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Utilization of powdered pig bone as a support for immobilization of lipase
JOURNALOF FERMENTATIONAND BIOENGINEERING Vol. 67, No. 5, 350-355. 1989
Utilization of Powdered Pig Bone as a Support for Immobilization of Lipase S A T O S H I N E G I S H I , * S E I G O S A T O , S U K E K U N I M U K A T A K A , ASP J O J I T A K A H A S H I
Institute of Applied Biochemistry, University of Tsukuba, Tsukuba-shi, lbaraki 305, Japan Received 12 December 1988/Accepted 4 March 1989 Pig bone was examined for its suitability as a support material for iipase immobilization. It was observed that pig bone (PB) particles dispersed readily in both polar and nonpolar solvents, and lipase was easily adsorbed. In particular lipase adsorbed on olive oil-soaked pig bone (OPB) particles exhibited a higher hydrolytic activity than that in lipase adsorbed on a selection of other representative supports, regardless of removing the presoaked olive oil from the particles after immobilization of lipase. The optimum pH and temperature for hydrolytic activity of OPB-adsorbed lipase were the same as those for free lipase, although thermal resistance was increased by immobilization. When OPB-adsorbed lipase was used for repeated batch reactions of olive oil hydrolysis, an activity of more than 80% of the initial activity of each run could be retained after 46 b reaction. The results suggest that PB is an excellent support material.
I m m o b i l i z a t i o n of enzymes is essential for application to industrial reaction processes. Immobilized enzymes allow easy separation from reaction mixtures as well as reuse o f the enzymes. Lipase is one o f the most extensively investigated enzymes due to its ability in fat splitting, esterizing and transesterizing. It has shown promise in such diverse fields as chemicals, foods, medicines and cosmetics, and many attempts have therefore been made to immobilize lipase (1-5). Representative methods p r o p o s e d for lipase immobilization include gel-entrapping using photo-crosslinkable resin (6) and a d s o r p t i o n on membranes (7-9) or resins (10). However, until now, almost all the supports used for lipase i m m o b i l i z a t i o n have been artificial, and the hydrolytic activities o f lipase immobilized on such supports have not proved adequate for industrial application. We consider that safety is a very i m p o r t a n t factor in applying immobilized enzymes, especially lipase, in industry, and that bio-substances are the safest materials for enzyme support. A n i m a l bone, which is generally discarded after the meat has been removed, is untoxic, mechanically strong and porous. It is composed of about 6 0 ~ organic and 40%0 inorganic substances. Most o f its organic c o m p o nent is collagen which contains a large number o f potential binding sites for enzyme attachment, while its inorganic c o m p o n e n t is close to h y d r o x y a p a t i t e (11). For such reasons, bone presents itself as having great potential as a support material for enzyme immobilization. This paper describes the utilization of waste animal bone, specifically pig bone, for the immobilization of lipase. The findings show pig bone to be an excellent enzyme support.
tein), were provided from Meito Sangyo Co. Ltd. In addition to pig bone particles, the following c o m m o n enzyme supports were tested for the immobilization o f lipase: Amberlite IRA-94 (Rohm & Haase), Dowex NWA-1 (Dow Chemical), Diaion HP-21 (Mitsubishi Chemical Industries), Chitopearl BCW-3001 (Fuji Spinning) and Celite R-630 (Johns Manville). A n immobilized lipase preparation named Lipozyme was also purchased from Novo Industries. Olive oil (saponification value, 195) was purchased from Yuro Yakuhin Co. Ltd. All other chemicals were o f reagent grade and obtained from commercial sources. Preparation of PB (Pig Bone) and OPB (Olive oil-soaked Pig Bone) particles PB and OPB particles were obtained from a pig thighbone by the following procedure. The thighbone was cut at both ends and cleaned. To remove fat, the bone was autoclaved at 120°C for 20 rain, and then ground into small particles. Particles ranging from 37 to 105/lm were selected by sifting. These were washed five times with ethanol and distilled water, and designated as PB support. The PB particles were soaked in olive oil for more than 12 h before use, and labelled OPB. Immobilization of lipase Ten grams o f support were added to 100ml o f 0.05 M phosphate buffer (pH 7.0) to which 5 g o f lipase powder had been dissolved. The mixture was shaken for 1 h at 0°C. These conditions for immobilization were determined by preliminary experiments as will be described later. The supports were separated from the mixture by filtration, and washed with a phosphate buffer until no more protein was released. In the case of OPB particles, further washing was performed with isooctane 3 times to wash out the olive oil. Assay of lipase activity The hydrolytic activities of the free and immobilized lipases were measured by the method o f F u k u m o t o et al. (12). The reaction mixture consisted o f 10 g lipase powder or 1-5 g o f immobilized lipase, 40 g o f olive oil and 300 ml of 0.05 M phosphate buffer (pH 7.7). The reactor consisted o f a separable flask with a capacity of 500 ml and inside diameter 8 cm. This flask was equipped with 4 baffle plates. The reaction mixture was agitated by a 6-blade flat turbine (5 cm in diameter). The reaction was carried out at 37°C for 3 0 m i n stirring at 500 rpm. One unit of hydrolytic activity was defined as the
MATERIALS AND METHODS E n z y m e and chemicals Lipase produced by Candida cylindracea was used t h r o u g h o u t the experiments. Two kinds o f lipase in powder form having different hydrolytic activities, Lipase-MY (14 U / r a g - p o w d e r , 140 U / r a g - p r o tein) and Lipase-OF (220 U / m g - p o w d e r , 2200 U / m g - p r o -
* Corresponding author. 350
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IMMOBILIZATION OF LIPASE
activity which would release one micromole o f free fatty acid in one minute.
Hydrolysis of olive oil and repeated use of immobilized lipase The reactor for the enzyme reaction was the same as that used for the activity assay. The reaction mixture consisted o f 35 g of olive oil, 300ml o f 0.05 M p h o s p h a t e buffer (pH 7.7) and 30 U / g - s u b s t r a t e o f free or immobilized lipase. Experimental conditions for the repeated batch reaction were the same as those described above. After 46 h had elapsed in each run, the immobilized lipase was recovered by filtration, and washed 3 times with the buffer solution and isooctane. The residual activity was then measured in accordance with the method mentioned above. Analysis Protein concentration was assayed by the
351
method o f Lowry et al. (13). The fatty acids p r o d u c e d in the reaction were titrated with 0.2 N or 0.025 N K O H using a p H meter as indicator. RESULTS AND DISCUSSION
Properties of pig bone(PB) particles The PB particles taken from pig thighbone proved to be a useful support material having the following properties. Its specific gravity was 1.7 g / c m 3 and it was observed that the PB particles dispersed well both in polar and n o n p o l a r solvents. These characteristics present a very i m p o r t a n t advantage in use as a support material for the immobilization o f lipase, because lipase is used not only for the hydrolysis of fats and oil in aqueous or aqueous-organic systems but also for
FIG. 1. Scanning electron micrographs of pig bone particles.
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NEGISHI ET AL.
J. FERMENT. BIOENG.,
esterification and interesterification in microaqueous systems. Figure 1 shows electron microscopic photographs of PB particles. The particles have irregular shapes and high porosity. It has been reported that the surface area of bone is more than 100m 2 per gram of inorganic component (11). This porosity is an additional advantage for the immobilization of enzymes. Optimum conditions for immobilization In order to obtain an immobilized lipase with high activity, the optimum conditions for immobilization were investigated using olive oil-soaked pig bone particles. To ascertain the time required for sufficient adsorption, lipase was kept in contact with OPB particles for various lengths of time at 0°C. After 30 min, 65% of maximum binding had occurred and the maximum amount of adsorbed enzyme was obtained after 60min. However, prolongation of the contact time beyond 60 min produced no increase in adsorption. Temperature had little influence on the adsorption of lipase on OPB particles. The activity of immobilized lipase obtained at 20°C was 85% of that obtained at 0°C. Optimum pH for immobilization was 7.0, although the free lipase showed its highest hydrolytic activity at pH 7.7. Immobilization at pH 6.0 and 8.0 gave 85%o and 90%o of the maximum activity obtained at pH 7.0, respectively. Enzyme concentration in an incubated solution for immobilization is also one of the factors which affects the amount of enzyme adsorbed on the supports. The effect of varying the lipase concentration is shown in Fig. 2. The incubation was performed at 0°C for 1 h. It is clear that the amount of adsorbed lipases reached a maximum at a concentration of 5 mg-protein/ml, although this figure is represented by the relative activity of immobilized lipase obtained after washing. TABLE
1.
Size
Support
-
8
.
0
-
~
-
1
O
8O v>-,6 0 >
4o
•
20 ¢Y
0
4.'0
0 Enzyme
61o
¢o¢~. (mg-prol. / ml)
F I G . 2. Effect o f e n z y m e c o n c e n t r a t i o n in i n c u b a t i o n s o l u t i o n o n i m m o b i l i z a t i o n o f lipase. T h e m i x t u r e c o n s i s t e d o f 10 g o f O P B a n d 100 ml o f e n z y m e s o l u t i o n , s h a k e n f o r 1 h at 0 ° C .
As a result of this investigation on the conditions for immobilization, we obtained lipase immobilized on OPB particles with a hydrolytic activity as high as 700-1000 U/g-dry support using crude lipase powder, Lipase-MY. Hydrolytic activity of lipase immobilized on various supports To compare the hydrolytic activity of lipase adsorbed on pig bone with those of other immobilized lipases, two lipases were immobilized on various representative supports. The immobilization was performed without any crosslinking agent for either kind of lipase powder. The hydrolytic activities of the two lipase powders (LipaseMY and Lipase-OF) were measured as 140 and 2200 U/rag-protein, respectively, by an agitation method (12). The results obtained using these lipase powders are presented in Tables 1 and 2. The amounts of proteins adsorbed on Celite, Amberlite
Immobilization of Lipase-MY on various supports Adsorbed protein (mg/g-support)
(,urn) Free enzyme Celite IRA-94 MWA- 1 HP-21 C h i t o p e a r l 3001 PB OPB L i p o z y m e ,~
O
100
-
8.5~9.0 t 0 - 14 2 . 0 - 2.5 4.0~4.5 15 ~ 18 0.7-0.9 5.0-8.0
0
400 ~ 500 290 ~ 830 210-600 100 37 4 105 3 7 ~ 105 300 4 600
Specific activity (U/mg-prot.)
Relative activity (%)
140 1.5 4 1.9 2.9 ~ 3.5 11 ~ 15 2.943.5 3 . 5 - 5.6 29435 1 1 0 ~ 140
100 1.1-1.4 2.1 ~ 2.5 7.1 4 11 2.1 - 2 . 5 2.5-4.0 21 ~ 2 5 8 0 - 100
-
-
-
-
Activity (U/g-support) -
-
10-15 35 ~ 40 15 ~ 20 1 0 - 15 5 0 - 60 2 5 - 30 7 0 0 - 1000 1 0 0 - 120
I m m o b i l i z e d lipase o b t a i n e d c o m m e r c i a l l y . P B , P i g b o n e p o w d e r ; O P B , olive o i l - s o a k e d pig b o n e p o w d e r .
TABLE Support Free enzyme Celite IRA-94 MWA-1 HP-21 C h i t o p e a r l 3001 PB OPB
2.
Immobilization of Lipase-OF on various supports
Adsorbed protein (mg/g-support)
Specific activity (U/mg-prot.)
Relative activity (0/0)
Activity (U/g-support)
-3.0~4.0 15~20 4 . 0 ~ 6.0 1 5 ~ 17 5.0 4 7.0 1.3 ~ 1.6 4.0 ~ 6.0
2200 31-34 11 - 17 31 - 3 6 4.4-6.6 66 ~ 88 330 - 440 330 - 440
100 1 . 4 ~ 1.6 0.5 4 0 . 8 1 . 4 ~ 1.7 0.2~0.3 3.0 4 4.0 15 - 20 15 - 20
-1 0 0 ~ 140 2 0 0 - 250 1 5 0 - 180 7 0 - 100 400-600 400 - 500 1500 - 2000
P B , P i g b o n e p o w d e r ; O P B , olive o i l - s o a k e d pig b o n e p o w d e r .
VOL, 67, 1989
I M M O B I L I Z A T I O N OF LIPASE TABLE 3.
IRA-94 and Chitopearl BCW-3001 respectively were larger than those of proteins adsorbed on PB and OPB particles, as can be seen in Table 1. However, the specific activities of proteins adsorbed on PB and OPB particles were remarkably higher than those on the other supports. In particular, the activity of proteins immobilized on OPB particles amounted to 80-100% of that of the free enzyme, in contrast to the relative activities of most proteins adsorbed on other supports which were less than 10%. Consequently OPB particles showed the highest activity per unit gram of support, 700-1000 U/g-support. This was 20-30 times higher thatn the activity of lipase adsorbed on other supports, and also about 7-8 times higher than that of Lipozyme, which is commercially sold immobilized lipase. As to the reason for obtaining immobilized lipase with such a high activity by using OPB particles, it was considered that OPB particles could adsorb lipase selectively from various proteins probably contained in lipase powder, because the OPB particles were presoaked in olive oil. This conjecture is supported by the data shown in Table 2. By using lipase powder with a 15-fold higher activity, every support showed higher activity in immobilized lipase. The activity of all other immobilized lipase except for OPB particles was 15-20 times that shown in Table 1 obtained using crude powder Lipase-MY. However, in the case of OPB particles, the increase of immobilized lipase activity was only 2-3 times, although this was still higher compared with others. This fact supported the idea that lipase proteins had been already adsorbed on OPB particles selectively, when the crude lipase powder was used for immobilization. The effect of olive oil presoaking of the support on lipase immobilization was tested using some other supports. The results are shown in Table 3. In Exp. A, where Lipase-MY was used, the lipase activity immobilized on each support increased ten or more times compared to that obtained with non olive oil-soaked supports. On the other hand, in Exp. B where Lipase-OF was used, the extent of increase in immobilized lipase activity of each support was not so significant as in the case of pig bone, although the effect of presoaking in olive oil was still recognized. This is because the selective adsorption effect was not sufficiently displayed owing to the use of powder with a high lipase content. From Table 3, however, it is worth noting that pig bone shows its greatest effect with presoaking in olive oil. As previously mentioned, big bone is quite porous and disperses well in both the aqueous and olive oil phases. Therefore, it absorbs a higher quantity of olive oil corn-
353
Effect of olive oil presoaking on immobilization of lipase Activity (U/g-support)
Exp.
Support
A
Celite MWA-1 HP-21 PB
180120140 4 700 -
200 150 170 1000
Celite MWA-1 HP-21 PB
180 ~ 400 ~ 400~ 1500 ~
200 500 500 2000
olive oil-soaked
non-soaked 10~ 15 15 - 2 0 10~ 15 25-30 100 4 150 4 70400-
140 180 100 500
Lipase-MY was employed (activity: 140 U/mg-prot.) in Exp. A and Lipase-OF (activity: 2200 U/mg-prot.) in Exp. B.
pared with other support materials. Besides, pig bone contains collagen which has a large number of potential binding sites for enzyme attachment. It can be supposed that these properties of pig bone are most advantageous in displaying selective adsorption and immobilizing lipase with the highest activity. It is also important to note that lipase was immobilized and remained in the pig bone itself, but not in the olive oil. Presoaked olive oil and its products, i.e. fatty acids and glycerol, which might be produced by immobilized lipase, were almost completely removed after immobilization by washing with buffer solution and isooctane. Therefore, only the lipases adsorbed solely on olive oil were washed away with it. Elimination of olive oil and the products was confirmed by two methods. The weight of pig bone increased about 35%0 after soaking in olive oil. However, it returned to its original weight by washing after the immobilization of lipase. Furthermore, we tried to extract the olive oil and fatty acids from the support after washing with buffer solution and isooctane by the methods of Folch et al. (14). No triglyceride and fatty acids could be discovered by TLC analysis. The effect of presoaking in other chemicals such as oleic acid, glycerol and poly vinyl alcohol (PVA) was examined. Although glycerol and PVA gave rather undesirable results, oleic acid showed a selective adsorption effect similar to olive oil. However, the activity of the immobilized lipase obtained was lower than that of OPB. From the results of various tests on the immobilization of lipase using pig bone particles, it was found that olive oil-presoaked pig bone (OPB) gave a hydrolytic activity which was higher than the activities of conventionally used
100
100
_8O >,60 .>__
~60
.~
N 20 a: 0
5
I
I
6
7
pH
Q: 0
8
•
20
i
30 40 50 60 Temperature ( *C )
FIG. 3. Comparison of pH and temperature dependence on hydrolytic activity of OPB-adsorbed lipase with those of free lipase. O, OPB-adsorbed lipase; zx, free lipase.
Symbols:
354
NEGISHI ET AL.
J. FERMENT. BIOEN(i.,
TABLE 4.
100
Run
;e .-. 8 0
Initial act. 1 2 3
.~
60
Residual activity after each ruw' (U/g-support) (%) 740 620 550 370
100 84 74 50
~ Each run was carried out for 46 h. Reaction mixture: olive oil, 35 g; buffer solution, 300 ml. Reaction conditions: agitator speed, 500 rpm; temperature, 37°(?.
~40 "6
20 0
Repeated use of lipase immobilized on OPB
I
o
,2
2;.
3;
48
Reaction time ( h ) FIG. 4. Comparison of time course of olive oil hydrolysis by OPB-adsorbed lipase with that by free lipase. The reaction mixture consisted of 35 g of olive oil and 300 ml of 0.05 M phosphate buffer. The reaction was carried out under optimum pH and temperature (7.7, 37°C). Symbols are the same as in Fig. 3.
supports. However, the problem concerning the activity yield of lipase still remains. The recovery yield of activity against the total lipase used for immobilization is still low, i.e., 15°/00and 2O//oofor Lipase-MY and Lipase-OF, respectively, under the conditions where the highest activity was obtained. Although, these values were increased to 70% (Lipase-MY) and 15% (Lipase-OF) by reducing the ratio of the enzyme solution to the supports to one-tenth that of the former case, the activities of immobilized lipase obtained decreased by 50O/oo and 25%, respectively. In the case of the adsorption method, however, the lipases which are not adsorbed on the supports can be reused by separating the supports from the enzyme solution after immobilization. Therefore, the activity yield as a whole can be improved by reusing the u n a d s o r b e d lipases, even if the yield value is low in one immobilization run. Effect o f p H and temperature on the activity o f lipase i m m o b i l i z e d on O P B The pH and temperature dependences of the activities of free and OPB-adsorbed lipase are shown in Fig. 3. The o p t i m u m pH and temperature of OPB-adsorbed lipase for displaying its hydrolytic activity were the same as those of free lipase. However, the thermal resistance was increased by immobilization. In contrast to free lipase, the activity of which was drastically reduced when the reaction temperature was raised to over 37°C, the OPB-adsorbed lipase showed 90% of the m a x i m u m activity at 45°C and remained 50%0 even at 60°C. It is considered that this increase in thermal resistance was due to conformational stabilization by immobilization. Hydrolysis o f olive oil and reuse of OPB-adsorbed iipase For comparison with hydrolysis by free lipase, olive oil was hydrolysed using OPB-adsorbed lipase. The olive oil concentration was 10°4 (w/v), and free or immobilized lipase was added in proportion with the activity of 30 U/g-oil. The progress of the hydrolyses is shown in Fig. 4. Although the lipase was equally added to both reaction systems, hydrolysis proceeded faster in the case of the OPB-adsorbed lipase. The OPB-adsorbed lipase dispersed well not only in the aqueous but also in the oil phase. In contrast to this, as free lipase disperses only in water, OPB-adsorbd lipase is superior for the hydrolysis of olive
oil. The results of repeated use of OPB-adsorbed lipase are shown in Table 4. Repeated batch reactions were carried out at an olive oil concentration of 10% (w/v) and initial lipase concentration of 30 U/g-oil. Each run was carried out for 46 h, and residual activity after each run was determined from the initial progress curve of hydrolysis in next run. Following the method discribed in 'Materials and Methods', immobilized lipase was washed with buffer solution and isooctane after each run to eliminate the remaining substrate and products. The activity of OPB-adsorbed lipase gradually decreased with each run. However, 8085% of the initial activity of each run was retained after the reaction and carried over to the next run. This reduction of activity included loss both in recovery and in washing, because the activity was measured after these operations. In the repeated batch experiments, although one batch reaction was continued for 46 h, the reaction time could be shortened by adding a higher quantity of immobilized lipase to the reaction system. In such a case, the activity loss of immobilized lipase in each run would be further reduced. Batch reaction is a suitable method for establishing a high extent of hydrolysis in a short period. Since the activity loss of OPB-adsorbed lipase in one batch operation is less than 20%, repeated batch hydrolyses can be performed with a small quantity of lipase supplement. In this paper, the utilization of waste pig bone for the immobilization of lipase is shown, with an application to the hydrolysis of olive oil. Although palm oil and beef tallow which are in solid form at the optimum temperature for hydrolytic activity of many lipases, are mainly utilized as raw materials for the industrial production of fatty acids and glycerol, we found in a previous study (15-17) that the hydrolysis of such solid lipids by lipase could be performed in the same way as that of liquid lipids by addition of a suitable organic solvent. Therefore, it may be possible to apply OPB-adsorbed lipase to hydrolyse industrial raw materials in the presence of an organic solvent. However, further investigation is necessary to develop industrial applications of OPB-adsorbed lipase.
ACKNOWLEI)GMENT The authors are grateful to Meito Sangyo Co. Ltd. for providing the lipase. REFERENCE
1. Cambou, B. and Klibanov, M.A.: Comparison of different strategies for the lipase-catalyzed preparative resolution of racemic acid and alcohol: asymmetric hydrolysis, esterification and transesterification. Biotechnol. Bioeng., 26, 1449-1454 (1984). 2. Lavaryre, J., Verrier, J., and Baratti, J.: Stereospecific
VoL. 67, 1989
3. 4.
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7.
8. 9.
hydrolysis by soluble and immobilized lipase. Biotechnol. Bioeng., 24, 2175-2187 (1982). Celebi, S.S., Uear, T., and Caglar, A.M.: The effect of solvents on the hydrolysis of lipids by free and immobilized microbial lipase. Adv. Biotechnol., 1, 691-697 (1981). Wisdom, A.R., Dunnill, P., and Lilly, D.M.: Enzymic interesterification of fats: factors influencing the choice of support for immobilized lipase. Enzyme Microb. Technol., 6, 443-446 (1984). Taylor, F., Panzer, C.C., Craig, C.J.Jr., and O'Brien, J.D.: Continuous hydrolysis of tallow with immobilized lipase in a microporous membrane. Biotechnol. Bioeng., 28, 1318-1322 (1986). Kimura, Y., Tanaka, A., Sonomoto, K., Nihira, T., and Fnkui, S.: Application of immobilized lipase to hydrolysis of triacylglyceride. Eur. J. Appl. Microbiol. Biotechnol., 17 107112 (1983). Hoq, M.M., Koike, M., Yamane, T., and Shimizu, S.: Continuous hydrolysis of olive oil by lipase in microporous hydrophobic hollow fiber bioreactor. Agric. Biol. Chem., 49, 3171-3178 (1985). Yamane, T., Hoq, M.M., and Shimizu, S.: Kinetics of continuous hydrolysis of olive oil by lipase in microporous hydrophobic membrane bioreactor. Yukagaku, 35, 10-17 (1986). Hoq, M.M., ¥amane, T., and Shimizu, S.: Role of oleic acid solubilized in buffer-glycerol solution on adsorbed lipase during continuous hydrolysis of olive oil in a microporous hydrophobic
IMMOBILIZATION OF LIPASE
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membrane bioreactor. Enzyme Microb. Technol., 8, 236-240 (1986). Omar, C.I., Saeki, H., Nishio, N., and Nagai, S.: Hydrolysis of triglycerides by immobilized thermostable lipase from Humicola lanuginosa. Agric. Biol, Chem., 52, 99-105 (1888). Beehtol, O.C.: Bone, p. 374-375. Technopia, vol. 16. Koudansha, Tokyo (1977). Fukumoto, J., Iwai, M., and Tsujisaka, Y.: Studies on lipase iv. Purification and properties of a lipase secreted by Rhizopus clelemar. J. Gen. Appl. Microbiol., 10, 257-265 (1964). Lowry, H.O., Rosebrough, J.N., Farr, L.A., and Randall, J.R.: Protein measurement with the folin phenol reagent. J. Biol. Chem., 193, 265-275 (1957). Folch, J., Less, M., and SIoanestanley, H.G.: A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem., 226, 497-509 (1957). Kobayashi, T., Mukataka, S., Kataoka, H., and Takahashi, J.: Enzymatic hydrolysis of fats in biphasic organic-aqueous systems. Hakkokogaku, 63,439-444 (1985). Mukataka, S., Kobayashi, T., and Takahashi, J.: Kinetics of enzymatic hydrolysis of lipids in biphasic organic-aqueous systems. J. Ferment. Technol., 63, 461-466 (1985). Mukataka, S., Kobayashi, T., Sato, S., and Takahashi, J.: Enzymatic hydrolysis of fats at high substrate concentration in biphasic organic-aqueous systems. J. Ferment. Technol., 65, 2329 (1987).
Utilization of Powdered Pig Bone as a Support for Immobilization of Lipase S A T O S H I N E G I S H I , * S E I G O S A T O , S U K E K U N I M U K A T A K A , ASP J O J I T A K A H A S H I
Institute of Applied Biochemistry, University of Tsukuba, Tsukuba-shi, lbaraki 305, Japan Received 12 December 1988/Accepted 4 March 1989 Pig bone was examined for its suitability as a support material for iipase immobilization. It was observed that pig bone (PB) particles dispersed readily in both polar and nonpolar solvents, and lipase was easily adsorbed. In particular lipase adsorbed on olive oil-soaked pig bone (OPB) particles exhibited a higher hydrolytic activity than that in lipase adsorbed on a selection of other representative supports, regardless of removing the presoaked olive oil from the particles after immobilization of lipase. The optimum pH and temperature for hydrolytic activity of OPB-adsorbed lipase were the same as those for free lipase, although thermal resistance was increased by immobilization. When OPB-adsorbed lipase was used for repeated batch reactions of olive oil hydrolysis, an activity of more than 80% of the initial activity of each run could be retained after 46 b reaction. The results suggest that PB is an excellent support material.
I m m o b i l i z a t i o n of enzymes is essential for application to industrial reaction processes. Immobilized enzymes allow easy separation from reaction mixtures as well as reuse o f the enzymes. Lipase is one o f the most extensively investigated enzymes due to its ability in fat splitting, esterizing and transesterizing. It has shown promise in such diverse fields as chemicals, foods, medicines and cosmetics, and many attempts have therefore been made to immobilize lipase (1-5). Representative methods p r o p o s e d for lipase immobilization include gel-entrapping using photo-crosslinkable resin (6) and a d s o r p t i o n on membranes (7-9) or resins (10). However, until now, almost all the supports used for lipase i m m o b i l i z a t i o n have been artificial, and the hydrolytic activities o f lipase immobilized on such supports have not proved adequate for industrial application. We consider that safety is a very i m p o r t a n t factor in applying immobilized enzymes, especially lipase, in industry, and that bio-substances are the safest materials for enzyme support. A n i m a l bone, which is generally discarded after the meat has been removed, is untoxic, mechanically strong and porous. It is composed of about 6 0 ~ organic and 40%0 inorganic substances. Most o f its organic c o m p o nent is collagen which contains a large number o f potential binding sites for enzyme attachment, while its inorganic c o m p o n e n t is close to h y d r o x y a p a t i t e (11). For such reasons, bone presents itself as having great potential as a support material for enzyme immobilization. This paper describes the utilization of waste animal bone, specifically pig bone, for the immobilization of lipase. The findings show pig bone to be an excellent enzyme support.
tein), were provided from Meito Sangyo Co. Ltd. In addition to pig bone particles, the following c o m m o n enzyme supports were tested for the immobilization o f lipase: Amberlite IRA-94 (Rohm & Haase), Dowex NWA-1 (Dow Chemical), Diaion HP-21 (Mitsubishi Chemical Industries), Chitopearl BCW-3001 (Fuji Spinning) and Celite R-630 (Johns Manville). A n immobilized lipase preparation named Lipozyme was also purchased from Novo Industries. Olive oil (saponification value, 195) was purchased from Yuro Yakuhin Co. Ltd. All other chemicals were o f reagent grade and obtained from commercial sources. Preparation of PB (Pig Bone) and OPB (Olive oil-soaked Pig Bone) particles PB and OPB particles were obtained from a pig thighbone by the following procedure. The thighbone was cut at both ends and cleaned. To remove fat, the bone was autoclaved at 120°C for 20 rain, and then ground into small particles. Particles ranging from 37 to 105/lm were selected by sifting. These were washed five times with ethanol and distilled water, and designated as PB support. The PB particles were soaked in olive oil for more than 12 h before use, and labelled OPB. Immobilization of lipase Ten grams o f support were added to 100ml o f 0.05 M phosphate buffer (pH 7.0) to which 5 g o f lipase powder had been dissolved. The mixture was shaken for 1 h at 0°C. These conditions for immobilization were determined by preliminary experiments as will be described later. The supports were separated from the mixture by filtration, and washed with a phosphate buffer until no more protein was released. In the case of OPB particles, further washing was performed with isooctane 3 times to wash out the olive oil. Assay of lipase activity The hydrolytic activities of the free and immobilized lipases were measured by the method o f F u k u m o t o et al. (12). The reaction mixture consisted o f 10 g lipase powder or 1-5 g o f immobilized lipase, 40 g o f olive oil and 300 ml of 0.05 M phosphate buffer (pH 7.7). The reactor consisted o f a separable flask with a capacity of 500 ml and inside diameter 8 cm. This flask was equipped with 4 baffle plates. The reaction mixture was agitated by a 6-blade flat turbine (5 cm in diameter). The reaction was carried out at 37°C for 3 0 m i n stirring at 500 rpm. One unit of hydrolytic activity was defined as the
MATERIALS AND METHODS E n z y m e and chemicals Lipase produced by Candida cylindracea was used t h r o u g h o u t the experiments. Two kinds o f lipase in powder form having different hydrolytic activities, Lipase-MY (14 U / r a g - p o w d e r , 140 U / r a g - p r o tein) and Lipase-OF (220 U / m g - p o w d e r , 2200 U / m g - p r o -
* Corresponding author. 350
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IMMOBILIZATION OF LIPASE
activity which would release one micromole o f free fatty acid in one minute.
Hydrolysis of olive oil and repeated use of immobilized lipase The reactor for the enzyme reaction was the same as that used for the activity assay. The reaction mixture consisted o f 35 g of olive oil, 300ml o f 0.05 M p h o s p h a t e buffer (pH 7.7) and 30 U / g - s u b s t r a t e o f free or immobilized lipase. Experimental conditions for the repeated batch reaction were the same as those described above. After 46 h had elapsed in each run, the immobilized lipase was recovered by filtration, and washed 3 times with the buffer solution and isooctane. The residual activity was then measured in accordance with the method mentioned above. Analysis Protein concentration was assayed by the
351
method o f Lowry et al. (13). The fatty acids p r o d u c e d in the reaction were titrated with 0.2 N or 0.025 N K O H using a p H meter as indicator. RESULTS AND DISCUSSION
Properties of pig bone(PB) particles The PB particles taken from pig thighbone proved to be a useful support material having the following properties. Its specific gravity was 1.7 g / c m 3 and it was observed that the PB particles dispersed well both in polar and n o n p o l a r solvents. These characteristics present a very i m p o r t a n t advantage in use as a support material for the immobilization o f lipase, because lipase is used not only for the hydrolysis of fats and oil in aqueous or aqueous-organic systems but also for
FIG. 1. Scanning electron micrographs of pig bone particles.
352
NEGISHI ET AL.
J. FERMENT. BIOENG.,
esterification and interesterification in microaqueous systems. Figure 1 shows electron microscopic photographs of PB particles. The particles have irregular shapes and high porosity. It has been reported that the surface area of bone is more than 100m 2 per gram of inorganic component (11). This porosity is an additional advantage for the immobilization of enzymes. Optimum conditions for immobilization In order to obtain an immobilized lipase with high activity, the optimum conditions for immobilization were investigated using olive oil-soaked pig bone particles. To ascertain the time required for sufficient adsorption, lipase was kept in contact with OPB particles for various lengths of time at 0°C. After 30 min, 65% of maximum binding had occurred and the maximum amount of adsorbed enzyme was obtained after 60min. However, prolongation of the contact time beyond 60 min produced no increase in adsorption. Temperature had little influence on the adsorption of lipase on OPB particles. The activity of immobilized lipase obtained at 20°C was 85% of that obtained at 0°C. Optimum pH for immobilization was 7.0, although the free lipase showed its highest hydrolytic activity at pH 7.7. Immobilization at pH 6.0 and 8.0 gave 85%o and 90%o of the maximum activity obtained at pH 7.0, respectively. Enzyme concentration in an incubated solution for immobilization is also one of the factors which affects the amount of enzyme adsorbed on the supports. The effect of varying the lipase concentration is shown in Fig. 2. The incubation was performed at 0°C for 1 h. It is clear that the amount of adsorbed lipases reached a maximum at a concentration of 5 mg-protein/ml, although this figure is represented by the relative activity of immobilized lipase obtained after washing. TABLE
1.
Size
Support
-
8
.
0
-
~
-
1
O
8O v>-,6 0 >
4o
•
20 ¢Y
0
4.'0
0 Enzyme
61o
¢o¢~. (mg-prol. / ml)
F I G . 2. Effect o f e n z y m e c o n c e n t r a t i o n in i n c u b a t i o n s o l u t i o n o n i m m o b i l i z a t i o n o f lipase. T h e m i x t u r e c o n s i s t e d o f 10 g o f O P B a n d 100 ml o f e n z y m e s o l u t i o n , s h a k e n f o r 1 h at 0 ° C .
As a result of this investigation on the conditions for immobilization, we obtained lipase immobilized on OPB particles with a hydrolytic activity as high as 700-1000 U/g-dry support using crude lipase powder, Lipase-MY. Hydrolytic activity of lipase immobilized on various supports To compare the hydrolytic activity of lipase adsorbed on pig bone with those of other immobilized lipases, two lipases were immobilized on various representative supports. The immobilization was performed without any crosslinking agent for either kind of lipase powder. The hydrolytic activities of the two lipase powders (LipaseMY and Lipase-OF) were measured as 140 and 2200 U/rag-protein, respectively, by an agitation method (12). The results obtained using these lipase powders are presented in Tables 1 and 2. The amounts of proteins adsorbed on Celite, Amberlite
Immobilization of Lipase-MY on various supports Adsorbed protein (mg/g-support)
(,urn) Free enzyme Celite IRA-94 MWA- 1 HP-21 C h i t o p e a r l 3001 PB OPB L i p o z y m e ,~
O
100
-
8.5~9.0 t 0 - 14 2 . 0 - 2.5 4.0~4.5 15 ~ 18 0.7-0.9 5.0-8.0
0
400 ~ 500 290 ~ 830 210-600 100 37 4 105 3 7 ~ 105 300 4 600
Specific activity (U/mg-prot.)
Relative activity (%)
140 1.5 4 1.9 2.9 ~ 3.5 11 ~ 15 2.943.5 3 . 5 - 5.6 29435 1 1 0 ~ 140
100 1.1-1.4 2.1 ~ 2.5 7.1 4 11 2.1 - 2 . 5 2.5-4.0 21 ~ 2 5 8 0 - 100
-
-
-
-
Activity (U/g-support) -
-
10-15 35 ~ 40 15 ~ 20 1 0 - 15 5 0 - 60 2 5 - 30 7 0 0 - 1000 1 0 0 - 120
I m m o b i l i z e d lipase o b t a i n e d c o m m e r c i a l l y . P B , P i g b o n e p o w d e r ; O P B , olive o i l - s o a k e d pig b o n e p o w d e r .
TABLE Support Free enzyme Celite IRA-94 MWA-1 HP-21 C h i t o p e a r l 3001 PB OPB
2.
Immobilization of Lipase-OF on various supports
Adsorbed protein (mg/g-support)
Specific activity (U/mg-prot.)
Relative activity (0/0)
Activity (U/g-support)
-3.0~4.0 15~20 4 . 0 ~ 6.0 1 5 ~ 17 5.0 4 7.0 1.3 ~ 1.6 4.0 ~ 6.0
2200 31-34 11 - 17 31 - 3 6 4.4-6.6 66 ~ 88 330 - 440 330 - 440
100 1 . 4 ~ 1.6 0.5 4 0 . 8 1 . 4 ~ 1.7 0.2~0.3 3.0 4 4.0 15 - 20 15 - 20
-1 0 0 ~ 140 2 0 0 - 250 1 5 0 - 180 7 0 - 100 400-600 400 - 500 1500 - 2000
P B , P i g b o n e p o w d e r ; O P B , olive o i l - s o a k e d pig b o n e p o w d e r .
VOL, 67, 1989
I M M O B I L I Z A T I O N OF LIPASE TABLE 3.
IRA-94 and Chitopearl BCW-3001 respectively were larger than those of proteins adsorbed on PB and OPB particles, as can be seen in Table 1. However, the specific activities of proteins adsorbed on PB and OPB particles were remarkably higher than those on the other supports. In particular, the activity of proteins immobilized on OPB particles amounted to 80-100% of that of the free enzyme, in contrast to the relative activities of most proteins adsorbed on other supports which were less than 10%. Consequently OPB particles showed the highest activity per unit gram of support, 700-1000 U/g-support. This was 20-30 times higher thatn the activity of lipase adsorbed on other supports, and also about 7-8 times higher than that of Lipozyme, which is commercially sold immobilized lipase. As to the reason for obtaining immobilized lipase with such a high activity by using OPB particles, it was considered that OPB particles could adsorb lipase selectively from various proteins probably contained in lipase powder, because the OPB particles were presoaked in olive oil. This conjecture is supported by the data shown in Table 2. By using lipase powder with a 15-fold higher activity, every support showed higher activity in immobilized lipase. The activity of all other immobilized lipase except for OPB particles was 15-20 times that shown in Table 1 obtained using crude powder Lipase-MY. However, in the case of OPB particles, the increase of immobilized lipase activity was only 2-3 times, although this was still higher compared with others. This fact supported the idea that lipase proteins had been already adsorbed on OPB particles selectively, when the crude lipase powder was used for immobilization. The effect of olive oil presoaking of the support on lipase immobilization was tested using some other supports. The results are shown in Table 3. In Exp. A, where Lipase-MY was used, the lipase activity immobilized on each support increased ten or more times compared to that obtained with non olive oil-soaked supports. On the other hand, in Exp. B where Lipase-OF was used, the extent of increase in immobilized lipase activity of each support was not so significant as in the case of pig bone, although the effect of presoaking in olive oil was still recognized. This is because the selective adsorption effect was not sufficiently displayed owing to the use of powder with a high lipase content. From Table 3, however, it is worth noting that pig bone shows its greatest effect with presoaking in olive oil. As previously mentioned, big bone is quite porous and disperses well in both the aqueous and olive oil phases. Therefore, it absorbs a higher quantity of olive oil corn-
353
Effect of olive oil presoaking on immobilization of lipase Activity (U/g-support)
Exp.
Support
A
Celite MWA-1 HP-21 PB
180120140 4 700 -
200 150 170 1000
Celite MWA-1 HP-21 PB
180 ~ 400 ~ 400~ 1500 ~
200 500 500 2000
olive oil-soaked
non-soaked 10~ 15 15 - 2 0 10~ 15 25-30 100 4 150 4 70400-
140 180 100 500
Lipase-MY was employed (activity: 140 U/mg-prot.) in Exp. A and Lipase-OF (activity: 2200 U/mg-prot.) in Exp. B.
pared with other support materials. Besides, pig bone contains collagen which has a large number of potential binding sites for enzyme attachment. It can be supposed that these properties of pig bone are most advantageous in displaying selective adsorption and immobilizing lipase with the highest activity. It is also important to note that lipase was immobilized and remained in the pig bone itself, but not in the olive oil. Presoaked olive oil and its products, i.e. fatty acids and glycerol, which might be produced by immobilized lipase, were almost completely removed after immobilization by washing with buffer solution and isooctane. Therefore, only the lipases adsorbed solely on olive oil were washed away with it. Elimination of olive oil and the products was confirmed by two methods. The weight of pig bone increased about 35%0 after soaking in olive oil. However, it returned to its original weight by washing after the immobilization of lipase. Furthermore, we tried to extract the olive oil and fatty acids from the support after washing with buffer solution and isooctane by the methods of Folch et al. (14). No triglyceride and fatty acids could be discovered by TLC analysis. The effect of presoaking in other chemicals such as oleic acid, glycerol and poly vinyl alcohol (PVA) was examined. Although glycerol and PVA gave rather undesirable results, oleic acid showed a selective adsorption effect similar to olive oil. However, the activity of the immobilized lipase obtained was lower than that of OPB. From the results of various tests on the immobilization of lipase using pig bone particles, it was found that olive oil-presoaked pig bone (OPB) gave a hydrolytic activity which was higher than the activities of conventionally used
100
100
_8O >,60 .>__
~60
.~
N 20 a: 0
5
I
I
6
7
pH
Q: 0
8
•
20
i
30 40 50 60 Temperature ( *C )
FIG. 3. Comparison of pH and temperature dependence on hydrolytic activity of OPB-adsorbed lipase with those of free lipase. O, OPB-adsorbed lipase; zx, free lipase.
Symbols:
354
NEGISHI ET AL.
J. FERMENT. BIOEN(i.,
TABLE 4.
100
Run
;e .-. 8 0
Initial act. 1 2 3
.~
60
Residual activity after each ruw' (U/g-support) (%) 740 620 550 370
100 84 74 50
~ Each run was carried out for 46 h. Reaction mixture: olive oil, 35 g; buffer solution, 300 ml. Reaction conditions: agitator speed, 500 rpm; temperature, 37°(?.
~40 "6
20 0
Repeated use of lipase immobilized on OPB
I
o
,2
2;.
3;
48
Reaction time ( h ) FIG. 4. Comparison of time course of olive oil hydrolysis by OPB-adsorbed lipase with that by free lipase. The reaction mixture consisted of 35 g of olive oil and 300 ml of 0.05 M phosphate buffer. The reaction was carried out under optimum pH and temperature (7.7, 37°C). Symbols are the same as in Fig. 3.
supports. However, the problem concerning the activity yield of lipase still remains. The recovery yield of activity against the total lipase used for immobilization is still low, i.e., 15°/00and 2O//oofor Lipase-MY and Lipase-OF, respectively, under the conditions where the highest activity was obtained. Although, these values were increased to 70% (Lipase-MY) and 15% (Lipase-OF) by reducing the ratio of the enzyme solution to the supports to one-tenth that of the former case, the activities of immobilized lipase obtained decreased by 50O/oo and 25%, respectively. In the case of the adsorption method, however, the lipases which are not adsorbed on the supports can be reused by separating the supports from the enzyme solution after immobilization. Therefore, the activity yield as a whole can be improved by reusing the u n a d s o r b e d lipases, even if the yield value is low in one immobilization run. Effect o f p H and temperature on the activity o f lipase i m m o b i l i z e d on O P B The pH and temperature dependences of the activities of free and OPB-adsorbed lipase are shown in Fig. 3. The o p t i m u m pH and temperature of OPB-adsorbed lipase for displaying its hydrolytic activity were the same as those of free lipase. However, the thermal resistance was increased by immobilization. In contrast to free lipase, the activity of which was drastically reduced when the reaction temperature was raised to over 37°C, the OPB-adsorbed lipase showed 90% of the m a x i m u m activity at 45°C and remained 50%0 even at 60°C. It is considered that this increase in thermal resistance was due to conformational stabilization by immobilization. Hydrolysis o f olive oil and reuse of OPB-adsorbed iipase For comparison with hydrolysis by free lipase, olive oil was hydrolysed using OPB-adsorbed lipase. The olive oil concentration was 10°4 (w/v), and free or immobilized lipase was added in proportion with the activity of 30 U/g-oil. The progress of the hydrolyses is shown in Fig. 4. Although the lipase was equally added to both reaction systems, hydrolysis proceeded faster in the case of the OPB-adsorbed lipase. The OPB-adsorbed lipase dispersed well not only in the aqueous but also in the oil phase. In contrast to this, as free lipase disperses only in water, OPB-adsorbd lipase is superior for the hydrolysis of olive
oil. The results of repeated use of OPB-adsorbed lipase are shown in Table 4. Repeated batch reactions were carried out at an olive oil concentration of 10% (w/v) and initial lipase concentration of 30 U/g-oil. Each run was carried out for 46 h, and residual activity after each run was determined from the initial progress curve of hydrolysis in next run. Following the method discribed in 'Materials and Methods', immobilized lipase was washed with buffer solution and isooctane after each run to eliminate the remaining substrate and products. The activity of OPB-adsorbed lipase gradually decreased with each run. However, 8085% of the initial activity of each run was retained after the reaction and carried over to the next run. This reduction of activity included loss both in recovery and in washing, because the activity was measured after these operations. In the repeated batch experiments, although one batch reaction was continued for 46 h, the reaction time could be shortened by adding a higher quantity of immobilized lipase to the reaction system. In such a case, the activity loss of immobilized lipase in each run would be further reduced. Batch reaction is a suitable method for establishing a high extent of hydrolysis in a short period. Since the activity loss of OPB-adsorbed lipase in one batch operation is less than 20%, repeated batch hydrolyses can be performed with a small quantity of lipase supplement. In this paper, the utilization of waste pig bone for the immobilization of lipase is shown, with an application to the hydrolysis of olive oil. Although palm oil and beef tallow which are in solid form at the optimum temperature for hydrolytic activity of many lipases, are mainly utilized as raw materials for the industrial production of fatty acids and glycerol, we found in a previous study (15-17) that the hydrolysis of such solid lipids by lipase could be performed in the same way as that of liquid lipids by addition of a suitable organic solvent. Therefore, it may be possible to apply OPB-adsorbed lipase to hydrolyse industrial raw materials in the presence of an organic solvent. However, further investigation is necessary to develop industrial applications of OPB-adsorbed lipase.
ACKNOWLEI)GMENT The authors are grateful to Meito Sangyo Co. Ltd. for providing the lipase. REFERENCE
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VoL. 67, 1989
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IMMOBILIZATION OF LIPASE
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