Doctoral Thesis

Lipase Selectivity in Lipid Modification

Arnar Halldorsson

Science Institute, University of Iceland, Dunhaga 3, IS-107 Reykjavík, Iceland

Supervisor: Prof. Gudmundur G. Haraldsson
The thesis was based on 7 papers, of which 6 are published and 1 is accepted for publication. The main objective of the research project was to synthesis of various lipids comprising n-3 polyunsaturated fatty acids, mainly EPA and DHA, using enzymes as well as traditional organic chemistry methods. The dissertation took place November 8 at the University of Iceland and the opponents were professor Patrick Adlercreutz from Lund University in Sweden and professor emeritus Frank D. Gunstone from St. Andrews University in Scotland. This was the PhD in Chemistry from the University of Iceland. Arnar is now jointly employed by Pronova Biocare in Norway and the Science Institute, University of Iceland as research scientist. His task is to transfer the enzyme processes he was working on in his PhD project to an industrial scale.

Arnar is well known among many of the Lipidforum members, and has attended and participated in three Lipidforum meetings. He received a PhD grant to the 21st Nordic Lipid Symposium in Bergen 2001 were he gave a presentation with the title “Lipase selectivity toward fish oil fatty acids”. At the 22nd Nordic Lipid Symposium in Naantali 2003 he gave another presentation entitled “Lipase-catalysed synthesis of structured triacylglycerol comprising EPA and DHA”. He also attended a meeting arranged by Lipidforum at the Blue Lagoon in Iceland 2000; Recent Developments in the Processing of Fats and Oils.

In May 2002 he received three awards at the 93rd AOCS meeting in Montreal: AOCS Honored Student Award, Frank C. Naughton Award and Biotechnology Student Excellence Award (1st place winner). At the conference he gave a presentation entitled “Regioselectivity of lipases toward glycerol and 1-O-alkylglycerols”

Summary

The beneficial health effects of marine fat can almost exclusively be attributed to n-3 polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). This has resulted in a growing demand for EPA and DHA as health supplements. Based on their selectivity, lipases can be used to enrich lipids with these valuable fatty acids.

There has been a growing demand for both environmentally friendly and economical methods for preparing various lipids. Lipases can contribute to the resolution of these issues with their selectivity and strong catalytic powers. The use of lipases and their selectivity in lipid modification for various purposes in the project are summarised below:

The synthesis of structured triacylglycerols requires a full regioselectivity control, which can hardly be accomplished by traditional methods of synthetic organic chemistry without resorting to multi-step protection-deprotection processes. A new and efficient chemoenzymatic approach to synthesise structured triacylglycerols comprising pure eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) at the mid-position and pure medium-chain fatty acid at the end-positions on the glycerol backbone was developed [1,2]. Lipase regioselectivity plays a vital role in this process, offering excellent yield and purity. Two types of related structured triacylglycerols were designed and synthesised in order to investigate whether the location of PUFA on the glycerol backbone can influence their oxidative stability [3]. Preliminary oxidative stability studies indicate that polyunsaturated fatty acids (PUFA) located at the mid-position are more stable than if located at the end-positions.

A process based on monoester hydrolysis was developed to investigate the fatty acid selectivity of numerous commercially available lipases towards fish oil fatty acids [4]. All the lipases tested discriminated strongly against the n-3 PUFA. Moreover, lipases displaying significant activity towards the n-3 PUFA preferred EPA to DHA. However, the two bacterial lipases from Pseudomonas preferred DHA to EPA. Digestive lipolytic enzymes isolated from salmon and rainbow trout intestines displayed reversed fatty acid selectivity [4]. Thus, n-3 PUFA esters were hydrolysed at a higher rate than more saturated fatty acid esters.

Lipase fatty acid selectivity was utilised to efficiently prepare concentrates of EPA and DHA by a kinetic resolution process [5]. The Rhizomucor miehei lipase discriminated between EPA and DHA, making it possible to concentrate them individually. Such processes make lipases an attractive alternative to existing concentrating techniques. The mild conditions under which the lipases work give them a clear advantage over the more harsh chemical processes, especially when working with the highly labile n-3 PUFA. When immobilised, lipases can be used repeatedly with little or no deterioration in their operating power. This suggests that the application of lipases represents an attractive option from an industrial point of view.

Astaxanthin diesters of varying fatty acid composition were prepared and screened in a hydrolysis reaction by various commercially available lipases [6]. The highest activity was observed with the Candida rugosa lipase, which discriminated against n-3 PUFA containing astaxanthin diesters. Digestive lipolytic enzymes isolated from salmon and rainbow trout intestines displayed a reverse fatty acid selectivity. Thus, astaxanthin diesters highly enriched with n-3 PUFA, including pure EPA and DHA, were observed to be hydrolysed at a considerably higher rate than the more saturated esters. Based on these results, in vivo studies of farmed salmon, conducted by Norsk Hydro, confirmed that astaxanthin deposition in fish muscle was considerably higher when using the astaxanthin diesters enriched with EPA and DHA as compared to unesterified astaxanthin. Since astaxanthin is a major cost ingredient in salmon fish feed these results could lead to significant cost reduction in salmon fish farming.

The highly valuable 1-O-alkyl-sn-glycerols can be prepared from the liver oil of various species of elasmobranch fish. However, due to the great variety of alkyl groups in the ether linkage moiety, they cannot be obtained as pure compounds from natural sources. Using a sequential acetylation process to resolve racemic 1-O-alkylglycerols kinetically made it possible to prepare pure chimyl, batyl and selachyl alcohols [7]. Comparison with pure enantiomers of solketal confirmed that these products had the same absolute configuration as the natural 1-O-alkyl-sn-glycerols, i.e. the S-configuration.

Publications:

  1. Halldorsson A., Magnusson C.D. and Haraldsson G.G. Chemoenzymatic synthesis of structured triacylglycerols. Tetrahedron Lett. 2001, 42, 7675-7677.
  2. Halldorsson A., Magnusson C.D. and Haraldsson G.G. Chemoenzymatic synthesis of structured triacylglycerols by highly regioselective acylation. Tetrahedron 2003, 59, 9101-9109.
  3. Haraldsson G.G., Halldorsson A. and Kulås E. Chemoenzymatic synthesis of structured triacylglycerols containing eicosapentaenoic and docosahexaenoic acids. J. Am. Oil Chem. Soc. 2000, 77, 1139-1145.
  4. Halldorsson A., Kristinsson B., Glynn C. and Haraldsson G.G. Separation of EPA and DHA in fish oil by lipase-catalyzed esterification with glycerol. J. Am. Oil Chem. Soc. 2003, 80, 915-921.
  5. Halldorsson A., Kristinsson B. and Haraldsson G.G. Lipase selectivity toward fish oil fatty acids. Eur. J. Lipid Sci. Techol. 2004, 106, 79-87.
  6. Halldorsson A. and Haraldsson G.G. Fatty acid selectivity of lipase towards astaxanthin diesters. J. Am. Oil Chem. Soc. 2004, 81, In Press.
  7. Haraldsson G.G., Thordarson P., Halldorsson A. and Kristinsson B. Kinetic resolution of 1-O-alkylglycerols by lipase. Tetrahedron: Asymmetry 1999, 10, 3671-3674.

Arnar Halldorsson

Phone: +354 525 4714,
E-mail: arnarha@hi.is

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