Kor. J. Appl. Microbiol. δloeng.
Vol.15, No.l,43-48! 19871
Characteristics of Lipases in Two Phase System
Kwon\ Dae Y.
,
Kee H. Kim2,
and Joon S. Rhee *Deþarlmιnt 01 Biological Sciι”“’ and EnginιènJZf[
Korea Advan c.ιd Iηstitute 01 S，‘Iιιι ηη d T，α Iz nolog\’
POB 150 Choη，gyaηg， Sιoul 131, Korea
이상계 내에서 리파제의
트서 --,=
권대영 김키혁 이준식*
한굿과학기술원 샘물공학괴
Characteristics of lipases from Candida rugosa and Rhizopus arrhizus during the hydrolysis of olive oil in two phase system were investigated for fat splitting and interesterification. Isooctane was u
‘
ed as an organic solvent for both Iipases. The lipid hydrolysis rate of olive oil by lipases in two phase system increased in proportion with the olive oil concentration up to 90%(v fv),
whereas in emul- sion system the rate increased only up to 5% of olive oil concentration and then decreased sub- stantia\ly thereafter with the increase of the substrate'concentration. The lipid hydrolysis rate for various pH of the aqueous phase(containing lipases) in two phase system was maximum at pH 6.0 and 6.5 for the lipase from C. rugosa and R. arrhizus,
respectively. The optimum temperature for the reaction was3rc
and 400C for the Candida and Rhizopus lipases,
respectively.Due to their keen substrate specifity, lipases were used as the catalysts in bioconversion of fats and oils, i.e., fat splitting and interesterification Recently, many attempts have been made to con- vert water-insoluble substrate(lipids or steroids)
to highly va.luable materials by using microbes or enzymes in the presence of organic solvents (1-5). Enzymatic fat splitting or interesterification of lipids in two phase system using organic solvents were advantageous for products separation or enzymes recovery, and application to the solid lipids (6).
In our papers reported earlier (7-9)
,
the efTects of organic solvents on the stability and catalytic activity of lipases from Candida rugosa and Rhizopus arrhizus were studied for fat splitting and interesterification in two phase system due to their specificity (10). The results showed that isooctane for the lipase from C. rugosa and isooctane or diisopropylether for the lipase fromR. arrhizus were the solvents most suitable in two phase system‘ Therefore, isooctane was used for comparative study of lipases from C. rugosa and R‘ arrhizus.
Kobayashi et al. (1) reported the hydrolysis of beef tallow, palm and olive oil by lipase from C. cylindracea(Syn. C. rugosa) in two phase system using isooctane (1). However, they studied the effects of isooctane, agitation speed, and baf- fles on lipase activity using only beef tallow as the substrate. Therefore, it is necessary to investigate such characteristics of lipases as temperature pro-
filε ， pH profile, and other kinetics in the hydroly- sis of olive oil in two phase system systematically In this paper, we described the characteristics of lipases from C. rugosa and R. arrhizus in two phase system using isooctane for the hydrolysis of olive oil by investigating the initial reaction rates of lipases to the olive oi!, pH profile, temperature.
Key words: Characteristics of lipase, Lipase, Two phase system, Hydrolysis
1. Present address: Applied Research Laboratory, Food Research Institutel AMC, Banwol, Korea 2. Present address: Division of Biotechnology, R & D Center, Lotte Group, Seoul, Korea
*
Corresponding author43
Materials and κ1ethods
κ1aterials
Lipases from C. rugosa and R. arrhizus, olive oil and tripalmitin were purchased from Sigma Chemical Co.(St. Louis, MO. USA).
Isooctane were purchased from Tokyo Kasei Chemical Co., Ltd.(Tokyo, Japa 쩌 AIl other re- agents and chemicals used were of analytical grade One unit of lipase activity was defìned, unless otherwise specifìed, as one micromole offauy acid produced per 1 hr under the analytical condi-
tlOns.
Reactor system
Lipid hydrolysis by lipases in two phase system was performed in the stirred tank reactor‘ Working volume of reaction was about 50 m/. Four baf- f1es wereconstructed in reactor to get rid of vortex formation and to help the formation of fìner dro- plets ofwater in solvent. Water droplets ofenzyme solution were dispersed to the solvent phase while the reaction was in progress. The reaction was performed by agitating the mixture with magnetic bar. Agitation speed was fìxed al 800 rpm. Glass vessels used were siliconized before using accQrd- ing to the method of lnman (11). Temperature was controlled al 300C by a thermomixer. The ratio ofsolvent phase to water phase in the reaction mixture were 8: 2(v /v), and 10% olive oil in isooctane was used.
Preparation of substrate emulsion
Emulsion of olive oil was prepared with gum arabic according to the method described by Lieberman and Ollis (12). Lipase activity in emulsion system was studied for the comparative purpose.
Lipase assay in standard assay system
Lipase activity in emulsion system or in two phase system were determined by the method of Kwon and Rhee(13).
Degree of hydrolysis
The degree of hydrolysis of olive oil was calcu- lated as follows;
Degree of hydrolysis (%) = (5. 608) ( μM FFA produced)[(saponifìcation value of oil)(g oil)J-1
where the saponifìcation value of olive oil deter- mined by AOCS method is 191 (14), which is consistent with the data reported (15).
Effecí of olive oil on Iipase activity
At constant agitation speed, pH, temperature, and proportion ratio of solvent to aqueous phase, the rates of olive oil hydrolysis by Iipase at vari- ous substrate concentration from 1% to 90%(v / v) olive oil in isooctane were observed. pH and temperature were adjusted to 7.0 and 350C, respectively.
Effect of pH
Variations ofreaction rate with pH for Iipases in two phase system were determined in proper buffers in the range of pH 3-9 at 30oC. Citrate buffer, pH 3-4; succinate buffer, pH 4-6 ; phosphate buffer, pH 6-7; tris buffer, pH 8-9 were used.
Effect of temperature
Hydrolysis rate at various temperature was determined from 100C to 50oC. The pH of aqueous solution was controlled at 6.0 and 6.5 for lipases from C. rugosa and R. arrhizus, respectively. The temperature of substrate solution and enzyme solution were controlled individually at given temperature before reaction. Reaction was performed by adding the enzyme solution to sub- strate solution at a given temperature.
Results and Discussion
Dependence of lipid hydrolysis on olive oil con- centration
To compare the differences in kinetic parameters of enzyme reaction between the two phase system and the emulsion system, Iipid hydrolysis rate of olive oil were measured in two phase system with various substrate concentrations. Fig.1 shows the effects of olive oil concentration on the Iipid hydrolysis rate by lipases from C. rugosa and R. arrhizus in two phase system. For the Can- dida lipase, the Iipid
hydr이ysis
rate was increasedtn proportion to the olive oil concentration up to 90%(v /v), whereas in emulsion system the rate was
Vol.15, No.l, 45
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Olive Oil Concentration, % Fig.2. Lipid hydrolysis to olive oil of lipase from R. arrhizus in two phase system and comparison of the two phase system to the emul- sion system.
Reaction volume is 5 ml. Symbols:
e
, emulsionsystem; 0 , two phase system.
25 0
30 60 90
Olive oil Concentration, %
Fig.l. Lipid hydrolysis ra te to olive oil by lipases from C. rugosa and R. arrhizus in two phase system.
Reaction conditions: pH of enzyme solution, 6 : temperature, 30.C ratio of solvent phase to aqueous phase, 8 : 2: agitation speed, 800 rpm;
reaction time, 10 min. The results are mean val ues of three replicates. These conditions are the same throughout the following figures, unless otherwise specified. Symbols:
e
, lipase from C.rugosa; 0 , lipase from R. arrhizμs.
0
The reason for the acidic shift in pH can be explained by the two possbilities as follows (20)
Firstly, one possible reason is that diffusion barrier of the intεrface in emulsion system affects the pH optimum (16), that is to say, the true optimum pH for the lipase reaction rate is near to that of two phase system rather than to that of
쪼 ι커↓ k \ .
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\ b
100
50
10
10%.
lipase
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Fig.3. Effects of pH on the lipase activity in two phase system.
Olive oil concentration in isooctane is Symbols: 0 , Iipase from C. rμ~gosa;
e
, from R. arrhizμs.4 8 0 2
increased up to only 5% of olive oil concentra- tion and decreased sharply thereafter (16). The same trend was observed with Rhizopus lipase(Fig.2). From the result, it was concluded that in the lipase reaction, substrate inhibition was eliminated by using water-immiscible organic solvent in two phase system(16).
Effect pH on lipase activity
Effect of pH on the olive oil hydrolysis rate by lipases from C. rugosa and R. arrhizus in two phase system were investigated as shown in Fig.3.
Lipid hydrolysis rate was maximum at pH 6. 0 and 6.5, for the Iipases from C. rugosa and R.
arrhizus, respectively. Optimum pH of the lipase reaction in emulsion system for the lipase from C. rugosa waspH7.0(16,17), andthatof lipase from R. arrhizus in normal aqueous sys- tem was reported as pH 7.0 or 8. 0 (18, 19). Compared to the profile of lipase in emulsion( or normal aqueous) system, optimum pH of lipases were shifted to acidic region by about one unit of pH.
emulsion system. Compared with emulsion system, there is no diffusion barrier in two phase system.
Since free fatty acids produced were diffused imme- diately' from the interface to solvent phase, the pH of aqueous phase was not affected by the products diffusion‘ The true optimum pH for lipase activity is near to that in two phase system. Therefore, pH optimum for the lipid hydrolysis in two phase system was shifted to acidic region from the alka- line region of emulsion system (16, 'W).
Secondly another possibility is the lower pH accomodates the nonionization process of fatty acids during the diffusion of the free fatty acids to solvent phase. We assume that only the nonionized form of a fatty acid(RCOOH) will dissolve in the solvent phase (20) . Therefore, lower pH in the aqueous phase is more favorable in the hydrolysis of olive oil thermodynamically in two phase system
(20) .
Effect of temperature 00 Iipase
Effects of temperature on the lipid hydrolysis rate by lipase from C. rugosa and R. arrhizus are shown in Fig.4. Olive oil in isooctane (10%
by volume) was used as a substrate. Phosphate buffers (0.05 M of pH 6. 0 and 6.5 for the lipase from C. rugosa and R. arrhizus, respectively) were used as enzyme solutions. Reaction time was 15 min, since the initial velocity was linear up to 15 min of reaction time. Maximum activities for Candida lipase and Rhizopus lipase were found to be 350C and 40oC, respectively. In emulsion system, the temperatures at which lipase showed the maximum activities were greater than those of two phase system for both microbial lipases (16, 21). This result must be due to the fact that the enzyme solution of lipase in organic solvents is thermally more unstable than that of aqueous system. We reported that destabilizing effect of organic solvent upon the lipase was greater at higher temperature (22).
Activation energy from Arrhenius plot, Ea, in two phase system is 11. 5 and 5.4 kcal/mole for the lipase from C. rugosa and R. arrhizus, respectively. Activation energy of Candida lipase was almost same between two phase systerp and emulsion system (16). The value of activation
100
~
종|겨
0 10 20 30
Temperature, 0 C
40 50
Fig.4. Effects of temperature on the Iipase in two phase system.
10% olive oil in isooctane was used. Symbols:
0 , lipase from C. rugosa; •• lipase from R.
arrhizμs.
energy for Rhizopus lipase is much smaller than that of Candida lipase due to its higher purity (8).
Hydrolysis of solid fats
One ofthe advantages in using two phase system is the broadening of the substrate spectrum to the solid fats. Tripalmitin was used as a model of solid fats. Due to its higher melting point, we were not able to prepare the emulsion with higher concen- tration ofsolid fats, thus tripalmitin was dispersed in aqueous phase for the reaction. However, lipid hydrolysis in organic solvent can be performed to the higher concentration of solid fats.
As shown in Fig.5. the tripalmitin dissolved in isooctane was hydrolyzed more rapidly two or three times by the lipase from C. rugosa than the tripalmitin dispersed in water system. Since the tripalmitin is solid state in aqueous system, the orientatÎon and accessibility of tripalmitin mole- cule at the interface cannot be freely controlled Therefore, thε lipase activity to the tripalmitin in water system was a half or a third ofthe activity in two phase system. In two phase system, tripalmitin was dissolved in isooctane up to 3%(v /v).
47
isooctane. Degree of lipid hydrolysis at various olive oil concentration was investigated as shown in Fig.6. With the decrease of olive oil concen- tration degree of olive oil hydrolysis was increased, although initial hydrolysis rate of olive oil was increased with the increase of olive oil concentra- tion. Using the 2. O%(v jv) olive oil concentra- tion, over 98% of olive oil was converted to fatty acids within 25 hr. At 10.0% olive oil concentra- tion 92% of olive oil was hydrolyzed within 48 hr.
Vol.15, No.l
6
4
2 10
ζ~~5~3성
그{〕 jRι
」。UQEagg 8이상계 (二相界) 내에서 올리브유를 가수분해 시키 띤서
Candida rugosa맞
Rhizoþus arrhizus의 리 파 제의 특성 을 조사했다. 지방 분해 속도는
emulsionsystem 에서는
olive oil농도 5%(v/v)까지 만 증가하 다가 그 이상의 농도에서 논 상당히 감소되었으나 이 상계 에서는
olive oil농도 90% 까지 계 속해서 증가 했다. 이 상계 에 서 러파제가 녹아 있는 수용액상의
pH
기 각 각
6.0,6.5일 때
C. rugosa와
R.arrhizμs 리파제의 지방 분 해 속도가 가장 높 았다.
또한
C. rugosa벚
R. arrhizus리 파 제 의 반응 최 적 온도는 각각 35'C 와 40'C 였다.
。 t
...,
요
.5 . 10 . 15 . 2 0 . 25 Tripalmitin Concentration, u mole/50 ml Fig.5. Fat hydrolysis rate by lipase from rugosa to tripalmitin in two phase system.
Reaction volume and reaction time is 50 ml and 1 hr, respectively. Symbols: 0, emulsion system;
.,
two phase system.C.
0
Batch hydrolysis of lipids in two-phase system At the optimized conditions, (pH, 6.0; tem- perature, 35'C agitation speed, 800 rpm), batch hydrolysis of olive oil in two phase system by lipase from C. rugosa was studied. The ratio of solvent phase to aqueous phase was 8: 2 for the 2.0 and 10.0%
이 ive
oil concentration in1. Kobayashi, T., S. Mukataka, H. Kataoka, and J. Takahashi, Hakkokogaku 63, 439 (1985) 2. Andersson, E., B κ1attiasson 、 and B. Hahn
Haegerdal, Enzyme Microb. Technol. 6, 301(1984)
3. Cremonesi, P., G. Carrea, L. Ferrara, and E Antonini, Biotechnol. Bioeng. 17, 1101 (1975) 4. Kimura, Y., A. Tanaka, K. Sonomoto, T. Ni-
hira, and S. Fukui, Appl. ιficrobiol. Biotechnol.‘
17, 107 (1983)
5. Kazandjian, R. Z 、 J.S. Dordick, and A.M KJibanov, Biotechnol. Bioeng. 28, 417 (1986) 6. Lilly, M‘D., J. Chem. Technol. Biotechnol.32,
162 (1982)
7. Kim, K.H., D.Y. Kwon, and J.S. Rhee, Lipids 19, 975(1984)
8. Kwon, D‘Y., and J.S. Rhee, Kor. 1. Food Sci.
Technol. 17, 490(1984)
9. Kwon, D.Y., and J.S. Rhee, Kor. J. Appl. Mi- References
100
영
-m【mE【。」{〕능}{
10 15 20 25 Reaction time, hr
Fig.6. Degree of lipid hydrolysis by lipase from C. rugosa at various olive oil concentration.
Reaction conditions: pH of enzyme solution, 6.
0: temperature, 35'C ratio of solvent phase to aqueous phase, 8 : 2: reaction volume, 50 ml Symbols: 0 - 0 , 2%: 0 --0, 5%: . - . , 10% olive oil concentration.
30 48 5
0
io!. Bioeng. 14, 57(1985)
10. Benzonana‘ G., and S. Esposito, Biochim. Bio- phys. ACla 231, 12 (1971)
11. Inman, J.K. in Melhods in Enzymology Vol 33, Academic Press, London, p.30 (1974) 12. Lieberman, R.B., and D.F. Ollis, Biotechnol.
Bioeng. 17, 1401 (1975")
13. Kwon, D‘Y., and 1.S. Rhee, JAOCS 63, 89(1986)
14. AOCS ~끼cial and Tentative Methods(edited by R.O. Walker), 3rd edition‘ Ill. Cd 3-25 (1978)
15. Weiss, T.J., in Food Oils and Their Uses, 2nd edition, A VI, Westport, p.47 (1983)
16. Kwon, D.Y., and ].S. Rhee, Kor. J. Chem
Eng. 1, 153 (1984)
17. Tomizuka, N., Y. Ota, and Yamada, Agric.
Biol. Chem.30, 576(1966)
18. Semeriva, M., and C. Dufour, Biochim. Bio- phys. Acta 260, 393(1972)
19. Laboureur, P., and M. Labrousse, Bull. Soc.
Chim. Biol.48, 747(1969)
20. Kwon, D.Y., pH D Thesis, KAIST, Seoul, p.
87(1986)
21. Canioni, P., R. Juien, J. Rathelot, and L.
Sarda, Lψids 12, 393 (1977)
22. Sohn, H.S., S.S. Chung, and J.S. Rhee, Biote- chno!. Lett. (submitted)
(Received January 9, 1987)
