Production of Omega-3 Fatty Acid Ethyl Esters from ...

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Abstract An organic solvent-stable lipase from Proteus vulgaris K80 was used to produce the omega-3 polyunsaturated fatty acid ethyl esters (ω-3 PUFA EEs).

Appl Biochem Biotechnol DOI 10.1007/s12010-016-1998-7

Production of Omega-3 Fatty Acid Ethyl Esters from Menhaden Oil Using Proteus vulgaris Lipase-Mediated One-Step Transesterification and Urea Complexation Soo-jin Kim 1 & Hyung Kwoun Kim 1

Received: 21 October 2015 / Accepted: 22 January 2016 # Springer Science+Business Media New York 2016

Abstract An organic solvent-stable lipase from Proteus vulgaris K80 was used to produce the omega-3 polyunsaturated fatty acid ethyl esters (ω-3 PUFA EEs). First, the lyophilized recombinant lipase K80 (LyoK80) was used to perform the transesterification reaction of menhaden oil and ethanol. LyoK80 produced the ω-3 PUFA EEs with a conversion yield of 82 % in the presence of 20 % water content via a three-step ethanol-feeding process; however, in a non-aqueous condition, LyoK80 produced only a slight amount of the ω-3 PUFA EEs. To enhance its reaction properties, the lipase K80 was immobilized on a hydrophobic bead to derive ImmK80; the biochemical properties and substrate specificity of ImmK80 are similar to those of LyoK80. ImmK80 was then used to produce ω-3 PUFA EEs in accordance with the same transesterification reaction. Unlike LyoK80, ImmK80 achieved a high ω-3 PUFA EE conversion yield of 86 % under a non-aqueous system via a one-step ethanol-feeding reaction. The ω-3 PUFA EEs were purified up to 92 % using a urea complexation method. Keywords Lipase . Omega-3 fatty acid ethyl esters . Immobilization . Transesterification . Urea complexation

Introduction Some fish oils contain a large amount of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) including eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), which are known to be physiologically essential fatty acids for humans. EPA and DHA play various physiological, structural, and therapeutic roles in the cardiovascular, immune, and

* Hyung Kwoun Kim [email protected]

1

Department of Biotechnology, The Catholic University of Korea, Bucheon, Gyeonggi-do 420-743, Korea

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nervous systems and developmental roles in the visual and neurological systems [1–5]. Because of their beneficial healthcare effect, ω-3 PUFAs can be used for functional food ingredients, dietary supplements, and pharmaceuticals [6]; therefore, researches to obtain pure ω-3 PUFAs from fish oils have been actively carried out in the interest of producing a valueadded product. As fish oils are mixtures of multifarious fatty acids with various chain lengths and saturation degrees [7], it is important to effectively separate the ω-3 PUFAs from the nonPUFAs. To obtain highly pure ω-3 PUFAs from fish oil, a variety of methods have been developed including chromatography, vacuum or molecular distillation, low-temperature crystallization, urea complexation, supercritical fluid fractionation, and enzymatic reactions [8–10]. Among these methods, lipase-catalyzed reactions such as esterification and transesterification are most widely used to produce ω-3 PUFA ethyl esters (EEs); these approaches have the following benefits: (1) lipase-assisted processes can be performed under mild conditions so that the processing cost is not high and products are not destroyed and (2) the substrate specificity of lipases can lead to the selective formation of a good-quality target product [11–13]. In addition, numerous advantages can be derived from lipase immobilization. Lipase immobilization can not only improve the activity and stability of a lipase, but the lipase can also be more easily separated from the reaction mixture for reuse [14, 15]. Some studies have therefore reported that ω-3 PUFA EEs can be synthesized by the ethanolysis of fish oil that has been catalyzed by an immobilized lipase [16, 17]. Lipase K80, an extracellular alkaline lipase produced from Proteus vulgaris, was isolated from soil collected near a sewage disposal plant in Korea [18]. This lipase showed a high specific activity in a hydrolysis reaction and a high stability in various organic solvents [19]. In a previous study, lipase K80 produced a biodiesel (fatty acid methyl ester) from olive oil and methanol via a three-step methanol-feeding process. When lipase K80 was immobilized onto methyl methacryl divinylbenzene (MA-DVB, Lewatit VP OC1600) via a hydrophobic interaction and was used for biodiesel production, its conversion yield reached approximately 90 % [19]. In this study, the production of ω-3 PUFA EEs from menhaden oil and ethanol was performed by using a transesterification reaction for which lipase K80 was the catalyst (Scheme 1). In addition, to improve the catalytic power and stability, lipase K80 was immobilized onto MA-DVB resin using hydrophobic adsorption and covalent cross-linking methods [20]. This immobilized form of lipase K80 was applied for the production of ω-3 PUFA EEs using a one-step ethanol-feeding transesterification reaction in a non-aqueous system. Finally, the produced ω-3 PUFA EEs were isolated from the reaction mixture using a urea complexation method. OH CH3CH2OH

O O

C O

R

O

C O

R

O

C

R

Menhaden oil

OH

ω-3 PUFA EE R; Eicosapentaenoic acid Docosahexaenoic acid

OH O

lipase CH3CH2O

C

ω-3 PUFA EE non-PUFA EE

R

non-PUFA EE R; Palmitic acid Oleic acid Other non-PUFA

Scheme 1 Lipase-catalyzed transesterification of menhaden oil with ethanol using LyoK80 and ImmK80

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Materials and Methods Chemicals Fish oil (from menhaden), olive oil, p-nitrophenyl palmitate, palmitic acid ethyl ester, linoleic acid ethyl ester, oleic acid ethyl ester, stearic acid ethyl ester, and glutaraldehyde were purchased from Sigma–Aldrich (St. Louis, MO, USA). MA-DVB resin was bought from GenoFocus Co. (Daejeon, Korea). Eicosapentaenoic acid ethyl ester and docosahexaenoic acid ethyl ester were purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). Ethanol was bought from Merck Chemical Co. (Darmstadt, Germany). Hexane and urea were purchased from Junsei Chemical Co. (Chuo-ku, Tokyo, Japan).

Production of P. vulgaris Lipase K80 in Escherichia coli The P. vulgaris lipase K80 gene was transformed into E. coli BL21 (DE3) cells. The transformed cells were cultured in an LB medium containing 100 μg/mL ampicillin. A seed culture was prepared at 37 °C overnight, and 1.5 L of main culture was also performed at 37 °C. When the optical density at 600 nm (OD600) reached 0.5, 1 mM isopropyl thio-β-Dgalactoside was added and the cells were cultured at 20 °C for an additional 22 h. The cultured cells were harvested using centrifugation (12,000×g, 10 min) and resuspended in 15 mL of Tris–HCl buffer (50 mM, pH 8.0). The cells were lysed by ultrasonication and the soluble fraction was obtained using centrifugation (10,000×g, 10 min). The cell-free extract was named Bsoluble K80^ for use in lyophilization or immobilization.

Immobilization of K80 Lipase To activate the bead, 1 g MA-DVB resin and 10 mL of methanol were mixed in a vial (30-mLsized) and treated for 1 h at 25 °C with shaking at 160 rpm. The methanol was then removed and 10 mL of potassium phosphate buffer (50 mM, pH 6.5) was added and incubated at 25 °C with shaking at 160 rpm. After 1 h, the buffer was removed and 10 mL of the cell-free extract (400 mg of protein) was added and incubated for 4 h at 25 °C with shaking at 160 rpm. After incubation, glutaraldehyde was added to the mixture to a final concentration of 25 mM, and the mixture was incubated for 20 h at 4 °C using a tube tumbler rotating mixer (Select BioProducts, SBS550-2). To remove the unbound enzyme, the bead was washed three times with 10 mL of potassium phosphate buffer (50 mM, pH 6.5); the duration of each washing was 10 min and the bead was subsequently dried on a centrifugal evaporator (EYELA, CVE2000). The immobilized K80 lipase preparation was named BImmK80^ and stored at 4 °C until use [20]. Immobilization yield (η) and activity retention (R) were calculated as follows: η ð%Þ ¼

Immobilized protein ðP f −Pi Þ  100 % Total loading protein ðP f Þ

where Pf is the protein amount of soluble lipase and Pi is that of the supernatant after immobilization. R ð%Þ ¼

Transesterification activity of immobilized lipase  100 % Transesterification activity before immobilization

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Hydrolytic Activity Assay The hydrolytic activity was measured using the pH stat method. A fish oil emulsion was prepared by emulsifying the mixture containing 1 % fish oil, 1 % arabic gum, 20 mM NaCl, and 5 mM CaCl2 for 2 min at the maximum speed in a Waring blender. After using a 10 mM NaOH solution to adjust the pH of the substrate emulsion (20 mL) to pH 8.0, 10 μL of soluble K80 (or 20 mg of ImmK80) was added. The fatty acid release rate was measured using a pH titrator (718 Stat Titrino, Metrohm, Herisau, Switzerland) for 3 min at 30 °C. One unit of lipase activity (U) was defined as the amount of enzyme required to liberate 1 μmol of fatty acid per minute.

Transesterification Activity Assay Enzyme samples (2 mg of LyoK80 or 20 mg of ImmK80) were added to 0.5 mL of 10 mM pnitrophenyl palmitate (pNPP) in hexane and 30 μL of ethanol. The reaction was performed at 30 °C for 10 min with 230 rpm shaking. After the settling of the lipase, 25 μL of clear supernatant was taken and mixed immediately with 1 mL of 0.1 M NaOH. The liberated pnitrophenol (p-NP) was extracted using the aqueous alkaline phase, and the amount of p-NP was determined by absorbance at 410 nm. One unit of lipase activity (U) was defined as the lipase amount needed to liberate 1 μmol p-NP per minute [21].

Effects of Temperature and pH on Free K80 and ImmK80 Lipases To determine the effects of temperature, the lipase activity was measured at various temperatures (20 to 80 °C) using the pH stat method. To evaluate temperature stability, the enzymes were pre-incubated for 30 min at 20 to 60 °C, after which the remaining activity was assayed at 30 °C. Enzyme activity was measured at various pH values to determine the optimal pH. The following buffers (50 mM) were used to characterize the pH effects: pH 4 to pH 6, acetic acid/ sodium acetate; pH 6 to pH 8, KH2PO4/K2HPO4; pH 8 to pH 9, Tris–HCl; pH 9 to pH 11, glycine–KCl–KOH; and pH 11 to pH 12, K2HPO4/K3PO4. To confirm the pH stability, the enzyme was incubated for 30 min in the previously mentioned pH buffers at 30 °C and then adjusted to pH 8.0; the residual activity of the enzyme was then evaluated under the conditions following the pH adjustment.

Substrate Specificity The hydrolysis rates of various substrates including tributyrin, tricaprylin, olive oil, and fish oil were measured using the previously described pH stat method.

Lipase-Catalyzed Transesterification The enzymatic transesterification of menhaden oil was conducted as follows. Menhaden oil and ethanol were mixed with a molar ratio of 1:3, whereby menhaden oil (1 mL) and ethanol (0.186 mL) were put in a glass vial (30 mL). Different amounts (0, 132, 298, and 508 μL) of water were added to give 0, 10, 20, and 30 % hydrated reaction mixtures, respectively. In case of 0 % reaction, 508 μL of hexane was added. The same unit (3 U) of the LyoK80 or ImmK80 was then added to the reaction mixture. In the case of LyoK80, 25 glass beads (3 mm, Superior

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Marienfeld, Germany) were added for efficient mixing. The transesterification reaction was conducted for 24 h at 30 °C in a shaking incubator (230 rpm). Ethanol was added in three steps for a LyoK80-mediated reaction and in one step for a ImmK80-mediated one. After enzyme reactions, the reaction mixtures were filtered and analyzed using thin-layer chromatography (TLC) and gas chromatography (GC).

Reusability To test the reusability of ImmK80, the immobilized lipase was separated by removing the reaction mixture. The immobilized lipase was then washed sequentially with ethanol and phosphate buffer (50 mM, pH 6.5) and finally dried using a rotary evaporator. The recovered immobilized lipase was used in the next transesterification batch with fresh substrates.

Urea Complexation The filtrated reaction product mixture (2 g) from the lipase-catalyzed transesterification process was dissolved in 20 mL of 95 % ethanol together with 8 g of urea. The mixture was heated at 60 °C with constant stirring until it turned into a clear homogeneous solution. The solution was allowed to crystallize at room temperature for 5 h by occasional shaking to promote the formation of urea complexes. It was then kept at 4 °C for 24 h. The crystals (UCF, urea complex fraction) were separated from the liquid (NUCF, non-urea complex fraction) using vacuum filtration at room temperature. To isolate fatty acids from the filtrate, an equal volume of distilled water was added to the filtrate (NUCF); subsequently, an equal volume of hexane was added and the mixture was stirred for 1 h. The hexane layer containing liberated fatty acids was separated from the aqueous phase containing urea. The hexane layer was washed with distilled water again and was then dried using anhydrous sodium sulfate; it was then concentrated on a rotary evaporator and further analyzed using TLC and GC [22].

Thin-Layer Chromatography Analysis of the Reaction Products At the predetermined time intervals, a small volume (0.1 mL) of the reaction mixture was taken and mixed with 1 mL of hexane for 2 min. Following centrifugal separation, 10 μL of the upper layer was applied on a TLC silica gel 60 F254 plate (Merck). As a developing solvent, hexane/ethyl acetate/acetic acid (90:10:1) was utilized, and methanol/sulfuric acid (1:1) was used as a visualization reagent. After development, the color reagent was sprayed over the silica gel plate and the plate was heated at a high temperature and analyzed.

Gas Chromatography Analysis of the Reaction Products A part of the reaction sample prepared for a TLC analysis was injected into a gas chromatograph (Hewlett-Packard 7890). The fatty acid ethyl ester was analyzed with a HP-5 column (cross-linked 5 % PH ME Siloxane, 0.32 mm × 30 m) and FID detector. The temperatures of the injector and detector were 230 and 250 °C, respectively. The column temperature was increased from 70 to 300 °C at a rate of 10 °C per minute and was maintained for 5 min at 300 °C. By comparing the retention times and peak areas of the standard fatty acid ethyl ester peaks, the total quantities (in mole) of the fatty acid ethyl ester in the reaction mixtures were calculated.

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Results and Discussion Production of ω-3 PUFA EEs Using Free Lipase K80 Recombinant lipase K80 was expressed in the E. coli BL21 (DE3) cells, and the lipase activity in the cell-free extract was determined as 622 U/mg protein. The cell-free extract was lyophilized to produce LyoK80. The ethanolysis of the menhaden oil was then carried out using LyoK80 with a three-step addition of ethanol. In this experiment, different amounts of water (0 to 30 %) were added to the reaction mixture to investigate the effect of water content on the reaction yield. The TLC analysis showed the consumption of menhaden oil with the time course when the water content was 10 to 30 % (Fig. 1a). Although the intensity of oil spot

B Water 0% (Hexane 30%)

Water 10%

non-PUFA EE ω-3 PUFA EE

Fish oil

100

0 2 4 6 24 Reaction time (h)

Conversion yield (%)

C

60 40 20 0

100

ω-3 PUFA EE

80 60 40 20

0 2 4 6 24 Reaction time (h)

100

non-PUFA EE

80

Water 30% Conversion yield (%)

Water 20%

Conversion yield (%)

A

0 0%

10% 20% 30% Water content

3rd

80 60

2nd 40 20 0 0

5

10 15 Reaction time (h)

20

1st Fig. 1 Ethanolysis of menhaden oil using LyoK80. a The effect of water content on the ethanolysis of fish oil was analyzed using thin-layer chromatography. b Conversion yields of non-PUFA EEs and ω-3 PUFA EEs after a 24-h reaction were measured by gas chromatography. c Time courses of ω-3 PUFA EE and non-PUFA EE production were measured under 20 % water condition. Black circle ω-3 PUFA EEs, white circle non-PUFA EEs. Reactions were performed at 30 °C for 18 h using LyoK80 lipase. Ethanol was added in three steps at 0, 2, and 4 h, as indicated by the arrows

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decreased most rapidly on the TLC plate at 30 % water content, a GC analysis revealed that the maximum yields of ω-3 PUFA EEs and non-PUFA EEs were observed at 10 % water content (Fig. 1b). This result implied that at high water content (20 and 30 %), the water molecules seem to induce the hydrolysis of the oil [23–25]. In a non-aqueous reaction condition, however, LyoK80 produced only a slight amount of ω-3 PUFA EEs (1.8 %) and non-PUFA EEs (9.3 %). Most probably, LyoK80 was prone to aggregation in the non-aqueous condition and the substrate accessibility therefore decreased [23, 26, 27]. Figure 1c shows the time course of the ethanolysis reaction at the 20 % water content condition. Both ω-3 PUFA EEs and non-PUFA EEs were produced continuously during the time course and reached a plateau after a 6-h reaction. The conversion yields of the ω-3 PUFA EEs and non-PUFA EEs were measured as 82 and 89 %, respectively. Although the LyoK80-mediated transesterification reaction was an efficient process with a high conversion yield, ethanol should be added in three steps because the enzyme has low stability in a high ethanol concentration. The development of a one-step ethanol-feeding reaction system is therefore necessary for the convenient production of ω-3 PUFA EEs.

Immobilization of K80 Lipase To improve the lipase activity and stability, lipase K80 was immobilized onto an MADVB bead, whereby a hydrophobic macroporous resin allows the lipase molecules to attach onto the outer surface or enter into the inner pore of the bead [20]. In our previous study, lipase K80 was immobilized onto the resin by hydrophobic adsorption; the immobilized product was then applied in the production of biodiesel from olive oil [19]. In this study, lipase K80 was immobilized onto MA-DVB resin using both covalent cross-linking and hydrophobic adsorption. It was reported that the reusability of the immobilized lipase was improved by adopting this covalent cross-linking method [20, 28]. As a result, the immobilization yield (η) was 77 %, which was calculated by measuring the protein amount attached to the bead after 4 h of incubation (Table 1). The total transesterification activities of the free K80 and ImmK80 were measured as 6.39 and 5.58 U, respectively, showing that the activity retention (R) is 87.3 %. The specific activity of ImmK80 was calculated as 18.1 U/g protein, which was higher than that (16.0 U/g protein) of free K80. This immobilization process is therefore an efficient process in our study, and we propose that ImmK80 should be used in transesterification reactions in organic solvents. Table 1 Immobilization yield and activity retention of lipase K80 Lipase type

Protein amount (mg)a

Immobilization yield (η, %)b

Transesterification activity of lipase (unit)c

Activity retention (R, %)

Free K80

400



6.39



Immobilized K80

308

77.0

5.58

87.3

a

Protein amounts of free and immobilized lipase K80 were both measured using the Bradford method

b

Immobilization yield was calculated based on protein amount in supernatant

c

Transesterification activity of free and immobilized lipase K80 was measured using the pNPP method

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Effects of Temperature and pH on Free and Immobilized Lipase K80 To identify the influence of immobilization on the biochemical properties of lipase, the effects of temperature and pH on lipase activity and stability were investigated. As a result, the optimal temperatures of catalysis by free K80 and ImmK80 were determined as 55 and 75 °C (Fig. 2a), respectively, implying that the immobilization onto the bead changed its active site and increased lipase activity at higher temperatures [29]. Their thermostabilities, however, are

A

B 100 Relative activity (%)

Relative activity (%)

100 80 60 40 20 0

20

30

40 50 60 70 Temperature (℃)

D

80 60 40 20 0

6

7

8

9 pH

E Relative Activity (%)

Relative activity (%)

Relative activity (%)

100

10

11

60 40 20 0

80

C

80

20

30 40 50 Temperature (℃)

60

100 80 60 40 20 0

4

5

6

7

8 9 10 11 12 pH

100 80 60 40 20 0

Fig. 2 Effect of immobilization on biochemical properties of lipase K80. The effects of temperature on activity (a) and stability (b) and the effects of pH on activity (c) and stability (d) were measured using the pH stat method. For stability test, the residual activity was measured after 30-min incubation under various temperatures and pH values. Hydrolysis activity toward various triglycerides (e) was also assayed. Error bars indicate standard deviations from more than three independent experiments. Black bar free lipase K80, white bar ImmK80

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almost the same, as both of them were fairly stable up to 40 °C and their activity decreased continuously over 40 °C. In addition, the effects of pH on free K80 and ImmK80 were almost the same and both enzymes showed an optimal pH of 10 (Fig. 2c). Their pH stabilities were also similar, as free K80 was stable at pH 6 to pH 11 and ImmK80 was fairly stable at pH 6 to pH 10 (Fig. 2b, d).

Substrate Specificity The substrate specificity toward various triglycerides was studied by measuring the hydrolytic activity (Fig. 2e). Both lipases showed high activity toward short-chain and medium-chain triglycerides, whereby they rapidly hydrolyzed tributyrin (C4) and tricaprylin (C8); however, both lipases showed low activity toward long-chain triglycerides. Meanwhile, the activity toward olive oil decreased after immobilization, whereas the activity toward menhaden oil increased slightly. These results suggest that the hydrolytic activity toward ω-3 PUFAs increased after immobilization.

Production of ω-3 PUFA EEs Using ImmK80 To check the performance of ImmK80 for ω-3 PUFA EE production, an ethanolysis reaction of the menhaden oil was carried out. First, to optimize the proper water content, the transesterification of the menhaden oil and ethanol was carried out under a variety of water concentrations (0 to 30 %). A TLC analysis showed that the menhaden oil spot intensity decreased with the time course for all of the water content levels used in this experiment. In particular, in contrast with LyoK80, ImmK80 achieved high conversion yields for both ω-3 PUFA EEs (83 %) and nonPUFA EEs (91 %) in a non-aqueous reaction condition (Fig. 3a, b). The reason why ImmK80 showed such high conversion yields in a water-free condition can be explained by the following. In the case of LyoK80, lipase enzymes aggregated easily in a non-aqueous condition; however, the immobilized lipase was spread evenly across the resin and the accessibility of the substrate to the lipase was therefore increased [27, 30]. Moreover, the lipase was fixed at its open conformation by forming a hydrophobic interaction between the hydrophobic resin and lipase [11, 30]; therefore, it can catalyze the reaction efficiently in a non-aqueous condition. A water-free condition has many advantages for a lipase-catalyzed reaction including the following: the increased solubility of the hydrophobic substrate, an ability to produce the target material without any corresponding by-product, the facility of product separation from the enzyme, and a reduced possibility of microbial contamination [31, 32]. Based on results shown in Fig. 3a, b, we concluded that a non-aqueous condition is a suitable condition for ImmK80 to perform the ethanolysis of fish oil. As shown in Fig. 3c, ImmK80 performed the transesterification reaction more rapidly than LyoK80 (Fig. 1c). The conversion reached the plateau within 4 h and the maximum yields of ω-3 PUFA EEs and non-PUFA EEs were 86 and 96 %, respectively. In the case of a free lipase K80-catalyzed reaction, ethanol was added according to a threestep feeding method due to the detrimental effect of ethanol on lipase K80 (Fig. 1c). The ImmK80-catalyzed reaction, however, was carried out by adding ethanol only once at the beginning, and a slightly higher conversion yield was consequently achieved due to an improved ethanol stability of the immobilized enzyme.

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B

Water 0% (Hexane 30%)

Water 10%

Fish oil

Water 20%

non-PUFA EE ω-3 PUFA EE

0 2 4 6 24 Reaction time (h)

Conversion yield (%)

Water 30%

D

80

Relative yield (%)

Conversion yield (%)

100

60 40 20 0

5

10 15 Reaction time (h)

20

non-PUFA EE

100 80 60 40 20 0 100

ω-3 PUFA EE

80 60 40 20 0

0 2 4 6 24 Reaction time (h)

C

0

Conversion yield (%)

A

0%

10% 20% 30% Water content

100 80 60 40 20 0

1

2 3 4 Operational cycle

5

1st Fig. 3 Ethanolysis of menhaden oil using ImmK80. a The effect of the water content on the ethanolysis of fish oil was analyzed by thin-layer chromatography, and b conversion yields of non-PUFA EEs and ω-3 PUFA EEs after a 24-h reaction were measured using gas chromatography. c Time courses of non-PUFA EE and ω-3 PUFA EE production were measured under non-aqueous condition. Black circle ω-3 PUFA EEs, white circle nonPUFA EEs. Reactions were performed at 30 °C for 18 h using ImmK80. d The reusability of ImmK80 was determined. The reactions were repeated by successive batch ethanolysis processes using fresh menhaden oil. The initial conversion yield was defined as 100 %

Reusability Figure 3d shows the conversion yield of an ethanolysis for which ImmK80 was used during five cycles of recovery. After each reaction cycle, ImmK80 was separated from the reaction mixture and then washed with ethanol to remove the remaining oil and fatty acid ethyl ester, followed by washing with a potassium phosphate buffer (50 mM, pH 6.5) to remove the remaining glycerol. After this two-step washing, the lipase was dried and then used for the next batch reaction. The conversion yield was maintained at the level higher than 90 % of the initial yield until the third cycle; however, after the fourth and fifth cycles, the yields

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drastically decreased to 60 and 10 %, respectively. This decrease of activity seemed to be caused by the entrapment, inside the resin, of the substrate and product that were not removed by the washing solvents, whereby they therefore prevented the access of new substrates [32, 33].

Purification of ω-3 PUFA EEs via a Urea Complexation Method The produced ω-3 PUFA EEs were purified from the reaction mixture using a urea complexation method. The menhaden oil contains the residues of approximately 20 % palmitic acid, 4 % stearic acid, 12 % oleic acid, 3 % linoleic acid, 15 % EPA, and 15 % DHA, and 31 % of the remaining fatty acids (product specification provided by Sigma–Aldrich). When the composition is calculated based on only the former six fatty acid residues, each percentage is recalculated as 29, 6, 17, 4, 22, and 22 %, respectively. The percentage of ω-3 PUFAs among them is approximately 44 %.

A

B 100

Fatty acid ethyl ester (%)

Non-PUFA EE ω-3 PUFA EE

7.69%

80 56.8% 60 Non-PUFA EE

92.3% 40

ω-3 PUFA EE 43.2%

20

0 1

Fatty acid ethyl ester (%)

C

Before urea complexation

2

After urea complexation

100

80

C16:0

C18:0

C18:1

C18:2

C20:5

C22:6

72

60

40

30

29 18

20 6

20

13

4

1

1

2

4

0 Before urea complexation

After urea complexation

Fig. 4 Purification of ω-3 PUFA EEs via urea complexation. a The enzymatic reaction mixture (lane 1) and the purified ω-3 PUFA EEs (lane 2) were analyzed by thin-layer chromatography. b Gas chromatography analysis data shows the composition of the enzymatic reaction mixture and purified ω-3 PUFA EEs. Gray bar ω-3 PUFA EEs (EPA, DHA), white bar non-PUFA EEs (palmitic acid, stearic acid, oleic acid, linoleic acid). c The fatty acid composition before and after urea complexation

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From the GC analysis, the percentage of ω-3 PUFA EEs after the enzymatic transesterification reaction is 43 %; through the urea complexation process, this was increased up to 92 % (Fig. 4a, b). Urea can form complexes with saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs), but it cannot make a complex with PUFAs. PUFAs can therefore be separated from the reaction mixture containing SFAs and MUFAs by using filtration and can be enriched with a high yield [10, 22, 25]. Figure 4c shows the fatty acid composition before and after the urea complexation process. Palmitic acid, stearic acid, and oleic acid can make complexes with urea, and a NUCF therefore contains only 1 % of each of those fatty acids. EPA and DHA were highly concentrated in the NUCF, with DHA accounting for 72 %. This result shows that ω-3 PUFA EEs can be purified and concentrated effectively using urea complexation.

Conclusions LyoK80 carried out the ethanolysis of menhaden oil with a high conversion yield; however, LyoK80 requires a three-step ethanol-feeding system due to its low ethanol stability. To overcome these problems, the immobilization technique was adopted and lipase K80 was successfully immobilized onto a hydrophobic bead, thereby forming ImmK80. When ImmK80 was applied for the ethanolysis reaction, it also performed the reaction with a high conversion yield, even in a non-aqueous system, according to a one-step ethanol-feeding process. The resulting ω-3 PUFA EEs of 92 % purity were separated using a urea complexation method. These results suggest that a methanolstable lipase from P. vulgaris can be immobilized covalently onto an MA-DVB bead and ImmK80-mediated one-step transesterification and subsequent urea complexation methods be employed to produce ω-3 PUFA EEs which is used in nutraceutical industry. Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (2014R1A2A2A01006978) and by the Research Fund 2015 of The Catholic University of Korea.

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