Ethyl esterification of docosahexaenoic acid in an organic solvent-free system with immobilized Candida antarctica lipase

JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 92, No. 1, 19-23. 2001

Ethyl Esterification of Docosahexaenoic Acid in an Organic Solvent-Free System with Immobilized

Candida antarctica Lipase YUJI SHIMADA,]* YOM1 WATANABE,’ AISIO SUGIHARA,’ TAKASHI BABA,2 TOMOAIU OOGURI,Z

SHIGERU MORIYAMA; TADAMASA TERAI,3 AND YOSHIO TOMINAGA’

Osaka Municipal Technical Research Institute, l-6-50 Joto-ku, Morinomiya, Osaka 536-8553,’ Central Research Institute, Maruha Corporation, 16-2 Wadai, Tsukuba, Ibaraki 300-4295,2 and Department of Applied Chemistry,

Osaka Institute of Technology, 5-16-I Omiya, Asahi-ku, Osaka 535-8585,’ Japan

Received 29 January 200UAccepted 3 April 2001

Ethyl docosahexaenoate (EtDHA) is regarded as a potentially useful pharmaceutical substance on account of its beneficial physiological activities. We attempted the ethyl esterification of docosahexaenoic acid (DHA) in an organic solvent-free system using Cizndidu antarctica lipase, which acts strongly on DHA and ethanol. Esterification of 88% was attained by shaking a mixture of DHAlethanol (l:l, mol/mol) and 2 wt% immobilized C. antarctica lipase at 30°C for 24 h. How- ever, even in the presence of an excess amount of ethanol, the extent of esterification could not be raised above 90%. To attain a higher level of esterification, a two-step reaction was found to be effective. The first step was performed in a mixture of DHA/ethanol (l:l, moYmol), and the reaction mixture was then dehydrated. In the second step, the resulting mixture was shaken at 30°C for 24 h with 5 molar equivalents of ethanol against the remaining DHA using 2 wt% immobilized lipase. By means of this two-step procedure, 96% esterification was attained. Repetition of the first and second reactions showed that the immobilized lipase was reusable for at least 50 cycles. In addition, DHA remaining in the second-step reaction mixture was removed by a conventional alkali refining process, giving purified EtDHA with a high yield.

[Key words: ethyl esterification, docosahexaenoic acid, Candida antarctica, lipase, immobilized enzyme]

Docosahexaenoic acid (22:6n-3; DHA) plays a role in the prevention of a number of human diseases, including car- diovascular disease (l-3),‘inflammation (4), and cancer (5, 6). DHA has also been reported to have important functions in the brain (7) and retina (8), and to accelerate the growth of preterm infants (9, 10). For these reasons, tuna oil, which contains DHA, has been used as a health food and as an ingredient of baby milk (11). In addition, the beneficial physiological activities of DHA have led to increasing at- tention being given to its medical applications.

We previously reported that the purity of DHA was raised to 90% by selective esterification of fatty acids originating from tuna oil with Rhizopus delemar lipase, which acted on DHA very weakly (12, 13). However, the form desired for medicinal purposes is the ethyl ester. DHA is chemically esterified with ethanol using an acid catalyst, but the process causes its isomerization. Lipases efficiently catalyze esterification as well as hydrolysis at ambient temperature under a nitrogen stream, and the mild conditions will not denature DHA. In addition, free fatty acids remaining in the reaction mixture can be removed by conventional alkali refining if the content is low. However, alkali refining in the

* Corresponding author. e-mail: [email protected] phone: +81-(0)6-6963-8073 fax: +81-(0)6-6963-8079

presence of larger amounts of free fatty acids emulsifies the mixture, and good recovery of the ethyl ester cannot then be expected. In general, alkali refining is adopted industrially, when the free fatty acid content is ~5%. We therefore attempted to achieve >95% ethyl esterification of DHA in an enzyme reaction system without an organic solvent, the inclusion of which would entail a risk of explosion.

Fatty acids can be esterified with ethanol using immobilized Rhizomucor miehei lipase, but the reaction requires an organic solvent (14, 15). In addition, although the esterification reached 99% when the water generated in ester synthesis was removed using a reflux trap packed with molecular sieves, DHA was not efficiently esterified in this reaction system (14, 15). We previously found that Candida antarctica lipase recognized polyunsaturated fatty acids and short-chain alcohols well as substrates (16, 17). Immobi- lized C. antarctica lipase was therefore used for ethyl esterification of DHA, but the water generated prevented the attainment of >90% esterification. In the present study, we show that a two-step reaction is effective in achieving of >95% esterification.

MATERIALS AND METHODS

Materials DHA (purity, 91.8%) was prepared as previously described (13) by Maruha Corp. (Tokyo). The preparation con-

19

20 SHIMADA ET AL. J. BIOSCI. BIOENG.,

tained 1.9% n-6 docosapentaenoic acid, 0.7% n-3 docosapentaenoic acid, 2.1% eicosapentaenoic acid, 1.9% arachidonic acid, and 0.8% oleic acid. Immobilized Cundidu antarctica lipase (Novo- zym 435) was obtained from Novozymes (Bagsvaerd, Denmark). The ester synthesis activity of the preparation was 10,300 PLU (propyl laurate m-&)/g. Ethanol (purity, 99.5%) was used as a substrate for ethyl esterification of DHA without dehydration. All other chemicals were of reagent grade.

Reaction Ethyl esterification of DHA was carried out at 30°C in a 20-ml screw-capped vessel sealed with nitrogen gas, with shaking at 130 oscillations/min. The first-step reaction was performed for 24 h in a vessel containing 10 g DHA/ethanol (1: 1, mol/mol) and 200 mg immobilized C. antarctica lipase. The reaction mixture was dehydrated for 45 min at 70°C and 5 mm Hg under a nitrogen stream. The second-step reaction was carried out for 24 h in an other vessel containing 10 g of the following reaction mixture: the dehydrated mixture from the first-step reaction, 5 molar equivalents of ethanol against the unesteritied DHA, and 200 mg immobilized lipase. The first- and second-step reactions were repeated by transferring the Iipase into a fresh substrate mixture every 24 h. The extent of esterification was calculated based on the acid values of the reaction mixture before and after incubation, which were determined by titrating with KOH solution.

0 1 2 3 4 5 6 7

Enzyme amount (%)

FIG 1. Effect of amount of immobilized C. antarctica lipase on ethyl esterification of DHA. The reaction was performed at 30°C in a mixture of 10 g DHA/ethanol (1 :l, mol/mol) and different amounts of lipase. Sym- bols: open circle, extent of DHA esteritication after 1 h; closed circle, after 7 h; open square, after 24 h.

The reaction for measuring the ester synthesis activity of C. antarctica lipase was performed according to the product sheet obtained from the supplier. A screw-capped vessel (20 ml) containing 7.7 g lauric acid, 2.3 g n-propanol, and 40 mg immobilized lipase was shaken at 60°C for 20 min. The ester formation was calculated based on the acid values of the reaction mixture measured before and after incubation. One PLU was defined as the amount of lipase preparation that synthesized 1 pmol propyl laurate per min. This reaction system was also employed in the control exper- iment to investigate the thermostability of the lipase.

9 60

5 60 ‘a 3 5 40

Y 20

0

0 1 2 4 7 10

Initial water content (%)

Analysis Free fatty acids were ethylated in ethanol dehydrated with molecular sieves using gaseous HCl as a catalyst. Fatty acid ethyl esters were analyzed on a DB-23 capillary column (0.25 mmx30 m; J&W Scientific, Folsom, CA, USA) connected to a Hewlett-Packard 5890 gas chromatograph (Avondale, PA, USA) as described previously (18). The water content in the oil layer was determined by Karl Fisher titration (moisture meter CA-07; Mitsubishi Chemical Corp., Tokyo). The weight contents of free fatty acids and ethyl fatty acids were analyzed by a TLC/FID analyzer (Iatroscan MK-5; Iatron Laboratories Inc., Tokyo) after development with a mixture of benzene/chloroform/acetic acid (50:20:0.7, by volume).

FIG 2. Effect of water content on ethyl esterification of DHA with immobilized C. antarctica lipase. A 10-g mixture consisting of DHA/ethanol (1: 1, mol/mol) and different amounts of water was shaken at 3O’C with 200 mg immobilized lipase. Symbols: open box, extent of DHA esterification after 1 h; hatched box, after 7 h; closed box, after 24 h.

tion was carried out with 2 wt% immobilized lipase, the esterification extent would reach nearly 90% after 24 h even when the activity was decreased to half by the repeated use. We thus set the lipase amount at 2 wt% of the substrate mixture.

RESULTS

First-step ethyl esterification of DHA We selected immobilized C. antarctica lipase as a catalyst for the ethyl esterification of DHA because this lipase was found to act strongly on DHA and short-chain alcohols (16, 17). The substrate DHA contained approximately 8% of other polyunsaturated fatty acids. However, since DHA was esterified with ethanol as strongly as the other fatty acids, the extent of DHA ethyl esterification was expressed as the overall extent of esterification.

Efict of water content A mixture of DHNethanol (1: 1, mol/mol) and various amounts of water was shaken at 30°C with 2 wt% immobilized C. antarctica lipase (Fig. 2). The reaction without water addition reached 49.7%, 83.2%, and 88.4% esterification after 1, 7, and 24 h, respectively. The extent of esterification after 24 h decreased only a little with increasing water content. If DHA is esterified completely, the amount of water generated will reach 4.8%. The result therefore showed that generated water hardly interfered in the reaction in which about 85% esterification was achieved.

Effect of lipase amount A mixture of DHA/ethanol (1: 1, mol/mol) was shaken at 30°C with different amounts of immobilized C. antarctica lipase. Figure 1 shows the extents of DHA esterification after 1, 7, and 24 h. The reaction velocity depended on the amount of lipase; 87-89% esterification was obtained after 1,7, and 24 h with 7,4, and 1 wt% immobilized lipase, respectively. If the esterification reac-

Effect of ethanol content DHA was esteritied at 30°C with 1 to 10 molar equivalents of ethanol using 2 wt% immobilized C. antarctica lipase (Fig. 3). When DHA was esterified with equimolar ethanol, the extents of esterification after 1 and 24 h were 50.3% and 87.9%, respectively. Increasing the amount of ethanol decreased the initial velocity, showing that ethanol inhibited esterification. Though an excess amount of ethanol slightly shifted the equilibrium to esterification, the extent of esterification after 24 h was 89.7% in the presence of 10 molar equivalents of ethanol.

VOL. 92,200l ETHYL ESTERIFICATION OF DHA 21

100

60 g

5 60 ‘z=

0 E 4o 9

20

0 1 2 3 5 10

EthanollDHA (mollmol)

FIG 3. Effect of ethanol content on esterilication of DHA with immobilized C. antarctica lipase. A 1 O-g mixture consisting of DHA and 1 to 10 molar equivalents of ethanol was shaken at 30°C with 200 mg immobilized lipase. Symbols: open box, extent of DHA esterilication after 1 h; hatched box, after 7 h; closed box, after 24 h.

Effect of temperature A mixture of DHA/ethanol (1: 1, mol/mol) was shaken with 2 wt% immobilized C. antarctica lipase at temperatures in the range from 30°C to 50°C. The esterification velocity depended on the temperature; the extents of esteritication after 1 h at 30°C 40°C and 50°C were 50.6%, 58.9%, and 64.2%, respectively. This result showed that the optimum temperature was ~50°C.

Repeated use of immobilized lipase may decrease the activity resulting from the effects of the amount of ethanol and the temperature and/or by release of enzyme from the support. In order to investigate the stability of immobilized lipase, the ethyl esterification of DHA as described above was repeated over 10 cycles by transferring the lipase into a fresh substrate mixture every 24 h. In addition, the reaction for measuring the esterification activity was repeated as a control without ethanol: a mixture of lauric acid/n-propanol (1: 1, mol/mol) and 40 mg immobilized lipase was shaken at 40°C and 50°C (the control reaction was not performed at 30°C because the substrate mixture did not melt at this temperature). Figure 4 shows the extent of esterification after 1 h in each cycle of the reaction relative to that in the first cycle. The activity did not decrease with repeated esterification of lauric acid at 40°C and 50°C. However, the activities of DHA ethyl esterification at 30°C 40°C and 50°C decreased to 8 l%, 73%, and 30% of those in the first cycle, respectively. These findings showed that the main factor in the inactivation of C. antarctica lipase was the presence of ethanol, and that the inactivation was accelerated at higher temperatures. Hence, the most appropriate temperature for DHA ethyl esterification was taken to be 30°C.

On the basis of the results obtained in the above experi- ments, the first-step reaction was carried out at 30°C for 24 h in a mixture of DHA/ethanol (1: 1, mol/mol) using 2 wt% immobilized C. antarctica lipase.

Second-step ethyl esterification of DHA The esterification of DHA reached 88% in the single reaction, but our target value was >95%. Hence, the reaction mixture obtained by the first-step reaction (EtDHA:DHA=87.7:12.3, by mol) was used as a substrate, and further esterification was carried out. First, the water layer was removed after letting the reaction mixture stand overnight. The resulting oil layer (water content, 3480 ppm) was allowed to react at

20 ’ 0 2 4 6 6 10

Cycle number

FIG 4. Stability of immobilized C. anrurcticu lipase in esterification reactions of DHA with ethanol and of lauric acid with n-propanol. Ethyl esteritication of DHA was carried out in a 1 O-g mixture of DHA/ethanol ( 1: 1, mol/mol) and 200 mg immobilized lipase at 30°C (closed circle), 40°C (closed square), and 50°C (closed triangle). Laurie acid was esterified with n-propanol in a 10-g mixture of lauric acid/n-propanol (1: 1, mol/mol) and 40 mg immobilized lipase at 40°C (open square) and 50°C (open triangle). The two reactions were repeated by transferring the lipase into a fresh substrate mixture every 24 h. The extent of esteritication after 1 h in each reaction is plotted relative to that of the first cycle reaction. The extents of DHA esteritication after 1 h at 3O”C, 4O”C, and 50°C were 49.5%, 59.3%, and 64.7%, respectively. The extents of lauric acid esterification after 1 h at 40°C and 50°C were 39.5% and 49.8%, respectively.

70 70

60 60

8 50 50

.E 40 40

E 30 30 iii $ 20 20

10 10

0 0 1 2 3 5 7 1 2 3 5 7

Ethanol/Free DHA (d/mol) Ethanol/Free DH4 (mol/~l)

FIG 5. Effect of ethanol content on the second-step esteriflcation of DHA with immobilized C. anfurctica lipase. The reaction mixture obtained by the first-step esterillcation was used as a substrate; the EtDHA content was 89.7 mol%. A 10-g mixture consisting of EtDHA/DHA and different amounts of ethanol was shaken at 30°C with 2 wt% immobilized lipase. The ethanol content is expressed as the molar equivalent against free DHA remaining in the first-step reaction mixture. (A) Substrate, oil layer after allowing the first-step reaction mixture to stand overnight (water content, 3480 ppm). (B) Substrate, dehydrated oil layer from the first-step reaction mixture (water content, 150 ppm). Symbols: open box, extent of DHA esteritication after 1 h; hatched box, after 7 h; closed box, after 24 h.

30°C with 1 to 7 molar equivalents of ethanol against the remaining DHA using 2 wt% immobilized lipase (Fig. 5A). The esterification of the remaining DHA after 1 h showed the maximum value, 23.8%, at 3 molar equivalents of ethanol. The esterification after 24 h depended on the ethanol amount, and was 44.6% at 5 molar equivalents of ethanol (total esterification, 93.2%). When esterification reaches 90%, the generated water content is 4.3%. DHA in the oil layer from the first-step reaction was esteritied with 5 molar equivalents of ethanol in the presence of 4.5% water using 2 wt% immobilized lipase. Only 18% of the remaining DHA was esterified after 24 h, indicating that an excess amount of water significantly suppressed the second-step esteritication.

22 SHIMADA ET AL. J. BIOSCI. BIOENG.,

To investigate the effect of the initial water content in the second-step reaction, the water in the oil layer obtained by the first-step esterification was removed by dehydration for 45 min at 70°C and 5 mm Hg. The water content in the resulting oil layer was reduced to 150 ppm. The dehydrated oil layer was allowed to react at 30°C with various amounts of ethanol using 2 wt% immobilized lipase (Fig. 5B). The esterification of the remaining DHA after 1 h showed the maximum value at 2 molar equivalents of ethanol, while the extent of esterification after 24 h increased with increasing ethanol amount and reached a constant value, 66.9%, at 5 molar equivalents of ethanol (total esterification, 95.9%). These findings showed that dehydration of the first-step reaction mixture was effective in increasing the extent of esteritication at the steady state.

On the basis of the above results, the substrate for the second-step esterification was prepared by adding 5 molar equivalents of ethanol against the remaining DHA into the dehydrated oil layer of the first-step reaction mixture. The reaction was performed at 30°C using 2 wt% immobilized C. antarctica lipase with shaking.

Repeated use of immobilized C. anlarctica lipase The first-step reaction was repeated by transferring immobilized lipase to a fresh substrate mixture every 24 h. Figure 6 shows the extents of esterification after 1, 7, and 24 h in each cycle. The extent of esterification after 1 h fell to half of the initial extent after 30 cycles. However, because an excess amount of lipase preparation was used as a catalyst, the extent of esteritication after 24 h decreased only a little, from 88.5% to 85.8%, over 50 cycles.

The first-step reaction mixture was collected every 7-10 cycles and dehydrated. Five molar equivalents of ethanol was added to the dehydrated mixture, and the resulting substrates were employed for the second-step reaction, which was repeated every 24 h. Table 1 shows the extents of esterification after 24 h in the first- and second-step reactions and the total esterification after both steps. In each step, esterification decreased only a little with the number of cycles, and the total esterification of DHA by two-step reaction was maintained at 95% even after 50 cycles.

Alkali refining of reaction mixture EtDHA was purified by alkali refining using 200 g of the second-step reaction mixture as the starting material (Table 2). The reaction

0’ I I ’ ’ 0 10 20 30 40 50

Cycle number

FIG 6. Durability of immobilized C. antarctica lipase in the first-step ethyl esterification of DHA. A mixture of 10 g DHA/ethanol (1: 1, mol/mol) was shaken at 30°C with 2% immobilized lipase. The reaction was repeated by transferring the lipase into a fresh substrate mixture every 24 h. Symbols: open square, esteritication extent in each cycle of the reaction after 1 h; closed circle, after 7 h; open circle, after 24 h.

TABLE 1. Repeated two-step DHA ethyl esterification with immobilized C. antarctica lipase

Cycle number Esterification (%)

First-step Second-step Total

1 88.5 66.8 96.2 5 87.7 65.2 95.7

10 87.1 66.3 95.7 15 88.3 66.9 96.1 20 87.5 65.6 95.7 30 87.4 66.1 95.7 50 85.8 65.1 95.0

The first-step reaction was carried out at 30°C in a vessel containing a mixture of 10 g DHA/ethanol (1: 1, mol/mol) and 200 mg immobilized lipase. The second-step reaction was performed in other vessel containing 10 g of the dehydrated first-step reaction mixture with 5 molar equivalents of ethanol against unesterified DHA. The first- and second-step reactions were repeated by transferring the lipase into a fresh substrate mixture every 24 h.

TABLE 2. Alkali refining of reaction mixture obtained by two-step ethyl esterification

Step Weight

(!z)

Acid value

(mz KOWa)

Reaction mixture 200 5.8 Evaporation 182 6.8 Alkali treatment (1) 166 2.2 Alkali treatment (2)b 158 0.4 Washing. with water 153 0.3

Alkali refining of the reaction mixture obtained by two-step esteritication of DHA was carried out as described in the text.

a Amount of NaOH, 0.7 molar equivalent. b Amount of NaOH, 1.5 molar equivalents.

mixture consisted of 89.8% ethyl ester, 3.5% free fatty acids, 6.3% ethanol, and 0.4% water. After removing ethanol from the mixture by a rotary evaporator, 2.5 N NaOH was gradu- ally added with stirring to give 0.7 molar equivalent against the remaining free fatty acid, and the resulting precipitate was then removed by centrifugation (6000 x g, 10 min). Ali- quots of 2.5 N NaOH were further added to the supernatant to give 1.5 molar equivalents, and the precipitate was removed by centrifugation. Alkali contaminating the supema- tant was removed by washing with water three times. Alkali refining decreased the acid value to 0.3 mg KOWg, and the ethyl ester was recovered with a yield of 85% (free fatty acid content, 0.17%). Unesterified DHA in the mixture could thus be efficiently removed by alkali refining.

DISCUSSION

We were unable to achieve >95% ethyl esteritication of DHA by a single reaction with immobilized C. antarctica lipase because the generated water interfered with the reaction. In general, such interference can be eliminated by adding a large amount of ethanol. However, in this study the extent of esteritication could not be raised above 90% even in the presence of 10 molar equivalents of ethanol (ethanol content, 58.4%). In the industrial process, water generated in esterification is removed at reduced pressure and/or by blowing nitrogen gas into the reaction mixture. However, ethanol is also evaporated under these conditions. The prob- lem can be basically solved by refluxing the reaction mix-

VOL. 92,200l ETHYL ESTERIFICATION OF DHA 23

ture and returning the ethanol separated from the refluxing vapor. An alternative is to use a two-step reaction; the generated water (lower layer) is withdrawn after the first step of the reaction, and the dehydrated oil layer is then allowed to react again with ethanol as second the step. While the former process is effective for a chemical reaction in which the refluxing reaction mixture is at a high temperature, an enzyme reaction at around 30°C must be carried out under reduced pressure to reflux the reaction mixture. In contrast, this study has shown that two-step reaction can be performed in a conventional reactor, with >95% esterification of DHA. C. antarctica lipase also acts strongly on other polyunsaturated fatty acids, such as y-linolenic, arachidonic, and eicosapentaenoic acids. The two-step reaction with immobilized C. antarctica lipase may thus be effective for the industrial ethyl esterification of polyunsaturated fatty acids without the use of an organic solvent.

Inactivation of C. antarctica lipase by ethanol In the ethanolysis of tuna oil, immobilized C. unturcticu lipase was significantly inactivated with a more than 2/3 molar equivalent of ethanol against the total fatty acids in tri- acylglycerols, in which a part of ethanol was not dissolved (17), while equimolar ethanol (methanol) against triacyl- glycerols was completely soluble in the presence of 30% ethyl (methyl) esters (16, 17) or n-hexane (19) and scarcely inactivated the lipase. In the present study, a high concentration of ethanol dissolved in DHA or EtDHA/DHA slightly inactivated the lipase (Fig. 4), although the esterification velocity decreased considerably (Figs. 3 and 5), indicating that dissolved ethanol did not significantly in- activate C. antarctica lipase even at a high concentration.

Effect of water The extent of esterification after 24 h in the first-step reaction decreased with increasing water content, but the esteritication remained high (84%) even in the presence of 10% water (Fig. 2). A similar phenomenon was observed in the esterification of fatty acids with lauryl alcohol or sterols (13, 20). We have clarified that a high degree of esterification can be obtained because lipases used in esterification reactions recognize fatty acids, lauryl alcohol, and sterols as substrates, but not their esters. When the EtDHA synthesized in this study was shaken at 30°C for 24 h with 2 wt% immobilized C. antarctica lipase in the presence of 10% water, the hydrolysis was only 13.7%. It was therefore concluded that a high level of esterification could be obtained in the first step because the product, EtDHA, was hydrolyzed very weakly.

ACKNOWLEDGMENTS

We thank Dr. Toshihiro Nagao of Osaka Municipal Technical Re- search Institute and Dr. Kazuaki Maruyama of Maruha Corp. for useful discussions, and Mr. Yoshinori Hirota and Mr. Noboru Ohyagi of Osaka Institute of Technology for their technical support.

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Documents

Ethyl esterification of docosahexaenoic acid in an organic solvent-free system with immobilized Candida antarctica lipase