Selective enrichment of omega-3 fatty acids in oils by phospholipase A1

Omega fatty acids are recognized as key nutrients for healthier ageing. Lipases are used to release ω-3 fatty acids from oils for preparing enriched ω-3 fatty acid supplements. However, use of lipases in enrichment of ω-3 fatty acids is limited due to their insufficient specificity for ω-3 fatty acids. In this study use of phospholipase A1 (PLA1), which possesses both sn-1 specific activity on phospholipids and lipase activity, was explored for hydrolysis of ω-3 fatty acids from anchovy oil. Substrate specificity of PLA1 from Thermomyces lenuginosus was initially tested with synthetic p-nitrophenyl esters along with a lipase from Bacillus subtilis (BSL), as a lipase control. Gas chromatographic characterization of the hydrolysate obtained upon treatment of anchovy oil with these enzymes indicated a selective retention of ω-3 fatty acids in the triglyceride fraction by PLA1 and not by BSL. 13C NMR spectroscopy based position analysis of fatty acids in enzyme treated and untreated samples indicated that PLA1 preferably retained ω-3 fatty acids in oil, while saturated fatty acids were hydrolysed irrespective of their position. Hydrolysis of structured triglyceride,1,3-dioleoyl-2-palmitoylglycerol, suggested that both the enzymes hydrolyse the fatty acids at both the positions. The observed discrimination against ω-3 fatty acids by PLA1 appears to be due to its fatty acid selectivity rather than positional specificity. These studies suggest that PLA1 could be used as a potential enzyme for selective concentrationof ω-3 fatty acids

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

Enrichment of omega-3 fatty acids (ɷ-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 ɷ-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 ɷ-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

Percent accumulation of different fatty acid classes in triglyceride fraction after 30% hydrolysis of anchovy oil by PLA1 (dark) and BSL (light).

<p>A, Percent accumulation of each fatty acid is calculated as (^UHFA_x-^TotalFA_x)/^TotalFA_x) X 100). Each of the fatty acid fractions were calculated based on peak area in the NMR spectra. B, Percent accumulation data plotted with positional information.</p

Percent hydrolysis of various fatty acids (from Fig 3) at 32% hydrolysis of anchovy oil by PLA1 (light) and BSL (dark).

<p>The relative proportions of fatty acids at other time points were also similar.</p

Time course of hydrolysis of anchovy oil and fatty acid distribution in the hydrolysate.

<p>Anchovy oil was subjected to hydrolysis by PLA1 (A) and BSL (B). The reaction product at various times was subjected to methylation and GC analysis. Percent hydrolysis of each fatty acid was calculated based on its hydrolysis at t_x as a fraction of t_o. Saturated (□), Monounsaturated (●), EPA (○), DHA (▲), Total hydrolysis (■).</p

Positional distribution of various fatty acids in anchovy oil.

<p>Positional distribution of various fatty acids in anchovy oil.</p

Phospholipase A1 activity towards <i>p</i>NP esters with different acyl chains.

<p>Activity of PLA1 on pNP-decanoate was taken as 100%.</p