Marine Lipid Biotechnology: Omega-3 Enriched Lipids by Use of Lipase

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1 The 6 th BIPRSP Conference Tromsö, Norway, February 20-22, 2013 Marine Lipid Biotechnology: mega-3 Enriched Lipids by Use of Lipase Gudmundur G. Haraldsson Science Institute, University of Iceland

2 utline Enrichment of TAG with omega-3 PUFA by lipase Concentration of EPA and DHA by lipase Structured lipids Summary and conclusion

3 EPA and DHA CH CH EPA DHA

4 The initial goal To develop methods to produce CL triglycerides highly enriched with EPA and DHA

5 Cod liver oil Virtually pure triglycerides More than 50 different fatty acids, saturated, monounsaturated and polyunsaturated Approximately 10% of EPA and 10% DHA

6 Most important features of lipase in biotechnology of fats and oils Selectivity High catalytic efficiency Mildness rganic medium

7 Lipase-catalyzed transesterification of cod liver oil Interesterification CL CL CL + PUFA Et (excess) 1,3-Specific Lipase PUFA CL PUFA + CL PUFA Et Et CL

8 Lipase-catalyzed transesterification of cod liver oil Conditions Mucor miehei lipase (MML) Solvent-free T = 65 C Three-fold excess of PUFA 10% Dosage of lipase

9 Lipase-catalyzed transesterification of cod liver oil bservations and results Incorporation rate much higher for EPA than DHA Randomization despite 1,3-regioselectivity Triglycerides of 70% EPA + DHA content Highly efficient

10 Homogeneous TAG of EPA and DHA EPA DHA EPA DHA EPA DHA Trieicosapentaenoin Tridocosahexaenoin

11 Esterification of glycerol with EPA H H H + 3 EPA H Lipase EPA EPA EPA + 3 H 2

12 Esterification of glycerol with EPA/DHA Conditions Candida antarctica lipase Solvent-free T = 65 C Stoichiometric amounts Vacuum ( Torr) 10% Dosage of lipase

13 Esterification of glycerol with EPA/DHA Results and observations Highly pure TAG homogeneous with EPA or DHA Excellent yields DHA reacted significantly slower than EPA Pure TAG of composition identical to concentrates

14 Direct esterification Reaction pathway Lipase (fast) CR H H Lipase (fast) CR H CR Lipase (slow) H H H 1-MAG Lipase (slow) 1,3-DAG Acylmigration Acylmigration CR CR CR Lipase (slow) H CR H Lipase (fast) CR CR H Lipase (fast) TAG 2-MAG 1,2-DAG

15 Lipase fatty acid selectivity Usually lipase prefers less unsaturated fatty acids EPA preferred over DHA Lipases from fish intestines prefer -3 PUFA over less unsaturated fatty acids

16 Reasons for lower lipase activity towards -3 PUFA Proximity of C=C to carboxyl group Shape of PUFA

17 Direct esterification of fish oil FFA Fish il H + H Lipase EPA + DHA H

18 Direct esterification of tuna oil FFA Time Conv. Ethyl esters Residual FFA h % EPA DHA EPA rec. EPA DHA DHA rec. % % % % % %

19 Structured lipids Lipids that have a predetermined composition and distribution of fatty acids at the glycerol backbone

20 Structured triacylglycerols PUFA

21 Structured triacylglycerols Why interesting? located at the end-positions of TAG are rapidly hydrolysed by Pancreatic lipase Rapidly absorbed into the intestines Rapidly carried to the liver a quick source of energy

22 Structured triacylglycerols Why interesting? Remaining 2-MAG contain the PUFA Absorbed through the intestinal wall Accumulate as TAG in Adipose Tissue Accumulate as PL in cell membranes Source of PUFA and Essential fatty acids

23 Structured TAG comprising EPA and DHA EPA DHA

24 Lipase advantages Regioselectivity Mild conditions to retard acyl-migration Mild conditions favourable for PUFA rganic conditions

25 Chemoenzymatic synthesis of structured triacylglycerols by lipase H PUFA H H CAL H PUFA H EDCI DMAP 1a-d 2a-d 3a-d = C 5 H 11 (a), C 7 H 15 (b), C 9 H 19 (c), C 11 H 23 (d) 2: PUFA = EPA 3: PUFA = DHA

26 Reaction conditions Enzymatic reaction Candida antarctica lipase Vinyl esters of (25% excess) Solvent: Dichloromethane Temperature: 0-4 Reaction time: 5 hours Purification: Crystallization (Hexane) Yields: Excellent (90-92%)

27 1,3-Diacylglycerols Enzymatic reaction H H H CAL 1a-d H Compound PUFA Yields (%) 1a -C 5 H b -C 7 H c -C 9 H d -C 11 H 23-90

28 Acyl-migration H H 1,3-DAG 1,2-DAG

29 Reaction conditions Coupling reaction EDCI (20% excess); DMAP (0.6 eq.) Stoichiometric amount of EPA and DHA Solvent: Dichloromethane Room temperature Reaction time: 2-3 hours Purification: Silica gel chromatography Yields: Excellent (90-95%)

30 Triacylglycerols Coupling reaction DHA 1a-d H DHA EDCI DMAP H 3a-d Compound PUFA Yields (%) 3a -C 5 H 11 DHA 94 3b -C 7 H 15 DHA 90 3c -C 9 H 19 DHA 94 3d -C 11 H 23 DHA 95

31 EDCI coupling agent H N C N + N Cl - CH 3 CH 3 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

32 Glyceryl ethers Major constituents in shark liver oil Chimyl alcohol (C 16:0 ) (S)-4 H Batyl alcohol (C 18:0 ) (S)-5 H H H Selachyl alcohol (C 18:1 ) (S)-6 H H H H H

33 Claimed beneficial health effects of ether lipids Shark liver oil used for a long time as a therapeutic and preventive agent Prevent radiation sickness from cancer X-ray therapy Stimulating for the allergic system Stimulate immune control Beneficial against asthma, psoriasis and arthritis Speed up the removal of heavy metals from the body

34 Structured ether lipids 1--Alkyl-2,3-diacyl-sn-glycerols R EPA R DHA

35 Synthesis of enantiopure structured EL R H H H CAL R H H PUFA EDCI DMAP H R H PUFA Chimyl: R = C 16 H 33 Batyl: R = C 18 H 37 Selachyl: R = C 18 H 35

36 Enantiopure structured PC EPA DHA P - + N P + N -

37 Reversely structured TAG and EL PUFA R PUFA PUFA

38 Activation of EPA and DHA as oxime derivatives PUFA H + N H EDCI PUFA N PUFA = EPA (94%) PUFA = DHA (95%)

39 Summary Regio- and enantiopure structured TAG, EL and PC utstanding regioselectivity of the lipases CAL: The best catalyst for TAG and EL RML: The best catalyst for PC Acyl migration eliminated by mild conditions Very high to excellent yields in all cases nly two steps Full characterization by 1 H, 13 C and 31 P NMR

40 Application Clinical research Individual fatty acid investigations Pure compounds useful as standards Isotopically labeled fatty acids Liposomes Focused libraries of lipid compounds

41 Acknowledgements Large number of my excellent students and postdocs at the University of Iceland University of Iceland Research Fund, Icelandic Research Fund and Lysi hf for financial support Avanti Polar Lipids for pure GPC Pronova Biocare in Norway for pure EPA and DHA Novozymes in Denmark for the lipase

42 The Blue Lagoon All year outdoor bathing Coctails served into the lagoon Thank you!