizomucor miehei lipase (RML) (Moroz et al., 2019). According to the configurations of lipase lid, the typical lid motion was assumed to follow a two‐step opening mechanism that first gives rise to a semi‐open conformation upon adsorption at the interface, followed by a full opening of the lid upon substrate binding (Kumar et al., 2015; Longhi et al., 2011) (Figure 4). Hinge‐type motion is similar to rotations around an articulated joint that allows major conformational changes without altering the internal packing of the individual domains (Papaleo et al., 2016).…”