Increased hydrolysis by Thermomyces lanuginosus lipase for omega-3 fatty acids in the presence of a protic ionic liquid

We report that the hydrolytic performance of Thermomyces lanuginosus lipase, TLL, and its selectivity towards concentrating clinically important omega 3 fatty acids was increased by the addition of a protic ionic liquid, pIL, Triethylammonium mesylate, TeaMs. We show that TeaMs has a structure altering effect on TLL, changing both the secondary and tertiary structure of TLL. The thermal activity of TLL was also significantly enhanced by the addition of TeaMs.

Understanding the influence of key ionic liquid properties on the hydrolytic activity of thermomyces lanuginosus lipase

Ionic liquids (ILs) are solvents with numerous properties, which have been recently used for enzyme catalysis. In this work, five different ILs based on primary, tertiary, and quaternary ammonium cations coupled with mesylate and propionate anions were used as media for hydrolysis by the industrially relevant enzyme Thermomyces lanuginosus lipase (TLL). We correlated the TLL activity with various key IL and IL-water properties, including ion concentration, water activity (aw), kosmotropicity, hydrogen-bond basicity (β), and pH. The ion concentration was associated with aw, and the molar ratio of water/IL 5:1 (aw≈0.6) was found to be the threshold for assured TLL activity. Triethylammonium mesylate was the best IL owing to its kosmotropicity and ideal intrinsic β. The pH of IL-water mixtures is a key parameter related to the conformational change of TLL. We demonstrated the pH effect of the IL-water mixtures can be overcome by buffering, and the buffered system displayed the greatest activity.

Lipase-catalysed incorporation of EPA into emu oil: formation and characterisation of new structured lipids

Partial hydrolysis of emu oil was performed using Thermomyces lanuginosus lipase to remove some shorter chain fatty acids. Then eicosapentaenoic acid (EPA) was incorporated into the modified emu oil using either Lipozyme RMIM or Lipozyme TLIM to produce new EPA enriched structured lipids. Using Isooctane as a reaction solvent increased the level of EPA incorporation, which was higher with RMIM than with TLIM. TLIM incorporated EPA almost exclusively into the sn-1,3 positions, whereas RMIM incorporated EPA at sn-1,3 and sn-2 positions in an almost statistical ratio. Both structured lipids were less oxidatively stable than emu oil.

Characterisation of lipase fatty acid selectivity using novel omega-3 pNP-acyl esters

 Lipases have applications for the industrial processing of lipids, including concentrating and/or modifying fish oil derived omega-3 fatty acids, widely used as nutritional supplement and functional food ingredients. A range of para-nitrophenol (pNP) acyl esters were synthesised as a means to rapidly screen lipases for fatty acid selectivity using spectrophotometric detection. The chosen esters were based primarily on the most abundant fatty acids present in anchovy and tuna oils. pNP derivatives of C16:1 n-7, C18:1 n-9 (OA), C18:2 n-6 (LA), C18:3 n-3 (ALA), C20:5 n-3 (EPA) and C22:6 n-3 (DHA) were synthesised. Storage stability of these pNP derivatives was shown to be at least 6 months and all pNP derivatives, including those of EPA and DHA, were shown to be stable throughout the conditions of the assay. We applied the new assay substrates for the determination of fatty acid selectivity of five widely utilised lipases. Results showed that the lipase from Candida rugosa was the most selective in terms of omega-3 specificity, preferentially hydrolysing all other medium– long chain substrates. Lipases from Rhizomucor miehei and Thermomyces lanuginosa also showed selectivity, with a significant preference for saturated fatty acids. Candida Antarctica lipase B and Aspergillus niger lipase were the least selective.

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.