Biotechnology Letters 26: 1077–1080, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.

1077

Phospholipase-mediated preparation of 1-ricinoleoyl-2-acyl-snglycero-3-phosphocholine from soya and egg phosphatidylcholine T. Vijeeta, J.R.C. Reddy, B.V.S.K. Rao, M.S.L. Karuna & R.B.N. Prasad∗ Division of Lipid Science and Technology, Indian Institute of Chemical Technology, Hyderabad-500 007, India ∗ Author for correspondence (Fax: +91-40-27193370; E-mail: [email protected]) Received 20 January 2004; Revisions requested 4 February 2004/10 March 2004; Revisions received 5 March 2004/29 April 2004; Accepted 29 April 2004

Key words: egg lecithin, phosphatidylcholine, phospholipase A1 , ricinoleic acid, soya lecithin

Abstract 1-Ricinoleoyl-2-acyl-sn-glycero-3-phosphocholine was prepared by incorporating ricinoleic acid completely in the sn-1 position of egg and soya phosphatidylcholine (PC) using immobilized phospholipase A1 as the catalyst. The optimum reaction conditions for maximum incorporation of ricinoleic acid into PC through transesterification were 10% (w/w) immobilized enzyme (116 mg), a 1:5 mol ratio of PC (soya, 387 mg; egg, 384 mg) to methyl ricinoleate (780 mg) at 50 ◦ C for 24 h in hexane.

Introduction Fatty acids in seed triacylglycerols are also present in the phospholipids. However, in case of castor (Ricinus communis), mustard (Arabis glabra), watercress (Nasturtium officinale), coriander (Coriandrum sativum), bitter gourd (Momordica charantia) and snake gourd (Trichosanthes anguina) unusual fatty acids such as 12-hydroxy cis-9-octadecenoic acid (ricinoleic), cis13-docosenoic acid (erucic), cis-6-octadecenoic acid (petroselinic) and conjugated trienoic acid (punicic) which are absent or present in minor amounts in their respective phospholipids (Vijayalakshmi & Rao 1972, Cahoon & Ohlrogge 1994, Hornung et al. 2002). However, diricinoleoyl-sn-glycero-3-phosphocholine is an intermediate in the oil formation in developing seeds of castor bean and is a substrate for important regulatory enzymes such as specific endoplasmic acyl- and phospho-transferases and phospholipase A (Harwood 1996). 1-Ricinoleoyl-2-oleoyl-glycero-3phosphocholine is also involved in the synthesis of oleate-β-12-hydroxylase (Bafor et al. 1991), a key enzyme for ricinoleic acid synthesis in Ricinus communis. The presence of hydroxy fatty acids in the phospholipids imparts hydrophilic properties and improves moisture retention of lecithin with increased

water dispersibility. Hydroxylated phospholipids are useful in baking applications where it can improve the dispersion of fats and retard staling (Schmidt & Orthoefer 1985). The existing chemical methods for the synthesis of ricinoleic acid containing phosphatidylcholine (PC) are complicated and involve many steps (Borsotti et al. 2001). Alternately, enzyme-mediated synthesis of phospholipids will have advantages because of mild reaction conditions and high stereochemical or positional specificities. Enzymatic methods to incorporate saturated and unsaturated fatty acids into PC using lipases and phospholipases have been reported (Haraldsson & Thorarensen 1999, Ghosh & Bhattacharyya 1997). However, there is no method for the incorporation of ricinoleic acid into PC. In the present investigation, we report the enzymatic incorporation of ricinoleic acid into egg and soya PC using phospholipase A1 (PLA1 ) catalyzed transesterification in hexane medium.

1078 (0.5 mmol, 387 mg) and methyl ricinoleate (2.5 mmol, 780 mg) were taken up in a 4 ml hexane screwcapped tube and immobilized PLA1 (10% w/w total substrates) was added. The contents were stirred at 50 ◦ C after tightly closing the tube. The reaction conditions were optimized by carrying out different sets of experiments by varying the concentration of methyl ricinoleate, dosage of enzyme and reaction time. Similar sets of experiments were carried out using egg PC (0.5 mmol, 384 mg). 1-Ricinoleoyl-2-acyl-sn-glycero3-phosphocholine (120 mg from soya PC and 118 mg from egg PC) and unreacted methyl ricinoleate were separated from the reaction product by silicic acid column chromatography using chloroform/methanol (80:20, v/v) and chloroform/methanol (55:45, v/v) as respective eluents. Fig. 1. Effect of methyl ricinoleate concentration on incorporation of ricinoleic acid into PC (0.5 mmol, soya, 387 mg; egg, 384 mg) in presence of immobilized PLA1 (10% w/w of total substrates, 116 mg), at 50 ◦ C for 24 h. Values are mean ± S.E. of three individual experiments.

Materials and methods Isolation of phosphatidylcholine from egg and soya lecithin Crude soya lecithin was procured from a local soybean oil refinery. Hen’s egg yolk was obtained locally. Crude soya lecithin (23 g) was dispersed in 100 ml acetone and slowly added to cold (10 ◦ C) acetone (1:5 w/v lecithin to acetone) while stirring. The contents were centrifuged at 5–10 ◦ C. The acetone insoluble material was taken up in ethanol (1:5, w/v) stirred magnetically for 1 h and centrifuged. The solvent was removed from the ethanol-soluble material to obtain a phospholipid mixture enriched with PC (5 g). Pure PC was isolated from this mixture by alumina column chromatography (Singleton et al. 1965). The yield of PC was 1.1 g. Similarly 2.59 g of pure PC was obtained from 70 g of hen’s egg yolk using this isolation procedure. Preparation of 1-ricinoleoyl-2-acyl-snglycero-3-phosphocholine Ten ml of Lecitase Novo solution (from geneticallymodified Aspergillus oryzae, gifted by M/s Novozymes South Asia Pvt. Ltd., Bangalore, India) with an activity of PLA1 of 4000 LENU ml−1 was added to Celite (5 g), stirred for 4 h and dried by lyophilization to obtain immobilized PLA1 . Soya PC

Enzymatic hydrolysis of 1-ricinoleoyl-2-acyl-snglycero-3-phosphocholine for the determination of positional distribution of fatty acids 1 - Ricinoleoyl- 2 - acyl- sn - glycero - 3-phosphocholine (100 mg) was dissolved in tert-butanol (0.5 ml), and hydrolyzed using Lecitase Novo solution (0.5 ml) in 1 ml water for 6 h. The hydrolyzed product was extracted with chloroform and dried over anhydrous Na2 SO4 . The free fatty acids and lyso-PC were separated by silicic acid column chromatography using chloroform and methanol as eluents respectively. The free fatty acids and lyso PC were converted to fatty acid methyl esters (Christie 1972). Fatty acid analysis The fatty acid methyl esters were analyzed by GC using cross-linked methyl siloxane column (HP-1, 30 m × 0.32 mm, id × 0.25 µm, film thickness). The oven was increased from 160 to 300 ◦ C at 8 ◦ C min−1 with a holding time of 5 min. The injector and detector were at 250 and 325 ◦ C, respectively. The fatty acid methyl esters prepared from modified PC with maximum incorporation of hydroxy fatty acids were silylated using bis (trimethylsilyl) trifluoroacetamide in pyridine and analysed using non-bonded cyanosilicone column (SP-2330, 30 m × 0.25 mm, id × 0.2 µm, film thickness). The oven was increased from 170 to 250 ◦ C at 5 ◦ C min−1 with a holding time of 5 min. The injector and detector were at 225 and 275 ◦ C, respectively.

1079 Table 1. Relative (% w/w) fatty acyl composition of egg and soya PC before and after incorporation of ricinoleic acid. Phospholipid

Fatty acid composition 16:0 16:1 18:0 18:1

18:2

18:3

20:4

18:1 OHa

Egg PC Modified PC

39 4

2 1

15 3

30 30

11 9

– –

3 3

– 50

Soya PC Modified PC

17 12

– –

2 2

21 19

56 14

4 3

– –

– 50

a Ricinoleic acid.

Fig. 2. Effect of enzyme dosage on incorporation of ricinoleic acid into PC at 50 ◦ C for 24 h using 1:5 molar ratio of PC (0.5 mmol, soya, 387 mg; egg, 384 mg) to methyl ricinoleate (2.5 mmol, 780 mg). Values are mean ± S.E. of three individual experiments.

Results and discussion

Fig. 3. Time course of the incorporation of ricinoleic acid into PC at 50 ◦ C using 1:5 mole ratio of PC (0.5 mmol, soya, 387 mg; egg, 384 mg) to methyl ricinoleate (2.5 mmol, 780 mg) in presence of immobilized PLA1 (10% w/w of total substrates, 116 mg). Values are mean ± S.E. of three individual experiments.

1- Ricinoleoyl- 2 - acyl- sn - glycero - 3 - phosphocholine was prepared by transesterification of egg and soya PC with methyl ricinoleate using sn-1 specific PLA1 immobilized on Celite. Highest conversions were obtained using a 1:5 molar ratio of PC to methyl ester (Figure 1) which yielded PC with 50% ricinoleic acid at 10% immobilized PLA1 (Figure 2). Using the above reaction conditions, maximum incorporation of ricinoleic acid was obtained after 24 h (Figure 3). Ricinoleic acid replaced mostly palmitic acid in egg PC, and linoleic acid in soya PC, since palmitic and linoleic acids are the respective major fatty acids in egg and soya PC (Table 1). To establish the ex-

clusive incorporation of ricinoleic acid in the sn-1 position, PC was hydrolyzed using Lecitase Novo solution which has PLA1 activity and the released fatty acid was found to be only ricinoleic acid. The fatty acid methyl esters of the sn-2 position of lyso PC did not contain ricinoleic acid. This clearly establishes that the sn-1 position of modified PC was completely replaced by ricinoleic acid. In conclusion, we report a simple enzymatic transesterification method for the preparation of 1ricinoleoyl-2-acyl-sn-glycero-3-phosphocholine using immobilized PLA1 .

1080 Acknowledgements This work (IICT Communication No. 031011) was supported by a financial grant from the Department of Biotechnology, Government of India, New Delhi (Grant No. BT/PR 2036/PID/23/62/2000 dated 20-32001). MSLK is grateful to the Council of Scientific and Industrial Research, Government of India for Senior Research Fellowship.

References Bafor M, Smith MA, Jonsson L, Stobart K, Stymne S (1991) Ricinoleic acid biosynthesis and triacylglycerol assembly in microsomal preparations from developing castor-bean (Ricinus communis) endosperm. Biochem. J. 280: 507–514. Borsotti G, Guglielmetti G, Spera S, Battistel E (2001) Synthesis of phosphatidylcholines containing ricinoleic acid. Tetrahedron 57: 10219–10227. Cahoon EB, Ohlrogge JB (1994) Metabolic evidence for the involvement of a -4-palmitoyl-acyl carrier protein desaturase in petroselinic acid synthesis in coriander endosperm and transgenic tobacco cells. Plant Physiol. 104: 827–837.

Christie WW (1972) The preparation of alkyl esters from fatty acid and lipids. In: Gunstone FD, ed. Topics in Lipid Chemistry, Vol. 3. London, UK: Paul Elek (Scientific Books) Ltd., Logos Press, pp. 171–198. Ghosh M, Bhattacharyya DK (1997) Soya lecithin-monoester interchange reaction by microbial lipase. J. Am. Oil Chem. Soc. 74: 761–763. Haraldsson GG, Thorarensen A (1999) Preparation of phospholipids highly enriched with n-3 polyunsaturated fatty acids by lipase. J. Am. Oil Chem. Soc. 76: 1143–1149. Harwood JL (1996) Recent advances in biosynthesis of plant fatty acids. Biochim. Biophys. Acta 1301: 7–56. Hornung E, Pernstich C, Feussner I (2002) Formation of conjugated 11 , 13 -double bonds by 12 -linoleic acid (1,4)-acyllipid-desaturase in pomegranate seeds. Eur. J. Biochem. 269: 4852–4859. Schmidt JC, Orthoefer (1985) Lecithin in baking applications. In: Szuhaj BF, List GR, eds. Lecithins. Champaign, IL: American Oil Chemists’ Society, pp. 203–211. Singleton WS, Gray MS, Brown ML, White JL (1965) Chromatographically homogeneous lecithin from egg phospholipids. J. Am. Oil Chem. Soc. 42: 53–56. Vijayalakshmi B, Rao SV (1972) Fatty acid composition of phospholipids in seed oils containing unusual acids. Chem. Phys. Lipids 9: 82–86.