Selective Enrichment of Omega-3 Fatty Acids in Oils by Phospholipase A1

Abstract

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

“…Among all the methods employed in concentrating ω-3 FAs in triglyceride form, selective hydrolysis of non-ω-3 FAs by lipases is considered to produce a cleaner product and at a less environmental load. In addition, ω-3 FAs concentrated in the form of triglycerides were shown to be biologically more valuable than free ω-3 FAs [39,[54][55][56]. For better yields and improved economics of the process, substrate selectivity of the lipase is the key factor for ω-3 FAs concentration.…”

“…In the semi-rational approach, we have identified the amino acids of GTL that interact with the substrate, i.e., triglyceride, by covalent docking of two representative substrates with GTL. Representative substrates were chosen based on the published information on fatty acid composition and distribution of fatty acids among the three carbon positions on the glycerol backbone [39]. Substrate 1 has EPA at sn-1, octadecanoic acid at sn-2 and 9-octadecenoic acid at sn-3 of a glycerol molecule and substrate 2 has octadecanoic acid at sn-1, DHA at sn-2 and 9-octadecenoic acid at sn-3 of a glycerol molecule.…”

“…In this procedure, the mixture of free fatty acid and glyceride was mixed with 30% ethanolic KOH (0.5M) where free fatty acids get solubilised as potassium salt and insoluble glycerides were extracted twice with hexane. Free fatty acids were regenerated by lowering the pH below 2 with HCl [39]. The purity of separated fatty acids, as well as glycerides, was tested on silica gel TLC using hexane: diethyl ether: acetic acid (60:40:1, v/v) as mobile phase.…”

“…Methylation of fatty acids from both the fractions was carried out by acid catalysis using hydrogen chloride-methanol as methylating reagent as described earlier [39], with one modification i.e., a commercial mixture of hydrogen chloride-methanol was used instead of preparing it by adding acetyl chloride in methanol. 1 μl of hexane containing fatty acid methyl esters (FAMEs) was analyzed by GC-MS (Agilent 6890).…”

“…The fatty acids were quantitated by using known quantities of an external fatty acid standard. Methyl nonadecanoate was used as an internal standard as described earlier [39]. Selectivity for a given fatty acid (Fax) was calculated by using the following equation:…”