Enzymatic fatty acid exchange in digalactosyldiacylglycerol

Enzymatic fatty acid exchange in digalactosyldiacylglycerol
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  Chemistry and Physics of Lipids104 (2000) 13–21 Enzymatic fatty acid exchange in digalactosyldiacylglycerol Mattias Persson *, Ingemar Svensson, Patrick Adlercreutz Department of Biotechnology ,  Center for Chemistry and Chemical Engineering  ,  Lund Uni   ersity ,  P . O Box  124  , S  - 221 00   Lund  ,  Sweden Received 12 February 1999; received in revised form 6 July 1999; accepted 16 July 1999 Abstract Six different lipases were screened for their ability of acidolysis between digalactosyldiacylglycerol (DGDG) andheptadecanoic acid in toluene. Lipases from  Geotrichum candidum ,  Alcaligenes  sp. and  Penicillium camembertii   did notcatalyse the acidolysis reaction.  Rhizopus arrhizus  and  Rhizomucor miehei   (Lipozyme) catalysed the acidolysis butproduced a mixture of DGMG, DGDG, acyl-DGMG and acyl-DGDG. The extra acyl group is bound to the primaryhydroxyl of the digalactosyl moiety.  Candida antarctica  also catalysed the acidolysis but the TLC analysis showedbands with higher R f   values than acyl-DGDG, these probably being different tetra and higher esters.  R .  arrhizus lipase was the most promising enzyme under the conditions used, with no tetra esters being formed and giving thehighest reaction rate of the enzymes investigated. Low water activity (0.06 or 0.11) and high fatty acid concentration(400 mM) increased the formation of acyl-DGDG whilst higher water activities (0.33 and 0.54) increased the amountof DGMG when  R .  arrhizus  lipase was used as catalyst. At a water activity of 0.11 and a fatty acid concentrationof 400 mM a yield of 24% modified DGDG was obtained. In this product the fatty acid srcinally present in the  sn -1position had been exchanged by heptadecanoic acid. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords :   Galactolipid; Digalactosyldiacylglycerol; Lipase; Lipid / locate / chemphyslip 1. Introduction Glycolipids are mainly found in the chloroplastmembrane in plants, algae and in the grains of cereals where they are involved in different mem-brane functions such as the electron transportchain in photosynthesis and also function as acarbohydrate reservoir (Hummel, 1975). One of the most abundant glycolipids in wheat and oat is1,2-diacyl-3- O -[  -galactopyranosyl]-(1-6)- O -[  -galactopyranosyl]-glycerol (DGDG). In contrast,DGDG is found only in small quantities inanimals.DGDG has many potential applications in thepharmaceutical and food industries. It enhancesthe baking quality of wheat flour (Prieto et al.,1993) and it also seems to have an anti tumourpromoting effect which depends on the fatty acidcomposition of the molecule (Shirahashi et al.,1993). DGDG has been isolated from a variety of sources, for example rye, wheat, oat and spinach(Bergqvist and Kaufmann, 1996). The fatty acid * Corresponding author. Tel.:  + 46-46-2224841; fax:  + 46-46-2224713. E  - mail address : (M. Persson)0009-3084 / 00 / $ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.PII: S0009-3084(99)00099-7  M  .  Persson et al  .  /   Chemistry and Physics of Lipids  104 (2000) 13–21 14 composition of DGDG differs considerably be-tween different sources. For some applications thenatural lipids are useful, but for other applica-tions it is better with another fatty acid composi-tion. Thus it is of interest to find ways to modifythe fatty acid composition in a controlled way.Fatty acid exchange reactions have been carriedout before with several types of lipids using enzy-matic catalysis (Adlercreutz, 1994). The most wellknown example is the enzymatic production of cocoa butter substitutes from cheaper raw materi-als (Macrae, 1985). Similar reactions have beencarried out with glycerophospholipids. Lipaseshave been used to exchange the fatty acid in the sn -1 position of phosphatidylcholine and otherglycerophospholipids (Svensson et al., 1992). Theselectivity for the  sn -1 position is achieved if lipases specific for the 1- and 3-positions intriglycerides are used. The most convenient typeof conversion is an acidolysis reaction between thelipid and the fatty acid to be incorporated. Inorder to exchange the fatty acid in the  sn -2 posi-tion of phospholipids, phospholipase A 2  has beenused in hydrolysis and esterification reactions (Eg-ger et al., 1997). Rhizopus arrhizus  lipase has been used for hy-drolysis of the acyl-residue in the  sn -1 position of DGDG and monogalactosyldiacylglycerol(MGDG) (Murakami et al., 1994). The  sn -1 re-gioselective acylation of monogalactosylglycerolhas been achieved with  Achromobacter  sp. lipase.Further acylation of the primary hydroxyl groupon the galactose moiety in monogalactosyl-1-acyl-glycerol was achieved using  Mucor ja  anicus  li-pase (Morimoto et al., 1995). Enzyme catalysedfatty acid exchange has to our knowledge notpreviously been described for galactolipids. Theaim of this work was to evaluate if lipases can beused to exchange the fatty acid in the  sn -1-posi-tion of DGDG. 2. Materials and methods 2  . 1 .  Chemicals Acetone, toluene, methanol, hexane, ethyl ac-etate, chloroform, acetic acid were of p.a gradeand purchased from Merck (Darmstadt, Ger-many). Heptadecanoic acid (99% purity) was ob-tained from Sigma Chemical Co (St. Louis, USA).Polypropylene powder EP 100 (200–400   m), wasfrom Akzo (Obernburg, Germany). Methyl ei-cosanoate was purchased from Larodan FineChemicals (Malmo¨, Sweden).TLC-plates (kieselgel60, 0.2 mm) and silica gel (kieselgel 60, 230–400mesh) were purchased from Merck (Darmstadt,Germany). Other chemicals were of analyticalgrade.Lipase from  R .  arrhizus  (lipase 80000) was agift from Gist -Brocades S.A (Delft, The Nether-lands). Immobilised lipase from  Rhizomucormiehei   (Lipozyme IM 20) and from  Candidaantarctica  (Novozyme SP 435) were from NovoNordisk A / S (Bagsvaerd, Denmark).Lipases from  Geotrichum candidum  (0.4 U / mg)and  Penicillium camembertii   (0.3 U / mg) camefrom Amano Pharmaceutical Co. (Nagoya,Japan).  Alcaligenes  sp. lipase (1.0 U / mg) was fromBiocatalysts Ltd (UK).The activity for lipases from  G  .  candidum ,  P . camembertii   and  Alcaligenes  sp was determined bypH-stat titration according to (Bosley and Clay-ton, 1994), with the exceptions that olive oil wasused instead of tributyrin, the pH was maintainedat 8 and the temperature was 25°C. One unit wasdefined as 1   mol fatty acid released per minutefrom olive oil. 2  . 2  .  Purification of digalactosyldiacylglycerol  (  DGDG   )  Commercial wheat flour (1 kg) was extractedtwice for 20 min with methanol. In the first ex-traction 1.5 l methanol was used and the secondtime 1 l was used. The mixture was filtered andthe methanol was removed using a rotary evapo-rator. The lipid mixture was dissolved in 1l of hexane. Remaining wheat flour and solids werefiltered off and the hexane evaporated off. Intotal, 7 g of extracted lipids was obtained from 1kg of wheat flour.To isolate DGDG, solid phase extraction wasapplied which has been used successfully by oth-ers (Prieto et al., 1993; Bergqvist and Herslo¨f,1995). The lipid mixture (2.5 g) was dissolved in  M  .  Persson et al  .  /   Chemistry and Physics of Lipids  104 (2000) 13–21  15 n-hexane / 2-propanol / 1-butanol / water (60 / 30 / 7 / 3)to a concentration of 250 mg / ml. Silica (30 g ) waspacked in a glass column and the column waspreconditioned with 200 ml n-hexane / 2-propanol / 1-butanol / water (60 / 30 / 7 / 3). 10 ml lipid samplewas applied. The following solvents were flushedthrough the column: hexane (200 ml) to removecarotenoids and neutral lipids, ethyl acetate (300ml) to remove remaining carotenoids, toluene / ace-tone 50 / 50 (200 ml) to remove monogalactosyldia-cylglycerol (MGDG) and finally acetone (2.5 l).After elution with 300 ml acetone pure DGDGfractions (97%) were collected. The purity of DGDG was checked with HPLC by Scotia LipidTeknik AB Stockholm Sweden. Pure DGDG (360mg) was obtained in the solid phase extractionfrom 2.5 g of lipid extract. 2  . 3  .  Preparation of immobilised enzyme byadsorption Lipases from  R .  arrhizus ,  G  .  candidum ,  Alcali  -  genes .sp and  P .  camembertii   were immobilised byadsorption onto a hydrophobic support.Solid support, polypropylene EP-100 (1 g) waswetted with 3 ml ethanol. Lipase powder (1 g) wasdissolved in 20 ml sodium phosphate buffer (20mM pH 6.0). The enzyme solution was mixedwith 1 g polypropylene support and incubated at25°C overnight. The preparation was filtered,washed with water and finally phosphate buffer(200 mM pH 7.0) was added and the preparationwas dried overnight under reduced pressure. 2  . 4  .  Preequilibration of water acti   ity Enzyme preparations and the substrate solu-tions were equilibrated over saturated salt solu-tions at 25°C to obtain a defined initial wateractivity. The salts used were LiBr (water activity( a w ) = 0.06) LiCl, ( a w = 0.11), MgCl 2  ( a w = 0.33)and Mg(NO 3 ) 2  ( a w = 0.54). Equilibration was per-formed for at least 16 h. 2  . 5  .  Enzyme reactions The reactions were performed in 4-ml contain-ers with teflon -lined septa. The reactions werestarted by mixing enzyme preparation (25 mg)and 2 ml of a solution of DGDG (10 mM) andheptadecanoic acid (400 mM unless otherwisestated) in toluene. The reactions were performedat 25°C on a reciprocal shaker, 175 rpm. 2  . 6  .  Thin - layer chromatography Samples, 50   l were withdrawn from the reac-tion vessel and applied as bands on thin-layerchromatography (TLC) plates. Digalactosyl-acyl-glycerol (DGMG), digalactosyldiacylglycerol(DGDG), acyl - digalactosyl - acylglycerol (acyl-DGMG) acyl - digalactosyldiacylglycerol (acyl-DGDG) and the fatty acid were separated withthe following solvent system: CHCl 3 / MeOH / H 2 O / HOAc (85:15:10:3.5). The plates were sprayedwith 0.1% 2,7-dichlorofluorescein in ethanol andthe bands were detected with UV light. The spotswere scraped off and the scrapings stored at − 20°C until fatty acid analysis. 2  . 7  .  Fatty acid analysis and quantification of DGMG  ,  DGDG  ,  acyl  - DGMG and acyl  - DGDG  Methyl esters were formed by adding 2 mlsodium-methoxide (3%) in methanol to the scrap-ings from the TLC-plates, 10   l internal standard(24.95 mM methyl eicosanoate) was added. Themixture was vortexed (3 × 10 s) and incubated for10 min in a water bath at 50°C. Hexane (600   l)was added and the tubes were vortexed (3 × 10 s).After that saturated NaCl solution (4 ml) wasadded and the tubes vortexed (3 × 10 s). Thetubes were centrifuged and samples were with-drawn from the upper layer for gas chromatogra-phy (GC) analysis.The amount of the different galactolipid specieswere calculated from the amount of fatty acidmethyl ester determined by GC. 2  . 8  .  GC analysis of fatty acid methyl esters Fatty acid methyl esters were analysed on aShimadzu gas chromatograph (GC 14-A) with aSP-2380 column (30 m, 0.32 id, 0.20   m filmthickness, Supelco, Bellefonte, PA). The columntemperature was raised from 150 to 190°C in 8  M  .  Persson et al  .  /   Chemistry and Physics of Lipids  104 (2000) 13–21 16 min and then held for 2 min at 190°C. Thetemperature of the injector was 220°C and thedetector 250°C. Fatty acid methyl esters weredetected using a flame ionisation detector. Re-sponse factors for the different methyl esterswere determined. 2  . 9  .  Purification and identification of acyl  - DGDG  Acyl-DGDG was purified by preparativeTLC. In this case the lipids were detected byI 2 -vapour. The lipid was then extracted with amixture of CHCl 3 / MeOH / H 2 O (2:2:1).The lipidwas dissolved in CDCl 3 / CD 3 OD and analysedwith one and two dimensional  1 H-NMR. Thespectrum were obtained with a Arx-500 (500MHz) spectrometer and the different carbohy-drate ring protons were assigned from a COSY-GRAD-SE spectrum. 3. Results and discussion 3  . 1 .  Screening with different lipases All six lipases (see Section 2) were investi-gated under identical conditions for their abilityto change the fatty acid in the  sn -1 position of DGDG with heptadecanoic acid. Lipases from P .  camembertii  ,  G  .  candidum  and  Alcaligenes  spdid not catalyse the acidolysis reaction underthe conditions used (Table 1). This could be ex-plained by the low loading (U / g support) of these enzymes on the support material. With  R . miehei   and  R .  arrhizus  lipase the reactions per-formed in similar ways. The two enzymescatalysed the acidolysis reaction but also es-terified the primary hydroxyl group of the sugarmoiety producing acyl-DGDG (Scheme 1).  C  . antarctica  lipase also catalysed the acidolysis butthe TLC analysis showed bands with higher  R f  values than acyl-DGDG. This indicates that theenzyme also esterified secondary hydroxylgroups of the sugar moiety to produce te-traesters and higher esters. It has been reported(Cao et al., 1998) that lipase B from  C  .  antarc - tica  is selective for the primary hydroxyl groupin the acylation of   D ( + )-glucose but is nonse-lective in the acylation of   D ( + )-galactose. . Rarrhizus  lipase seemed to be the most promisingenzyme with a higher acidolysis rate comparedto lipases from  P .  camembertii  ,  G  .  candidum ,  Al  - caligenes  sp. and  R .  miehei  . The  Rhizopus  en-zyme did not form any tetraesters as  C  . antarctica  lipase did and was therefore used inall the subsequent experiments. 3  . 2  .  Identification of acyl  - DGDG  Acyl-DGDG was purified by preparative TLCfrom the reaction mixture and analysed by  1 H-NMR. The spectrum was compared with the re-ported spectrum for DGDG (Hauksson et al.,1995).The ratio between the methyl triplet at 0.90ppm and the anomeric doublet at H-1   was 9which indicates that the lipid contains three acylchains. The signal for the two protons at theH-6   position were located at 4.2 ppm in acyl-DGDG compared to 3.6 in DGDG indicatingthat the primary hydroxyl group at the terminalgalactose unit had been acylated. Otherwise thespectrum agreed with the spectrum reported forDGDG (Hauksson et al., 1995). Table 1Screening of lipases in their ability to incorporate heptade-canoic acid into DGDG a Interesterification rate (  mol fatty acid / hSource of li-pase g enzyme preparation)3.0 Rhizopusarrhizus 0.4 Candidaantarctica 0.2 Rhizomucormiehei  0 Penicilliumcamembertii  0 Geotrichumcandidum 0 Alcaligenes  sp. a Reaction conditions: 10 mM DGDG, 100 mM heptade-canoic acid and 25 mg enzyme preparation in toluene at wateractivity 0.11.  M  .  Persson et al  .  /   Chemistry and Physics of Lipids  104 (2000) 13–21  17Scheme 1. Reaction scheme for the acidolysis of DGDG catalysed by lipases from  R .  miehei   or  R .  arrhizus . X = digalactosyl, R1 andR2  = acyl residues naturally occurring in DGDG, R = heptadecanoyl residue.
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