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Enzymatic hydrolysis of fats at high substrate concentrations in biphasic organic-aqueous systems
[J. Ferment. Technol., Vol. 65, No. 1, 23-29. 1987]
Enzymatic Hydrolysis of Fats at High Substrate Concentrations in Biphasic Organic-Aqueous Systems SuKPKum MmO, TAr~, TETSUO KO~AYASm, S~IGO SATO, and J o j I TAr,~aASHI
Institute of Applied Biochemistry, University of Tsukuba, Sakura-mura, Niihari-gun, Ibaraki 305, Japan The hydrolysis of palm oil and beef tallow by lipase has been studied for practical applications in a biphasic isooctane-aqueous system using a high substrate concentration. The effective lipase concentration for the hydrolysis was found to be about 120 IU per g of substrate. The addition of twenty percent isooctane brought about the most rapid reaction and produced the highest percentage of hydrolysis. For both palm oil and beef tallow, a percentage of hydrolysis higher than 98% was achieved in the 20% isooctane system at a higher concentration of 50%. However, when the substrate concentration was higher than 50 %, the final value of hydrolysis decreased as the concentration of the substrate increased. Utilization of recycled lipase was attempted using an ultrafiltration membrane reactor. Approximately 60% of the lipase activity was recoverable after each reaction.
T h e e n z y m e hydrolysis oflipids has attracted m u c h attention for the industrial production o f fatty acids and glycerol u n d e r n o r m a l temperature a n d pressure. M a n y studies on the e n z y m a t i c hydrolysis of lipids have been carried out using liquid lipids such as olive oil and tributyrin as the substrate.l-4) At the present time, however, beef tallow and p a l m oil, w h i c h are in a solid form at n o r m a l temperature, are the m a i n raw materials for the industrial p r o d u c t i o n of fatty acids and glycerol. I n o u r previous papers,5,e) the advantages o f using lipase in a biphasic organic-aqueous system for the hydrolysis of fats were d e m o n strated, and the kinetics o f the enzymatic hydrolysis in this system were studied. T h e experiments in these studies were performed at low fat concentrations because the effects o f physical a n d chemical factors on the hydrolysis could be determined relatively easily. However, the establishment of a process h a v i n g a high rate of productivity is needed for the practical application of this reaction system. I t is necessary to carry out the reaction at high substrate concentrations, as well as to achieve a high percentage of hydrolysis. Generally, the hydrolysis of solid lipids
(fats) is difficult c o m p a r e d with that of liquid lipids, such as olive oil. I n addition, the fatty acids and glycerol must be of a high quality if they are to be utilized as raw materials for the m a n u f a c t u r e o f cosmetics, paint a n d other products. T h e p u r i t y as well as the rate o f productivity o f fatty acids and glycerol are therefore very i m p o r t a n t . I n the present paper, the hydrolysis o f fats by lipase in a biphasic isooctane-aqueous system was studied at high substrate concentrations, and the o p t i m u m percentage o f isooctane to be a d d e d a n d the a p p r o p r i a t e concentration of the e n z y m e were determined to achieve an effective hydrolysis system where fatty acids a n d glycerol are produced at high productivity a n d purity. Furthermore, the possibility of the recovery and reuse o f the e n z y m e was e x a m i n e d using a m e m b r a n e reactor. Materials and Methods Chemicals The chemicals used for the hydrolysis were virtually the same as those described in a previous paper, 5) except that palm oil was used in addition to beef tallow as a substrate. The specificities and fatty acid compositions of beef tallow and palm oil used in this study are shown in Table 1. Candida cylindracea lipase was used as the enzyme. The organic solvent added to the reaction system was isooctane because
24
MUKATAKA et al.
Table 1. Specificities and fatty acid compositions for beef tallow and palm oil. Beef tallow
Palm oil
AV SV
0. 35 194. 7
0. 18 199. 5
c~
o
Ct4 Ct8 CIs C18 : 1 Cls : ~
(~)
O
[J. Ferment. Technol., ;4
5
6
a
II--II~
o.4 ( ~ )
3.6 26.6 20. 2 43. 2
1.0 40. 4 3.0 42.9
6.4
12.3
AV, Acid value; SV, Saponification value. it was recognized in the previous report as the most suitable solvent for hydrolysis by lipase. Apparatus The reactor consisted of a separable flask having a capacity of 500 ml and an inside diameter of 8 cm. This flask was equipped with four baffle plates. The reaction mixture was agitated by a 6blade flat turbine (5 cm in diameter). An ultrafiltration membrane reactor (Toyo UHP-76) was also used in order to investigate the potentiality for the recovery and reuse (recycling) of the enzyme. The reactor was equipped with a Diaflo-YM5 membrane (molecular weight cut-off 5000, Amicon Co. Ltd.) and it was maintained at 37°C in a water bath (Fig. 1). Hydrolysis Hydrolysis for the determination of the optimum reaction condition was carried out according to the method previously reported, unless otherwise noted. 5) The procedure for the repeated reactions in the membrane reactor was as follows. The reaction mixture was prepared so as to produce a total volume of 150 ml, and the hydrolysis was performed under the prescribed conditions. After each reaction, 50 ml of isooctane was added to the reaction mixture in order to completely dissolve the fatty acids which had been produced. Following this, two phase separations were done. The isooctane phase was recovered using an aspirator. Oltrafiltration of the aqueous phase was performed by increasing the pressure in the reactor to 1.5 arm gauge pressure with nitrogen gas and then relieving the pressure when the residual liquid volume had decreased to 5 ml. The fresh medium, which consisted of fat, isooctane and M/lfi phosphate buffer (pH 7.7) at the predetermined ratio, was then added to the reactor. Analysis To monitor the percentage of hydrolysis of the substrate, the fatty acids produced were titrated with 0.5 N KOH. The percentage of hydrolysis was calculated on the basis of the saponification values of the substrates.
Fig. 1. Schematic diagram of the tltrafiltration membrane reactor system. 1, Nitrogen gas cylinder; 2, Water bath; 3, Drain; 4, Membrane; 5, Valve; 6, Sampling port; 7, Reaction mixture; 8, Magnetic stirrer; 9, Speed controll. The final hydrolysis value was also confirmed by determining the amount of residual glycerides in the reaction mixture. The amount of residual glycerides was measured by thin-layer chromatography (TLC) using a silica gel plate (Type 60, Merck). The composition of the developing solvent was hexane : ethyl ether : acetic acid (80 : 30 : 1). The spots on the plate were visualized by spraying 50% sulfuric acid and heating at l l0°C for 5 rain. The charred spots were analyzed with a densitometer (Asuka Kogyo, Model OZ-802) and estimated quantitatively from the calibration curve for which cholesteryl palmitate was used as the authentic material.
Results and Discussion Proper hydrolysis
enzyme
concentration
for
In enzyme reactions, the rate of reaction greatly depends on the enzyme concentration. T h u s , to d e t e r m i n e a o p t i m a l l i p a s e c o n c e n t r a t i o n f o r t h e h y d r o l y s i s o f fats at a high substrate concentration the hydrolyses o f 5 0 % ( w / v ) p a l m o i l a n d b e e f t a l l o w were carried out at various enzyme conc e n t r a t i o n s (Figs. 2 a n d 3). Figure 2 illustrates the time course of the hydrolysis of 50% palm oil at different enzyme concentrations. Palm oil was hydrolyzed almost completely, even when the l i p a s e c o n c e n t r a t i o n w a s 15 I U p e r g o f substrate, although a very long reaction time (24 h) w a s n e c e s s a r y for c o m p l e t e h y d r o l y s i s . Hydrolysis time decreased with an increase in the enzyme concentration. At an enzyme c o n c e n t r a t i o n o f 120 I U p e r 1 g o f s u b s t r a t e , 95% of the palm oil was hydrolyzed after 4 h
Vol. 65, 1987]
Enzymatic Hydrolysisof Fats
25
~" 75
i 50
~o 50 ~
0[ 0
I 6
I
12
I 18
I 2q
I
R e a c t l o n time (h) Fig. 2. Effects of enzyme concentration on the hydrolysis of 50% palm oil in the 20% isooctaneaqueous system. Lipase (IU/g-substrate): 120 (O); 60 (A); 30 (O);
15 ([]). and the value approached 100% after about 6h. The results for the hydrolysis of 50% beef tallow are presented in Fig. 3. The hydrolysis rate of beef tallow was slower than that for palm oil. The percentage of hydrolysis at 2 4 h was only 92%, even at an enzyme concentration of 30 IU/g-substrate, while at 60 IU/g-substrate and 24 h, the percentage of hydrolysis reached 98%. A percentage hydrolysis of 98% was achieved in 12 h when the enzyme was at a concentration of 120 I U per 1 g of substrate. Based on these results, it is evident that the rate of hydrolysis increased with an increase in the enzyme concentration. However, judging from the time courses for the hydrolyses of both palm oil and beef tallow (Figs. 2 and 3), a significant increase cannot be expected, even if the enzyme concentration is increased to greater than 120 IU/g-substrate. Furthermore, the use of lipase above this concentration is not economical. Thus, it is considered that a lipase concentration of about 120 IU/gsubstrate is the most suitable for the hydrolysis of palm oil and beef tallow. O p t i m u m percentage of added isooctane It was demonstrated in a previous paperS) that the appropriate percentage of isooctane to be added for the hydrolysis of 12% beef tallow or palm oii was within the
25 0
0
6
12
18
Reaction time (h)
Fig. 3. Effects of enzyme concentration on the hydrolysis of 50% beef tallow in the 20% isooctaneaqueous system.
Lipase (IU/g-substrate): 120 (O); 60 (A); 30 (El). range of 10 to 20%. Therefore, a 20% isooctane system was adopted for determining the most suitable enzyme concentration. It may be considered, however, that the optimum percentage of isooctane changes with the concentration of the substrate. T o identify the optimum reaction system, the hydrolyses of 50%(w/v) palm oil and beef tallow were performed under various percentages of added isooctane and at a lipase concentration of 120 IU/g-substrate. Figure 4 shows the results for palm oil. The hydrolysis of palm oil was facilitated by the addition ofisooctane which was within
i00 ~
--
-
•
Ily .,<-..-
0
6
12 18 214 Re0cti0n time (h) Fig. 4. Effects of the isooctane fraction on the hydrolysis 50% palm oil. Lipase: 120 IU/g-substrate, Isooctane (%) ; 0 (O) ; 10 (A); 20 (O); 45 (~); 65 (A); 85 (c-l). The isooctane fraction is represented by the volume percent in the reaction mixture without substrate.
26
MLrKATAKAet
the range of I0 to 45%(v/v). In particular, the 20% isooetane system produced the most rapid reaction and gave the highest percentage of hydrolysis. Similar results were also obtained for the hydrolysis of beef tallow (data are abbreviated). Based on these results, it is concluded that the optimum percentage of isooctane to be added is 20%(v/v) for the hydrolysis of 50% palm oil and beef tallow. (Proviso: percentage of isooctane is expressed by the volume percent in the mixture without substrate.) Final percentage of hydrolysis at high substrate concentrations An almost
complete hydrolysis of the substrate in a 20% isooctane system was observed for both the hydrolyses of 50% palm oil and of beef tallow. However, it is essential to increase the substrate concentration in order to improve the production of fatty acids and glycerol. At the same time, a high level of conversion of the substrate into its products is also required from a practical application standpoint. Therefore, hydrolysis at a substrate concentration higher than 50% was carried out in order to investigate its applicability. The results of the hydrolysis of 60 and 70% palm oil are shown in Fig. 5-a, b. These hydrolyses were carried out in three systems, I0, 20 and
lOO [-
"~ I| "S 5 0 II ioo
{a) 60 Z(W/V) i l I I
I
°
75 50
(b) 70 %(W/V) I
6
I
I
12 18 Reoctlon time (h)
t I
24
Fig. 5-(a), (b). Hydrolysisof 60 and 70% palm oil with varing percentages of isooctane added. Lipase: 120 IU/g-substrate, Isooctane (%): I0 (@) ; 20 (O) ; 40 (A).
[J. Ferment. Technol.,
al.
40% isooctane respectively. For both substrate concentrations of 60 and 70%, the 20% isooctane system produced the most rapid reaction and the highest percentage of hydrolysis, as in the case of hydrolysis at a 50% substrate concentration. In this system, the final percentages of hydrolysis were 96 and 90% at substrate concentrations of 60 and 70%, respectively. Hydrolyses were also performed with higher enzyme concentrations of 200 and 300 IU/g-substrate, however, the final percentages of hydrolysis were not further improved by increasing the enzyme concentration (Fig. 6-a, b). Hydrolysis at various substrate concentrations was also performed with beef tallow. The final values obtained for the hydrolyses of beef tallow and palm oil are summarized in Table 2. The results for the hydrolysis of beef tallow were founded to be inferior to those for palm oil. These results suggest that it is difficult to exceed a hydrolysis value of 98% at a substrate concentration higher than 50%. T h a t is, the upper limit of the substrate concentration, at which 98% hydrolysis was obtained, was 50% for both palm oil and beef tallow. Residual glycerides Fatty acids and glycerol produced by the hydrolysis of fats and oil have numerous uses as industrial materials, and in many cases, a high quality is required for the manufacture of other products, such as cosmetics. In biphasic isooctane-aqueous reaction systems, both beef Table 2. Final values for the hydrolysisof palm oil and beef tallow at various substrate concentrations. Substrate % (w/v)
Degree of hydrolysis (%) Palm oil Beef tallow
40
--
99
50
99
98
60
96
94
70
90
--
Isooctane: 20% (v/v)
Lipase: 120 IU/g-substrate
Voi. 65, 1987]
Enzymatic Hydrolysis of Fats
1
W/V) 50 o
[]
t
27
I
100
~ 7s 50
(b) 70 %(W/V) I
6
I
!
12 18 Renctlon time (h)
I 24
Fig. 6-(a), (b). Hydrolysis of 60 and 70% palm oil in 20% isooctane-aqueous systems with various lipase concentrations. Lipase (IU/g-substrate): 100 (0); 200 (A); 300 (O). tallow and p a l m oil were hydrolyzed above 98% by lipase at a substrate concentration of 50%. T h e final percentage of hydrolysis strongly influences the quality of the products. In our experiments, the time course of hydrolysis and its final percentages were determined based on the amount of fatty acids produced. Therefore, to confirm the findings, residual glycerides were analyzed by thin-layer chromatography. Figure 7 shows the thin-layer chromatog r a m of four samples of the reaction mixture for the hydrolysis of 50% p a l m oil. The first sample was a mixture prepared before the reaction, the second sample was a reaction mixture to which isooctane had not been added, while the third and fourth samples included the 20 and 65°/0 isooctane systems, respectively. T h e second, third and fourth samples were prepared after the reactions and final values of 95, 99, and 90% were estimated, respectively, from the titration of the fatty acids produced in each hydrolysis. T h e amount of glycerides in each sample was determined by a densitometer. In the hydrolysis of triglycerides b y C. cylindracea lipase, diglycerides and monoglycerides formed during the intermediate stages of the reaction are also hydrolyzed to
Fig. 7. Thin layer chromatograms of the reaction mixture in various hydrolysis. 1, sample before reaction; 2-4, samples after reaction (2, no isooctane; 3, 20% isooctane; 4, 65% isooctane). A, Sterol ester (internal standard) ; B, Trlglyceride; C, Fatty acid; D, Di- and monoglycerides; E, Origin. fatty acids and glycerol. Although the T L C analysis using densitometer showed traces of di- and monoglycerides, the exact amounts of these compounds could not be quantified for the samples prepared after the reactions had taken place (2, 3 and 4). Therefore, in Table 3, only the results of the determination of the spots for triglycerides on the T L C plates are presented. These values are in agreement with those estimated from the final Table 3. Amount of residual triglycerides in the reaction mixtures determined by TLC-densitometry. Isooctane
Residual triglycerldes
(%)*
(%)
0
3.3
20
0.7
65
7.2
Palm oil: 50% (w/v) Lipase: 120 IU/g-substrate * These values are expressed as volume percent in the reaction mixture without substrate.
MUKATAKAet al.
28
values of hydrolysis. For the hydrolysis of 50% palm oil in the 20% isooctane system, particularly, the very high value of 99.3% was also confirmed based on the amount of the residual glycerides. Repeated use o f llpase by u s i n g a m e m b r a n e reactor For the industrial application of enzymatic reaction systems, recovery and reuse (recycling) of the enzyme are essential cost reduction measures. In biphasic organic-aqueous reaction systems, the substrate (fat) is dissolved in the organic solvent phase while the lipase is dissolved in the aqueous phase. The fatty acids and glycerol produced by the hydrolysis of fat are also dissolved in the organic and aqueous phases, respectively. After the reaction, therefore, the fatty acids and the remaining substrate can be removed from the reactor by withdrawing the organic solvent. Furthermore, the glycerol dissolved in the aqueous phase is a low molecular weight substance. So, taking these fact into consideration, the use ofultrafiltration membrane is an effectual method for the recovery of the enzyme used in a biphasic organic-aqueous system. When the substrate concentration is high, the reaction mixture becomes viscous making |
i00 F 2~
~
-
,~
[J. Ferment. Technol.,
the separation of the two layers (aqueous and organic phases) rather lengthy. In our experiments, 50 ml of isooctane were added to the 150 ml reaction mixture at the end of the reaction in order to completely dissolve the fatty acids, and also to accelerate the separation into the two phases. The reaction mixture separated into two distinct phases within one hour. Figure 8 shows the time course of the hydrolysis of 50% palm oil using recycled lipase. Lipase was added to the reaction system (120 IU/g-substrate) only at the start of the first run. Hydrolysis in each run was performed at the optimal value of 20% isooctane. For comparison, the broken line in Fig. 8 indicates the time cource of hydrolysis at a lipase concentration of 30 IU/gsubstrate. Although the degree of hydrolysis attained was almost 100% for each run, the reaction rate decreased with successive repetitions of the reaction. This implies a gradual reduction in the lipase activity during the processes of reaction and recovery. The residual lipase activity in each reaction was estimated by comparing with the time courses of the hydrolyses shown in Fig. 8 and in Fig. 2. It was roughly estimated that 60% of the lipase activity was retained in each reaction. In order to confirm this, each repeated
|
7 5 ~ 1
~1°°
50
.~ 75
I O0|
}50 i
6
l
I
12 18 Reaction time (h)
l
24
Fig. 8. Repeated use of lipase for the hydrolysis of 50% palm oil in an ultrafiltration m e m b r a n e reactor. Lipase was added to the reaction system at 120 I U per g of substrate only at the start of the first run. Repetition number: 1st (@); 2nd (O); 3rd (A); 4th (V1); 5th (Ill). Dotted line indicates the progress of hydrolysis with 30 I U lipase/g-substrate.
O0
~ 25 0
0
[ I 5I 10 15 Reactlontime(h)
I 20
Fig. 9. Hydrolysis of 50% palm oil using recycled lipase in an ultrafiltration membrane reactor wlth 40% lipase supplements added. Repetition number; 1st (@); 3rd ((~); 5th (O).
Vol. 65, 1987]
Enzymatic Hydrolysis of Fats
hydrolysis was performed with a 40% supplement of lipase added to the reaction system (Fig. 9). Since the time courses of both the 3rd and 5th reactions were very similar to that of the first, it is confirmed that approximately 60% of the lipase activity was recovered after each reaction. These results indicate that the ultrafihration m e m b r a n e reactor is useful for the hydrolysis of fats in biphasic organic-aqueous systems. Recently, stabilizers for lipase have been developed,7, s) therefore, for recycled lipase, the use of these enzyme-stabilizing agents m a y improve the efficiency of enzyme recovery. Acknowledgments
This research was largely supported by a Grant from the Special Project on Tropical Agricultural Resources, University of Tsukuba.
29 References
1) Blain, J.A., Akhtar, M.W., Patterson, J. D. E.: Pak. J. Biochem., 10, 41 (1976). 2) Kimura, Y., Tanaka, A., Sonomoto, K., Nihira, T., Fukui, S.: Eur. J. Appl. Microbiol. Biotechnol., 17, 107 (1983). 3) Hoq, M. M., Koike, M., Yamane, T., Shimizu, S.: Agric. Biol. Chem., 49, 3171 (1985). 4) Han, D., Rhee, J.S.: Biotechnol. Lett., 7, 651 (1985). 5) Kobayashi, T., Mukataka, S., Kataoka, H., Takahashi, J.: Hakkokogaku, 63, 439 (1985). 6) Mukataka, S., Kobayashi, T., Takahashi, J.: J. Ferment. Technol., 63, 461 (1985). 7) Murakami, S., Funada, T., Ishida, S.: Yukagaku, 32,493 (1983). 8) Murakami, S., Funada, T., Ishida, S.: Yukagaku, 33, 148 (1984). (Received August 8, 1986)
Enzymatic Hydrolysis of Fats at High Substrate Concentrations in Biphasic Organic-Aqueous Systems SuKPKum MmO, TAr~, TETSUO KO~AYASm, S~IGO SATO, and J o j I TAr,~aASHI
Institute of Applied Biochemistry, University of Tsukuba, Sakura-mura, Niihari-gun, Ibaraki 305, Japan The hydrolysis of palm oil and beef tallow by lipase has been studied for practical applications in a biphasic isooctane-aqueous system using a high substrate concentration. The effective lipase concentration for the hydrolysis was found to be about 120 IU per g of substrate. The addition of twenty percent isooctane brought about the most rapid reaction and produced the highest percentage of hydrolysis. For both palm oil and beef tallow, a percentage of hydrolysis higher than 98% was achieved in the 20% isooctane system at a higher concentration of 50%. However, when the substrate concentration was higher than 50 %, the final value of hydrolysis decreased as the concentration of the substrate increased. Utilization of recycled lipase was attempted using an ultrafiltration membrane reactor. Approximately 60% of the lipase activity was recoverable after each reaction.
T h e e n z y m e hydrolysis oflipids has attracted m u c h attention for the industrial production o f fatty acids and glycerol u n d e r n o r m a l temperature a n d pressure. M a n y studies on the e n z y m a t i c hydrolysis of lipids have been carried out using liquid lipids such as olive oil and tributyrin as the substrate.l-4) At the present time, however, beef tallow and p a l m oil, w h i c h are in a solid form at n o r m a l temperature, are the m a i n raw materials for the industrial p r o d u c t i o n of fatty acids and glycerol. I n o u r previous papers,5,e) the advantages o f using lipase in a biphasic organic-aqueous system for the hydrolysis of fats were d e m o n strated, and the kinetics o f the enzymatic hydrolysis in this system were studied. T h e experiments in these studies were performed at low fat concentrations because the effects o f physical a n d chemical factors on the hydrolysis could be determined relatively easily. However, the establishment of a process h a v i n g a high rate of productivity is needed for the practical application of this reaction system. I t is necessary to carry out the reaction at high substrate concentrations, as well as to achieve a high percentage of hydrolysis. Generally, the hydrolysis of solid lipids
(fats) is difficult c o m p a r e d with that of liquid lipids, such as olive oil. I n addition, the fatty acids and glycerol must be of a high quality if they are to be utilized as raw materials for the m a n u f a c t u r e o f cosmetics, paint a n d other products. T h e p u r i t y as well as the rate o f productivity o f fatty acids and glycerol are therefore very i m p o r t a n t . I n the present paper, the hydrolysis o f fats by lipase in a biphasic isooctane-aqueous system was studied at high substrate concentrations, and the o p t i m u m percentage o f isooctane to be a d d e d a n d the a p p r o p r i a t e concentration of the e n z y m e were determined to achieve an effective hydrolysis system where fatty acids a n d glycerol are produced at high productivity a n d purity. Furthermore, the possibility of the recovery and reuse o f the e n z y m e was e x a m i n e d using a m e m b r a n e reactor. Materials and Methods Chemicals The chemicals used for the hydrolysis were virtually the same as those described in a previous paper, 5) except that palm oil was used in addition to beef tallow as a substrate. The specificities and fatty acid compositions of beef tallow and palm oil used in this study are shown in Table 1. Candida cylindracea lipase was used as the enzyme. The organic solvent added to the reaction system was isooctane because
24
MUKATAKA et al.
Table 1. Specificities and fatty acid compositions for beef tallow and palm oil. Beef tallow
Palm oil
AV SV
0. 35 194. 7
0. 18 199. 5
c~
o
Ct4 Ct8 CIs C18 : 1 Cls : ~
(~)
O
[J. Ferment. Technol., ;4
5
6
a
II--II~
o.4 ( ~ )
3.6 26.6 20. 2 43. 2
1.0 40. 4 3.0 42.9
6.4
12.3
AV, Acid value; SV, Saponification value. it was recognized in the previous report as the most suitable solvent for hydrolysis by lipase. Apparatus The reactor consisted of a separable flask having a capacity of 500 ml and an inside diameter of 8 cm. This flask was equipped with four baffle plates. The reaction mixture was agitated by a 6blade flat turbine (5 cm in diameter). An ultrafiltration membrane reactor (Toyo UHP-76) was also used in order to investigate the potentiality for the recovery and reuse (recycling) of the enzyme. The reactor was equipped with a Diaflo-YM5 membrane (molecular weight cut-off 5000, Amicon Co. Ltd.) and it was maintained at 37°C in a water bath (Fig. 1). Hydrolysis Hydrolysis for the determination of the optimum reaction condition was carried out according to the method previously reported, unless otherwise noted. 5) The procedure for the repeated reactions in the membrane reactor was as follows. The reaction mixture was prepared so as to produce a total volume of 150 ml, and the hydrolysis was performed under the prescribed conditions. After each reaction, 50 ml of isooctane was added to the reaction mixture in order to completely dissolve the fatty acids which had been produced. Following this, two phase separations were done. The isooctane phase was recovered using an aspirator. Oltrafiltration of the aqueous phase was performed by increasing the pressure in the reactor to 1.5 arm gauge pressure with nitrogen gas and then relieving the pressure when the residual liquid volume had decreased to 5 ml. The fresh medium, which consisted of fat, isooctane and M/lfi phosphate buffer (pH 7.7) at the predetermined ratio, was then added to the reactor. Analysis To monitor the percentage of hydrolysis of the substrate, the fatty acids produced were titrated with 0.5 N KOH. The percentage of hydrolysis was calculated on the basis of the saponification values of the substrates.
Fig. 1. Schematic diagram of the tltrafiltration membrane reactor system. 1, Nitrogen gas cylinder; 2, Water bath; 3, Drain; 4, Membrane; 5, Valve; 6, Sampling port; 7, Reaction mixture; 8, Magnetic stirrer; 9, Speed controll. The final hydrolysis value was also confirmed by determining the amount of residual glycerides in the reaction mixture. The amount of residual glycerides was measured by thin-layer chromatography (TLC) using a silica gel plate (Type 60, Merck). The composition of the developing solvent was hexane : ethyl ether : acetic acid (80 : 30 : 1). The spots on the plate were visualized by spraying 50% sulfuric acid and heating at l l0°C for 5 rain. The charred spots were analyzed with a densitometer (Asuka Kogyo, Model OZ-802) and estimated quantitatively from the calibration curve for which cholesteryl palmitate was used as the authentic material.
Results and Discussion Proper hydrolysis
enzyme
concentration
for
In enzyme reactions, the rate of reaction greatly depends on the enzyme concentration. T h u s , to d e t e r m i n e a o p t i m a l l i p a s e c o n c e n t r a t i o n f o r t h e h y d r o l y s i s o f fats at a high substrate concentration the hydrolyses o f 5 0 % ( w / v ) p a l m o i l a n d b e e f t a l l o w were carried out at various enzyme conc e n t r a t i o n s (Figs. 2 a n d 3). Figure 2 illustrates the time course of the hydrolysis of 50% palm oil at different enzyme concentrations. Palm oil was hydrolyzed almost completely, even when the l i p a s e c o n c e n t r a t i o n w a s 15 I U p e r g o f substrate, although a very long reaction time (24 h) w a s n e c e s s a r y for c o m p l e t e h y d r o l y s i s . Hydrolysis time decreased with an increase in the enzyme concentration. At an enzyme c o n c e n t r a t i o n o f 120 I U p e r 1 g o f s u b s t r a t e , 95% of the palm oil was hydrolyzed after 4 h
Vol. 65, 1987]
Enzymatic Hydrolysisof Fats
25
~" 75
i 50
~o 50 ~
0[ 0
I 6
I
12
I 18
I 2q
I
R e a c t l o n time (h) Fig. 2. Effects of enzyme concentration on the hydrolysis of 50% palm oil in the 20% isooctaneaqueous system. Lipase (IU/g-substrate): 120 (O); 60 (A); 30 (O);
15 ([]). and the value approached 100% after about 6h. The results for the hydrolysis of 50% beef tallow are presented in Fig. 3. The hydrolysis rate of beef tallow was slower than that for palm oil. The percentage of hydrolysis at 2 4 h was only 92%, even at an enzyme concentration of 30 IU/g-substrate, while at 60 IU/g-substrate and 24 h, the percentage of hydrolysis reached 98%. A percentage hydrolysis of 98% was achieved in 12 h when the enzyme was at a concentration of 120 I U per 1 g of substrate. Based on these results, it is evident that the rate of hydrolysis increased with an increase in the enzyme concentration. However, judging from the time courses for the hydrolyses of both palm oil and beef tallow (Figs. 2 and 3), a significant increase cannot be expected, even if the enzyme concentration is increased to greater than 120 IU/g-substrate. Furthermore, the use of lipase above this concentration is not economical. Thus, it is considered that a lipase concentration of about 120 IU/gsubstrate is the most suitable for the hydrolysis of palm oil and beef tallow. O p t i m u m percentage of added isooctane It was demonstrated in a previous paperS) that the appropriate percentage of isooctane to be added for the hydrolysis of 12% beef tallow or palm oii was within the
25 0
0
6
12
18
Reaction time (h)
Fig. 3. Effects of enzyme concentration on the hydrolysis of 50% beef tallow in the 20% isooctaneaqueous system.
Lipase (IU/g-substrate): 120 (O); 60 (A); 30 (El). range of 10 to 20%. Therefore, a 20% isooctane system was adopted for determining the most suitable enzyme concentration. It may be considered, however, that the optimum percentage of isooctane changes with the concentration of the substrate. T o identify the optimum reaction system, the hydrolyses of 50%(w/v) palm oil and beef tallow were performed under various percentages of added isooctane and at a lipase concentration of 120 IU/g-substrate. Figure 4 shows the results for palm oil. The hydrolysis of palm oil was facilitated by the addition ofisooctane which was within
i00 ~
--
-
•
Ily .,<-..-
0
6
12 18 214 Re0cti0n time (h) Fig. 4. Effects of the isooctane fraction on the hydrolysis 50% palm oil. Lipase: 120 IU/g-substrate, Isooctane (%) ; 0 (O) ; 10 (A); 20 (O); 45 (~); 65 (A); 85 (c-l). The isooctane fraction is represented by the volume percent in the reaction mixture without substrate.
26
MLrKATAKAet
the range of I0 to 45%(v/v). In particular, the 20% isooetane system produced the most rapid reaction and gave the highest percentage of hydrolysis. Similar results were also obtained for the hydrolysis of beef tallow (data are abbreviated). Based on these results, it is concluded that the optimum percentage of isooctane to be added is 20%(v/v) for the hydrolysis of 50% palm oil and beef tallow. (Proviso: percentage of isooctane is expressed by the volume percent in the mixture without substrate.) Final percentage of hydrolysis at high substrate concentrations An almost
complete hydrolysis of the substrate in a 20% isooctane system was observed for both the hydrolyses of 50% palm oil and of beef tallow. However, it is essential to increase the substrate concentration in order to improve the production of fatty acids and glycerol. At the same time, a high level of conversion of the substrate into its products is also required from a practical application standpoint. Therefore, hydrolysis at a substrate concentration higher than 50% was carried out in order to investigate its applicability. The results of the hydrolysis of 60 and 70% palm oil are shown in Fig. 5-a, b. These hydrolyses were carried out in three systems, I0, 20 and
lOO [-
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{a) 60 Z(W/V) i l I I
I
°
75 50
(b) 70 %(W/V) I
6
I
I
12 18 Reoctlon time (h)
t I
24
Fig. 5-(a), (b). Hydrolysisof 60 and 70% palm oil with varing percentages of isooctane added. Lipase: 120 IU/g-substrate, Isooctane (%): I0 (@) ; 20 (O) ; 40 (A).
[J. Ferment. Technol.,
al.
40% isooctane respectively. For both substrate concentrations of 60 and 70%, the 20% isooctane system produced the most rapid reaction and the highest percentage of hydrolysis, as in the case of hydrolysis at a 50% substrate concentration. In this system, the final percentages of hydrolysis were 96 and 90% at substrate concentrations of 60 and 70%, respectively. Hydrolyses were also performed with higher enzyme concentrations of 200 and 300 IU/g-substrate, however, the final percentages of hydrolysis were not further improved by increasing the enzyme concentration (Fig. 6-a, b). Hydrolysis at various substrate concentrations was also performed with beef tallow. The final values obtained for the hydrolyses of beef tallow and palm oil are summarized in Table 2. The results for the hydrolysis of beef tallow were founded to be inferior to those for palm oil. These results suggest that it is difficult to exceed a hydrolysis value of 98% at a substrate concentration higher than 50%. T h a t is, the upper limit of the substrate concentration, at which 98% hydrolysis was obtained, was 50% for both palm oil and beef tallow. Residual glycerides Fatty acids and glycerol produced by the hydrolysis of fats and oil have numerous uses as industrial materials, and in many cases, a high quality is required for the manufacture of other products, such as cosmetics. In biphasic isooctane-aqueous reaction systems, both beef Table 2. Final values for the hydrolysisof palm oil and beef tallow at various substrate concentrations. Substrate % (w/v)
Degree of hydrolysis (%) Palm oil Beef tallow
40
--
99
50
99
98
60
96
94
70
90
--
Isooctane: 20% (v/v)
Lipase: 120 IU/g-substrate
Voi. 65, 1987]
Enzymatic Hydrolysis of Fats
1
W/V) 50 o
[]
t
27
I
100
~ 7s 50
(b) 70 %(W/V) I
6
I
!
12 18 Renctlon time (h)
I 24
Fig. 6-(a), (b). Hydrolysis of 60 and 70% palm oil in 20% isooctane-aqueous systems with various lipase concentrations. Lipase (IU/g-substrate): 100 (0); 200 (A); 300 (O). tallow and p a l m oil were hydrolyzed above 98% by lipase at a substrate concentration of 50%. T h e final percentage of hydrolysis strongly influences the quality of the products. In our experiments, the time course of hydrolysis and its final percentages were determined based on the amount of fatty acids produced. Therefore, to confirm the findings, residual glycerides were analyzed by thin-layer chromatography. Figure 7 shows the thin-layer chromatog r a m of four samples of the reaction mixture for the hydrolysis of 50% p a l m oil. The first sample was a mixture prepared before the reaction, the second sample was a reaction mixture to which isooctane had not been added, while the third and fourth samples included the 20 and 65°/0 isooctane systems, respectively. T h e second, third and fourth samples were prepared after the reactions and final values of 95, 99, and 90% were estimated, respectively, from the titration of the fatty acids produced in each hydrolysis. T h e amount of glycerides in each sample was determined by a densitometer. In the hydrolysis of triglycerides b y C. cylindracea lipase, diglycerides and monoglycerides formed during the intermediate stages of the reaction are also hydrolyzed to
Fig. 7. Thin layer chromatograms of the reaction mixture in various hydrolysis. 1, sample before reaction; 2-4, samples after reaction (2, no isooctane; 3, 20% isooctane; 4, 65% isooctane). A, Sterol ester (internal standard) ; B, Trlglyceride; C, Fatty acid; D, Di- and monoglycerides; E, Origin. fatty acids and glycerol. Although the T L C analysis using densitometer showed traces of di- and monoglycerides, the exact amounts of these compounds could not be quantified for the samples prepared after the reactions had taken place (2, 3 and 4). Therefore, in Table 3, only the results of the determination of the spots for triglycerides on the T L C plates are presented. These values are in agreement with those estimated from the final Table 3. Amount of residual triglycerides in the reaction mixtures determined by TLC-densitometry. Isooctane
Residual triglycerldes
(%)*
(%)
0
3.3
20
0.7
65
7.2
Palm oil: 50% (w/v) Lipase: 120 IU/g-substrate * These values are expressed as volume percent in the reaction mixture without substrate.
MUKATAKAet al.
28
values of hydrolysis. For the hydrolysis of 50% palm oil in the 20% isooctane system, particularly, the very high value of 99.3% was also confirmed based on the amount of the residual glycerides. Repeated use o f llpase by u s i n g a m e m b r a n e reactor For the industrial application of enzymatic reaction systems, recovery and reuse (recycling) of the enzyme are essential cost reduction measures. In biphasic organic-aqueous reaction systems, the substrate (fat) is dissolved in the organic solvent phase while the lipase is dissolved in the aqueous phase. The fatty acids and glycerol produced by the hydrolysis of fat are also dissolved in the organic and aqueous phases, respectively. After the reaction, therefore, the fatty acids and the remaining substrate can be removed from the reactor by withdrawing the organic solvent. Furthermore, the glycerol dissolved in the aqueous phase is a low molecular weight substance. So, taking these fact into consideration, the use ofultrafiltration membrane is an effectual method for the recovery of the enzyme used in a biphasic organic-aqueous system. When the substrate concentration is high, the reaction mixture becomes viscous making |
i00 F 2~
~
-
,~
[J. Ferment. Technol.,
the separation of the two layers (aqueous and organic phases) rather lengthy. In our experiments, 50 ml of isooctane were added to the 150 ml reaction mixture at the end of the reaction in order to completely dissolve the fatty acids, and also to accelerate the separation into the two phases. The reaction mixture separated into two distinct phases within one hour. Figure 8 shows the time course of the hydrolysis of 50% palm oil using recycled lipase. Lipase was added to the reaction system (120 IU/g-substrate) only at the start of the first run. Hydrolysis in each run was performed at the optimal value of 20% isooctane. For comparison, the broken line in Fig. 8 indicates the time cource of hydrolysis at a lipase concentration of 30 IU/gsubstrate. Although the degree of hydrolysis attained was almost 100% for each run, the reaction rate decreased with successive repetitions of the reaction. This implies a gradual reduction in the lipase activity during the processes of reaction and recovery. The residual lipase activity in each reaction was estimated by comparing with the time courses of the hydrolyses shown in Fig. 8 and in Fig. 2. It was roughly estimated that 60% of the lipase activity was retained in each reaction. In order to confirm this, each repeated
|
7 5 ~ 1
~1°°
50
.~ 75
I O0|
}50 i
6
l
I
12 18 Reaction time (h)
l
24
Fig. 8. Repeated use of lipase for the hydrolysis of 50% palm oil in an ultrafiltration m e m b r a n e reactor. Lipase was added to the reaction system at 120 I U per g of substrate only at the start of the first run. Repetition number: 1st (@); 2nd (O); 3rd (A); 4th (V1); 5th (Ill). Dotted line indicates the progress of hydrolysis with 30 I U lipase/g-substrate.
O0
~ 25 0
0
[ I 5I 10 15 Reactlontime(h)
I 20
Fig. 9. Hydrolysis of 50% palm oil using recycled lipase in an ultrafiltration membrane reactor wlth 40% lipase supplements added. Repetition number; 1st (@); 3rd ((~); 5th (O).
Vol. 65, 1987]
Enzymatic Hydrolysis of Fats
hydrolysis was performed with a 40% supplement of lipase added to the reaction system (Fig. 9). Since the time courses of both the 3rd and 5th reactions were very similar to that of the first, it is confirmed that approximately 60% of the lipase activity was recovered after each reaction. These results indicate that the ultrafihration m e m b r a n e reactor is useful for the hydrolysis of fats in biphasic organic-aqueous systems. Recently, stabilizers for lipase have been developed,7, s) therefore, for recycled lipase, the use of these enzyme-stabilizing agents m a y improve the efficiency of enzyme recovery. Acknowledgments
This research was largely supported by a Grant from the Special Project on Tropical Agricultural Resources, University of Tsukuba.
29 References
1) Blain, J.A., Akhtar, M.W., Patterson, J. D. E.: Pak. J. Biochem., 10, 41 (1976). 2) Kimura, Y., Tanaka, A., Sonomoto, K., Nihira, T., Fukui, S.: Eur. J. Appl. Microbiol. Biotechnol., 17, 107 (1983). 3) Hoq, M. M., Koike, M., Yamane, T., Shimizu, S.: Agric. Biol. Chem., 49, 3171 (1985). 4) Han, D., Rhee, J.S.: Biotechnol. Lett., 7, 651 (1985). 5) Kobayashi, T., Mukataka, S., Kataoka, H., Takahashi, J.: Hakkokogaku, 63, 439 (1985). 6) Mukataka, S., Kobayashi, T., Takahashi, J.: J. Ferment. Technol., 63, 461 (1985). 7) Murakami, S., Funada, T., Ishida, S.: Yukagaku, 32,493 (1983). 8) Murakami, S., Funada, T., Ishida, S.: Yukagaku, 33, 148 (1984). (Received August 8, 1986)