enrichment of ethyl docosahexaenoate by selective alcoholysis with immobilized rhizopus delemar...

JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 84, No. 2, 138-143. 1997

Enrichment of Ethyl Docosahexaenoate by Selective Alcoholysis with Immobilized Rhizopus delemar Lipase

YUJI SHIMADA,‘* AK10 SUGIHARA,’ SHIZUKA YODON0,2 TOSHIHIRO NAGAO,’ KAZUAKI MARUYAMA,3 HIROFUMI NAKANO,’ SADAO KOMEMUSHI,2 AND

YOSHIO TOMINAGA’

Osaka Municipal Technical Research Institute, I-6-50 Morinomiya, Joto-ku, Osaka 536,’ Department of Agricultural Chemistry, School of Agriculture, Kinki University, Nakamachi, Nara 631,2 and Maruha Corp., 16-2

Wadai, Tsukuba, Ibaraki 30&42,3 Japan

Received 13 January 1997/Accepted 27 May 1997

We attempted to purify ethyl docosahexaenoate (E-DHA) by the alcoholysis of fatty acid ethyl esters with lipase. Fatty acid ethyl esters originating from tuna oil (E-DHA content, 23 mol%; E-tuna-231 were used as starting materials, and Rhizopus delemar lipase immobilized on a ceramic carrier was used as a catalyst which acted only very weakly on E-DHA. Because the immobilized lipase did not exhibit the alcoholysis activity, it was activated by shaking at 30°C for 24 h in the E-tuna-23/lauryl alcohol mixture to which water (2%) was added. The alcoholysis activity of the lipase increased markedly as a result of this pretreatment, but the hydrolysis activity also increased. The hydrolysis activity was completely repressed by repeating the reaction after trans- ferring the immobilized enzyme into a fresh E-tuna33/lauryl alcohol mixture without adding additional water. Several factors affecting the alcoholysis of E-tuna-23 were investigated to determine the optimum reaction conditions. When alcoholysis was conducted at 30°C with shaking in a reaction mixture containing E-tuna- 23/lauryl alcohol (1: 3, mol/mol) and the activated lipase (4% of the mixture volume), E-DI-IA was efficiently enriched in the ethyl ester fraction. By alcoholyzing E-tuna-23 with lauryl alcohol for 50 h under these reaction conditions, the E-DHA content was increased from 23 mol% to 52 mol% in a 90% yield. In addition, when the fatty acid ethyl esters, of which the E-DHA contents were 45 mol% and 60 mol%, were alcoholyzed for 50 h, the contents of E-DHA were increased to 72 mol% and 83 mol%, respectively. In these reactions, the recovery of E-DHA in the ethyl ester fraction was greater than 90%. We termed this new reaction system selective alcoholysis because advantage of the fatty acid specificity of the lipase was taken in this reaction system. To investigate the stability of the immobilized lipase, continual batch reactions were carried out by replacing the reaction mixture with a fresh E-tuna-23/lauryl alcohol mixture every 24 h. The decrease in the extent of alcoholysis was only 15% even after the 47th batch reaction.

[Key words: enrichment, ethyl docosahexaenoate, selective alcoholysis, Rhizopus delemar, lipase, immobi- kzed enzyme]

The n-3 series of fatty acids, in particular eicosapen- taenoic acid (EPA) and docosahexaenoic acid (DHA), exhibit various physiological functions on being incor- porated into phospholipids (1, 2). The ethyl ester of EPA (E-EPA) has been used in the treatment of arteriosclerosis obliterans (3). Oil containing DHA has been used as a food material, a component in infant formulas, and a health food (4), and the medical application of DHA has been receiving increasing attention. The method of purification of DHA by the formation of a complex with silver was recently reported (5), but this method has not been industrially accepted because of residual silver and high cost. Therefore, other suitable purification methods are desired.

Polyunsaturated fatty acids (PUFAs) are very unstable against heat and oxidation. Enzyme reactions for the processing of PUFA-containing oil have drawn attention, because they proceed efficiently at ambient temperature and pressure and under a nitrogen stream. Several lipases do not act to an appreciable degree on PUFAs, and PUFAs can be enriched by taking advantage of this property. For example, when tuna oil, borage oil and arachidonic acid-containing oil from Mortierella were hydrolyzed with Candida rugosa or Geotrichum candi-

* Corresponding author.

dum lipase, DHA, y-linolenic acid and arachidonic acid could be enriched in glycerides (termed selective hydrolysis) (6-11). Furthermore, when free fatty acids originating from tuna and borage oils were esterified with alcohol in a reaction mixture containing organic solvent using a lipase, DHA and y-linolenic acid could be enriched in the free fatty acid fraction, respectively (termed selective esterification) (12, 13).

Recently, we noted that a lipase esterified fatty acid with fatty alcohol but did not hydrolyze the ester generated, and thus discovered a new method of selective esterification which did not require any organic solvent. When lauryl alcohol was used as a substrate, Rhizopus delemar lipase catalyzed esterification efficiently and acted only very weakly on DHA. As a result, DHA could be enriched from 23 wt% to approximately 90 wt% by the selective esterification of fatty acids originating from tuna oil with lauryl alcohol (14). It is well known that lipases catalyze transesterification as well as hydrolysis and esterification. When fatty acid ethyl esters were alcoholyzed with lauryl alcohol using lipase, higher extent of the alcoholysis can be expected because the fatty acid lauryl esters generated are very poor substrates of the lipase. In addition, since Rhizopus lipase acts only very weakly on DHA, it is presumed that ethyl docosahexaenoate (E-DHA) can be enriched in the unreacted ethyl

138

VOL. 84, 1997 ENRICHMENT OF ETHYL DHA BY SELECTIVE ALCOHOLYSIS 139

ester fraction. In this paper, we describe a new method of enriching E-DHA by the selective alcoholysis, with lauryl alcohol, of fatty acid ethyl esters originating from tuna oil using immobilized Rhizopus delemar lipase as a catalyst.

MATERIALS AND METHODS

Fatty acid ethyl esters and alcohol Fatty acid ethyl esters, of which the E-DHA contents were 23 mol%, 45 mol%, and 60 mol%, were prepared from tuna oil using the oil processing line of Maruha Corp. (Tokyo), and were designated as E-tuna-23, E-tuna-45, and E- tuna-60, respectively. Lauryl alcohol was purchased from Wako Pure Chemical Ind. Ltd. (Osaka).

Preparation of immobilized lipase and alcoholysis R. delemar lipase (Ta-lipase, 120,000 U/g; Tanabe Seiyaku Co. Ltd., Osaka) was immobilized on a ceramic carrier, SM-10, a gift from NGK Insulators (Aichi), as described in our previous paper (15). After the ceramic carrier (50 g) was suspended in 200ml of 10% lipase solution, 400 ml of cold acetone (-80°C) was gradually added with stirring, and the precipitate was dried in vacua. Approxi- mately 90% of the lipase was immobilized on the carrier using this procedure, and the activity of the lipase was 34,000 u/g.

Unless otherwise specified, the activation of the immobilized lipase and the alcoholysis were conducted as follows. The immobilized enzyme (480 mg) was activated in 12 g of a fatty acid ethyl esters/lauryl alcohol mixture (1 : 3, mol/mol) containing 240 mg of water with shaking (140oscillations/min) at 30°C in a screw-capped vessel (20 ml) for 24 h. The activated enzyme was transferred into the same amount of a fresh fatty acid ethyl esters/ lauryl alcohol mixture (1 : 3, mol/mol) without adding additional water, and then the alcoholysis was conducted under the same conditions as those for the activation of the immobilized lipase.

Analysis Lipase activity was measured by titrating fatty acids from olive oil (Wako Pure Chemical) with 0.05 N KOH as described previously (16). The reaction was carried out at 30°C for 60min with stirring at 500 rpm. One unit (U) of lipase activity was defined as the amount of the enzyme that liberated 1 pmol of fatty acid/min.

Ethyl and lauryl esters of fatty acids were analyzed on a Hewlett-Packard 5890 Plus gas chromatograph (Avon- dale, PA, USA) connected to a DB-5 capillary column (0.25 mm x lOm, J&W Scientific, Folsom, CA, USA). The column temperature was raised from 150°C to 300°C at a rate of lO”C/min, and maintained for 10 min at 300°C. The temperatures of the injector and detector (FID, flame-ionization detector) were set at 250°C. The carrier gas was helium at a flow rate of 25 cm/s. Fatty acid ethyl esters separated on the DB-5 capillary column were identified by comparison with ethyl esters from tuna oil, whose fatty acid composition was determined by analysis with a DB-23 capillary column (0.25 mm x 30 m, J&W Scientific) according to the method outlined in our previous paper (9), used as standards. Fatty acid lauryl esters used as standards were synthesized by esterification of fatty acids from tuna oil with lauryl alcohol using Rhizopus lipase as described elsewhere (14), and purified by silica gel 60 (Merck, Darmstadt, Ger- many) column chromatography using a mixture of n- hexaneiethyl acetate (98 : 2, v/v) as an eluent. The fatty

acid composition (mol%) of the resulting lauryl esters was determined by analysis using the DB-23 capillary column after methylation of the esters in methanol using Na-methylate as a methylation reagent according to the method outlined in our previous paper (9). By comparison with the lauryl esters used as standards, fatty acid lauryl esters separated on the DB-5 column were identified. When a mixture of ethyl and lauryl esters of fatty acids (1 : 1, w/w) was analyzed using the DB-5 capillary column, the total peak areas of ethyl and lauryl esters were equal. Thus, the quantitative analysis was carried out based on the peak area of each fatty acid ester.

The extent of hydrolysis was measured from the acid value of the reaction mixture and the saponification value of the original ethyl esters. The extent of alcoholysis was calculated from the molar ratio of lauryl esters to ethyl and lauryl esters, which were analyzed by gas chromatography.

The water content in the reaction mixture was determined by Karl Fischer titration (MKC-210, Kyoto Elec- tric Kogyo Co. Ltd., Kyoto).

RESULTS

Activation of immobilized lipase When immobilized Rhizopus lipase was used without any pretreatment, it did not catalyze the alcoholysis of fatty acid ethyl esters with lauryl alcohol. Therefore, the lipase was incubated in a mixture of E-tuna-231lauryl alcohol (1 : 2, mol/mol) containing various amounts of water (Table 1). When the lipase was shaken in the mixture to which water (0.8 to 2.0%) was added, the alcoholysis activity effectively increased but the hydrolysis activity also increased. The immobilized lipase did not exhibit the activity in presence of 4.0% water because it aggregated and did not disperse well in the reaction mixture.

The reaction was continued by replacing the reaction mixture every 24 h with a fresh mixture of E-tuna- 23/lauryl alcohol (1 : 2, mol/mol) without adding additional water (Table 2). When the immobilized lipase was activated by shaking in the mixture containing 2% water, the hydrolysis was not observed after the fourth reaction. In addition, the lipase activated by pretreatment in a reaction mixture containing 4% water did not exhibit the alcoholysis activity prior to the third reaction. In the fourth reaction, however, the alcoholysis activity was fully expressed, because the enzyme was dispersed well in the reaction mixture. In addition, the hydrolysis activity was completely repressed in the fifth reaction. On the basis of these results, the immobilized lipase that had been used four times after the pretreat-

TABLE 1. Effect of water content in the reaction mixture on the activation of immobilized R. delemar lipase

Water content (%)

Alcoholysis (%)

Hydrolysis (%)

0 3.3 1.2 0.2 17.3 2.9 0.4 31.3 3.5 0.8 37.9 5.4 2.0 36.8 7.4 4.0 1.3 3.2

The reaction mixture containing 12 g of an E-tuna-23/lauryl alcohol mixture (1 : 2, mol/mol), 0.48 g of immobilized Rhizopus lipase and various amounts of water was shaken in a screw-capped vessel (20 ml) at 30°C for 24 h.

140 SHIMADA ET AL. J. FERMENT. BIOENG.,

TABLE 2. Reuse of activated immobilized lipase in the reaction mixture without adding additional water

Water content (%)

Reaction number Alcoholysis (%)

Hydrolysis (%)

2.0 First Second Third Fourth Fifth

36.8 1.4 47.7 3.0 49.8 0.4 49.5 ND 48.4 ND

4.0 First 1.3 3.2 Second 1.6 2.7 Third 4.4 5.1 Fourth 50.9 1.4 Fifth 49.6 ND

The first reaction (pretreatment) was carried out in the reaction mixture containing 2% and 4% of water, and subsequent reactions were carried out in the mixture without adding additional water.

ment in the mixture containing 2% water was chosen to determine the optimum reaction conditions for the alcoholysis of fatty acid ethyl esters with lauryl alcohol.

Several factors affecting alcoholysis Effect of amount of enzyme Alcoholysis was con-

ducted at 30°C for 24 h in 12 g of an E-tuna-23/lauryl alcohol mixture (1 : 2, mol/mol) using different amounts of the immobilized lipase. Figure 1 shows the extent of alcoholysis and the fatty acid contents in the ethyl ester fraction. The extent of alcoholysis was increased by increasing the amount of enzyme in the mixture, but a significant increase was not observed when the enzyme was present at a level greater than 4%. When the amount

0 2 4 6 8 10

f I I

0' I 1 1 I I (

0 2 4 6 8 10 Enzyme amount (%)

FIG. 1. Effect of the amount of immobilized R. delemar lipase on the extent of alcoholysis of fatty acid ethyl esters originating from tuna oil (E-tuna-23) with lauryl alcohol. (A) Extent of alcoholysis; (B) fatty acid contents in the ethyl ester fraction. The fatty acid contents were expressed relative to those in the original ethyl esters. Symbols: 0, ethyl pahnitate; 0, ethyl oleate; 0 , ethyl eicosapentaenoate; n , ethyl docosahexaenoate.

-20 30 40 50

I I

30 40

Temperature (“C)

FIG. 2. Effect of temperature on the alcoholysis of fatty acid ethyl esters originating from tuna oil (E-tuna-23) with lauryl alcohol using immobilized R. delemar lipase. (A) Extent of alcoholysis; (B) fatty acid contents in the ethyl ester fraction. The fatty acid contents were expressed relative to the original contents. Symbols: 0, ethyl palmitate; 0, ethyl oleate; 0 , ethyl eicosapentaenoate; n , ethyl docosahexaenoate.

of enzyme was less than 4%, the contents of ethyl palmitate (E-PA) and ethyl oleate (E-OA) decreased, and that of E-EPA increased. When the amount of enzyme exceeded 4%, the content of E-EPA decreased but those of E-PA and E-OA did not change. E-DHA was enriched in the ethyl ester fraction by increasing the amount of enzyme in the mixture, In addition, the amount of lauryl docosahexaenoate (L-DHA) was approximately 10 mol% of the initial E-DHA content, and this amount scarcely changed even when the extent of alcoholysis was increased as a result of using a large amount of the enzyme.

Eflect of temperature A mixture containing 12 g of E-tuna-23/lauryl alcohol (1 : 2, mol/mol) and 0.48 g of immobilized lipase (4% of the reaction mixture) was shaken for 24 h at a range of temperatures from 21°C to 50°C (Fig. 2). The extent of alcoholysis hardly changed at temperatures greater than 3O”C, although it was slightly dependent on temperature below 30°C. The content of E-DHA increased somewhat and that of E-EPA decreased with increasing temperature, but the contents of E-PA and E-OA were hardly affected by the reaction temperature.

Efect of amount of lauryl alcohol Alcoholysis was carried out at various ratios of lauryl alcohol to E-tuna- 23 (Fig. 3). The extent of alcoholysis depended on the amount of lauryl alcohol, and reached an almost constant value at a ratio of 2. The contents of E-DHA and E-EPA in the ethyl ester fraction increased, and those of E-PA and E-OA decreased with increasing the extent of alcoholysis. While the E-DHA content increased slightly


0 2 4 6 8 10

0

l

0 2 4 6 8 10 LautylalcohollEthylesters(mol/mol)

FIG. 3. Effect of lauryl alcohol content in the reaction mixture on the alcoholysis of fatty acid ethyl esters originating from tuna oil (E-tuna-23). (A) Extent of alcoholysis; (B) fatty acid contents in the ethyl ester fraction. The fatty acid contents were expressed relative to the original contents. Symbols: 0, ethyl palm&ate; 0, ethyl oleate; q , ethyl eicosapentaenoate; n , ethyl docosahexaenoate.

at the ratio of more than 2, a ratio of 3 was chosen from the view-point of industrial production.

Time course of alcoholysis On the basis of the above results, the alcoholysis was conducted at 30°C in a mixture containing 12g of E-tuna-23/lauryl alcohol (1 : 3, mol/mol) and 0.48 g of immobilized Rhizopus lipase. Figure 4 shows a typical time course of the reaction. The extent of alcoholysis increased rapidly until 10 h. The contents of E-PA and E-OA in the ethyl ester fraction decreased with increasing the extent of alcoholysis, and reached almost constant values after 25 h. The E-DHA content increased in the early stage of the reaction, and increased gradually even after 25 h. The E- EPA content increased until 20 h, and then decreased gradually. E-DHA was gradually alcoholyzed with lauryl alcohol, and 89mol% of the initial content was reco- vered in the ethyl ester fraction after 53 h.

Stability of immobilized lipase Alcoholysis of E- tuna-23 was continued by replacing the reaction mixture with a fresh ethyl fatty acids/lauryl alcohol mixture every 24 h as outlined in Materials and Methods (Fig. 5). In the first reaction, a mixture containing 2% water was used for the activation of the immobilized lipase, but subsequent reactions were carried out in a mixture without adding additional water. The extent of alcoholysis decreased linearly, and was 85% of the initial value in the 47th batch reaction. The content of E-DHA decreased in proportion to the extent of alcoholysis, but the E-EPA content hardly changed. This phenomenon agreed with the findings that the E-EPA content did not change between 20 h and 30 h and that the E-DHA content increased with the reaction time (Fig. 4B).

0-J-J 0 10 20 30 40 50 60

0 10 20 30 40 50 60

Reaction time (h)

FIG. 4. Time course of the alcoholysis of fatty acid ethyl esters originating from tuna oil (E-tuna-23) with lauryl alcohol. (A) Extent of alcoholysis; (B) fatty acid contents in the ethyl ester fraction. The fatty acid contents were expressed relative to the original contents. Symbols: 0, ethyl palmitate; 0, ethyl oleate; q , ethyl eicosapentaenoate; m, ethyl docosahexaenoate.

After the 47th batch reaction, the reaction time was extended to 48 h and the reaction was continued. As a result, the extent of alcoholysis returned to the original level (Fig. 5B). The 53rd reaction was conducted after adding water (0.8%) to the reaction mixture, and then the subsequent reactions were carried out for 24 h in the mixture without adding additional water. The extent of hydrolysis in the 53rd reaction was 5.2% and that in the subsequent reactions decreased gradually. However, the extent of alcoholysis did not recover. This result suggested that the decrease in the extent of alcoholysis could not be attributed to the release of bound water from the lipase molecule. Therefore, the decrease in the extent of alcoholysis was due to either the inactivation of the lipase or the release of the lipase from the ceramic support. In addition, because the extent of alcoholysis and the E-DHA content in the ethyl ester fraction decreased linearly but not exponentially, the decrease may be due to the release of enzyme from the support.

Effect of water content in the substrate mixture on alcoholysis Continuous alcoholysis batch reaction was carried out using a substrate mixture from which the water was not removed. Thus, the water content changed from 150 to 5OOppm, but the extent of hydrolysis associated with alcoholysis was less than 0.5%. Since a large amount of water (17,000 ppm) was contained in the mixture after shaking it with the equal amount of water, the effect of water content on the extent of alcoholysis was examined (Table 3). The original mixture of E-tuna-23/lauryl alcohol (1 : 3, mol/mol) contained 310 ppm of water, which was reduced to 87 ppm by the

142 SHIMADA ET AL.

80

Z A

$ 60 E 0 2 40 Q

20 0 10 20 30 40 50 60

2.5 8 E 1

g 5 2.0 E 8 n

8 1.5

9 .= m al a: 1.0

24h-+-48h I

0 10 20 30 40 50 60 Cycle number

FIG. 5. Stability of immobilized lipase in the alcoholysis of fatty acid ethyl esters originating from tuna oil (E-tuna-23) with lauryl alcohol. The reactions until 47 times were done for 24 h, and subsequent reactions were carried out for 48 h. (A) Extent of alcoholysis; (B) fatty acid contents in the ethyl ester fraction. The fatty acid contents were expressed relative to the original contents. Symbols:O, ethyl eicosapentaenoate; 0, ethyl docosahexaenoate.

addition of molecular sieves 3A. When this low-water content mixture was used as a substrate, the extents of alcoholysis and hydrolysis were the same as those obtained on using the original mixture. However, the extent of hydrolysis was increased by increasing the water content in the substrate, and reached 1.1 and 7.0% on using mixtures containing 1,200ppm and 17,OOOppm water as substrates, respectively. These results indicated that the water content in the mixture should be less than 1,000 ppm to suppress the hydrolysis associated with alcoholysis below 1%.

Alcoholysis of fatty acid ethyl esters with different docosahexaenoic acid contents When E-tuna-23, E- tuna-45, and E-tuna-60 were alcoholyzed with lauryl

J. FERMENT. BIOENG.,

TABLE 3. Effect of water content in substrate mixture on alcoholysis of E-tuna-23 with lauryl alcohol

Water content Alcoholysis (%)

Hydrolysis (ppm) (%)

B7a 52.8 0.3 310” 54.8 0.4 800 52.9 0.7

1200 54.7 1.1 2400 52.7 1.4 8600 53.3 4.3

17Oooc 52.3 7.0

Immobilized lipase, which was used four times in a mixture of E- tuna-23/lauryl alcohol (1 : 3, mol/mol) without adding additional water after the activation, was used as a catalyst. The reaction was carried out for 24 h as described in Materials and Methods.

B In a substrate mixture from which the water was removed using molecular sieves 3A.

b In the original substrate mixture. c In the substrate mixture saturated with water.

alcohol for 50 h, the E-DHA contents were enriched to 52 mol%, 72 mol%, and 83 mol%, respectively (Table 4). The recovery of E-DHA was greater than 90% in all cases. In addition, the E-EPA content in the ethyl ester fraction was decreased in the alcoholysis of E-tuna-60, although it was increase in that of E-tuna-23. This result suggested that E-EPA was alcoholyzed more efficiently by decreasing the contents of the preferred lipase substrates (E-PA, E-OA, etc.) in the reaction mixture.

DISCUSSION

We have described a novel method of concentrating E- DHA in the ethyl ester fraction by alcoholysis taking advantage of the fatty acid specificity of Rhizopus lipase. This method does not require any organic solvent and can be used to purify E-DHA continually without pre- cise control of the water content in the reaction mixture. In addition, when E-tuna-60 was used as a substrate, E-DHA was purified to 83 mol% in high yield (94%) (Table 3). Therefore we believe that this method is very effective for purification of E-DHA. Hydrolysis and esterification taking advantage of the fatty acid specificity of lipases are called selective hydrolysis and selective esterification, respectively. There has been no report regarding the high-level processing of oils and fats or their related compounds by alcoholysis. Thus, we termed

TABLE 4. Alcoholysis, with lauryl alcohol, of ethyl esters of which docosahexaenoic acid (DHA) contents are different

Substrate Reaction time Alcoholysis Fatty acid content (mol%)

(h) (%) 16:O 18: 1 20 : 5 22 : 6 Recover{ of DHA

(X)

E-tuna-23a od 0 21.1 13.9 25 56.1 9.7 5.4 50 62.7 9.1 6.7

E-tuna-45b od 0 2.3 10.8 2.5 33.6 0.7 2.4 50 41.4 NDe 2.1

E-tuna-60c Od 0 0.9 5.0 25 26.3 ND 0.8 50 31.7 ND 0.7

The reaction was carried out as described in Materials and Methods. a Ethyl esters of which the ethyl docosahexaenoate (E-DHA) content was 23 mol%. b Ethyl esters of which the E-DHA content was 45 mol%. c Ethyl esters of which the E-DHA content was 60 mol%. d Before reaction. e Not detected.

9.2 22.7 loo 14.9 47.9 92.6 12.5 52.2 90.4 12.5 44.9 100 14.6 65.6 97.0 12.7 71.9 93.8 8.6 60.1 100 8.9 78.7 96.5 7.1 83.1 94.4


this new technique selective alcoholysis. From the time course of the alcoholysis of E-tuna-23

(Fig. 4), the activity of the lipase on each fatty acid was found to be in the following order: PA, OA>EPA> DHA. This order agreed with the fatty acid specificity of Rhizopus lipase in hydrolysis (11). When tuna oil was hydrolyzed with soluble Rhizopus lipase, 12% of the initial DHA was released in 29% hydrolysis (9). When fatty acids originating from tuna oil were esterified with lauryl alcohol using the soluble lipase, 16% of the initial DHA changed to lauryl ester in 72% esterification (14). When the alcoholysis of ethyl esters originating from tuna oil (E-tuna-23) was conducted using the immobilized lipase, 10% of the initial DHA was alcoholyzed with lauryl alcohol in 63% alcoholysis (Table 4). In addition, when tuna oil was acidolyzed with caprylic acid using the immobilized lipase, DHA at 1,3-position was hardly exchanged for caprylic acid in 74% acidolysis extent (15). From these results, it was suggested that the fatty acid specificity of Rhizopus lipase was strict in the order of hydrolysis > esterification > alcoholysis > acidolysis.

The acidolysis activity of immobilized Rhizopus lipase was activated by shaking in the reaction mixture containing water (2%), but the hydrolysis activity was simultane- ously occurred. This simultaneous hydrolysis was completely repressed by repeating the reaction in a mixture without adding additional water, and only acidolysis occurred efficiently (15, 17). The alcoholysis activity was also activated using the same procedure: the lipase was shaken in a mixture containing more than 0.8% water (Table 1). These facts may suggest that the lipase can trap water molecule(s) in or near the catalytic site by catalyzing hydrolysis during the pretreatment. This may be the mechanism of the activation of acidolysis and alcoholysis activities.

When the alcoholysis of E-tuna-23 was conducted in a reaction mixture containing molecular sieves 3A (25%), the initial rate and the extent of the alcoholysis after 24 h decreased to 53 and 75% of those on using a mixture without molecular sieves, respectively (data not shown). These decreases may be attributed to the release of the water bound to the immobilized enzyme. However, the bound water was not released from the lipase molecule in the continual batch reaction, because the alcoholysis activity which had decreased did not recover upon by addition of water to the substrate mixture. Therefore, a small amount of water contained in the substrate mixture may suppress the release of bound water.

REFERENCES

1. Fischer, S.: Dietary polyunsaturated fatty acids and eicosanoid formation in humans. Adv. Lipid Res., 23, 169-198 (1992).

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

Harris, W.S.: Fish oils and plasma lipid and lipoprotein metabolism in humans: a critical review. J. Lipid Res., 30, 785-809 (1989). Hara, K.: Pharmaceutical application of icosapentaenoic acid. Yushi, 46, 91-99 (1993). (in Japanese) Maruyama, K. and Nishikawa, M.: Physiological function of a fish oil component and its application to foods. Food Chemi- cals, No. 4, 31-37 (1995). (in Japanese) Yamaguchi, M., Tanaka, I., and Ohtsu, Y.: New method for separation and purification of polyunsaturated fatty acids using a silver ion-exchanged spherical clay mineral. Yukagaku, 40, 959-964 (1991). (in Japanese) Hoshino, T., Yamane, T., and Shimizu, S.: Selective hydrolysis of fish oil by lipase to concentrate n-3 polyunsaturated fatty acids. Agric. Biol. Chem., 54, 1459-1467 (1990). Tanaka, Y., Hiraao, .I., and Funada, T.: Concentration of docosahexaenoic acid in glyceride by hydrolysis of fish oil with Can&da cylindracea lipase. J. Am. Oil Chem. Sot., 69, 1210- 1214 (1992). RahmatuIIah, M. S. K. S., Shukla, V. K. S., and Mukhejee, K. D.: Enrichment of i-linolenic acid from evening primrose oil and borage oil via lipase-catalyzed hydrolysis. J. Am. Oil Chem. Sot., 71, 569-573 (1994). Shimada, Y., Maruyama, K., Okazaki, S., Nakamura, M., Sugihara, A., and Tominaga, Y.: Enrichment of polyunsaturated fatty acids with Geotrichum cundidum lipase. J. Am. Oil Chem. Sot., 71, 951-954 (1994). Shimada, Y., Maruyama., K., Nakamura, M., Nakayama, S., Sugihara, A., and Tominaga, Y.: Selective hydrolysis of polyunsaturated fatty acid-containing oil with Geotrichum candi- dum lipase. J. Am. Oil Chem. Sot., 72, 1577-1581 (1995). Shimada, Y., Sugihara, A., Maruyama, K., Nagao, T., Nakayama, S., Nakano, H., and Tominaga, Y.: Enrichment of arachidonic acid: selective hydrolysis of a single-cell oil from Mortierella with Candida cylindracea lipase. J. Am. Oil Chem. Sot., 72, 1323-1327 (1995). Hill, M. J., Kiewitt, I., and Mukherjee, K. D.: Enzymatic frac- tionation of fatty acids: enrichment of r-linolenic acid and docosahexaenoic acid by selective esterification by lipases. J. Am. Oil Chem. Sot., 67, 561-564 (1990). Foglia, T. A. and Sonnet, P. E.: Fatty acid selectivity of lipases: r-linolenic acid from borage oil. J. Am. Oil Chem. Sot., 72, 417-420 (1995). Skimada, Y., Sugihara, A., Nakano, H., Kuramoto, T., Nagao, T., Gemba, M., and Tominaga, Y.: Purification of docosahexaenoic acid by selective esterification of fatty acids from tuna oil with Rhizopus delemar lipase. J. Am. Oil Chem. Sot., 74, 97-101 (1997). Shimada, Y., Suglhara, A., Maruyama, K., Nagao, T., Nakayama, S., Nakano, H., and Tominaga, Y.: Production of structured lipid containing docosahexaenoic and caprylic acids using immobilized Rhizopus delemar lipase. J. Ferment. Bioeng., 81, 299-303 (1996). Sugihara, A., Tani, T., and Tominaga, Y.: Purification and characterization of a novel thermostable lipase from Bacillus sp. J. Biochem., 109, 211-216 (1991). Sbimada, Y., Suglhara, A., Nakano, H., Yokota, T., Nagao, T., Komemushi, S., and Tominaga, Y.: Production of structured lipids containing essential fatty acids by immobilized Rhizopus delemar lipase. J. Am. Oil Chem. Sot., 73, 1415- 1420 (1996).

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