Substrate structure and computation guided engineering of a lipase for omega-3 fatty acid selectivity

Abstract

Enrichment of omega-3 fatty acids (ω-3 FAs) in natural oils is important to realize their health benefits. Lipases are promising catalysts to perform this enrichment, however, fatty acid specificity of lipases is poor. We attempted to improve the fatty acid selectivity of a lipase from Geobacillus thermoleovorans (GTL) by two approaches. In a semi-rational approach, amino acid positions critical for binding were identified by docking the substrate to the GTL and best substitutes at these positions were identified by site saturation mutagenesis followed by screening to obtain a variant of GTL (CM-GTL). In the second approach based on rational design, a variant of GTL was designed (DM-GTL) wherein the active site was narrowed by incorporating two heavier amino acids in the lining of acyl-binding pocket to hinder access to bulky ω-3 FAs. The affinities DM-GTL with designed substrates were evaluated in silico. Both, CM-GTL and DM-GTL have shown excellent ability to discriminate against the ω-3 FAs during hydrolysis of oils. Engineering the binding pocket of an enzyme of a complex substrate, such as a triglyceride, by incorporating the information on substrate structure and computationally derived binding modes, has resulted in designing two efficient lipase variants with improved substrate selectivity.

Altering the Chain Length Specificity of a Lipase from Pleurotus citrinopileatus for the Application in Cheese Making

“…Considering the criteria that more suitable binding modes of small- to medium-chain length substrates should be found and the binding affinity of those variants should be increased for those substrates, compared to long-chain pNPP, 35 mutants were predicted to have a higher selectivity for short- to medium-chain fatty acids ( Table 1 ). A similar approach was followed for the lipase from Geobacillus thermoleovorans, where the mutants were ranked and analyzed by their calculated binding affinity [ 34 ]. With the used workflow, the laboratory screening effort for the PCI_Lip mutants was thus reduced to about 23% compared to an approach where all eight positions were fully saturated.…”

Microalgal polyunsaturated fatty acids: Hotspots and production techniques

“…Lipase with high specificity must be extensively screened, which can increase the cost. To enhance the functional specificity of lipase, computer-aided design can be employed (Moharana and Rao, 2020).…”

Molecular characterization of transesterification activity of novel lipase family I.1

“…Validation of the resulting structure based on the Ramachandran plot using MolProbity [ 23 ]. Protein minimization was not performed to prevent amino acid residues from being present in the dissallowed area on the basis of the Ramachandran plot [ 24 ]. PubChem data were used to create the ligand structure ( https://pubchem.ncbi.nlm.nih.gov/ ) [ 25 ].…”