The lipase/lipoxygenase bienzyme system in AOT reversed micelles in octane

Article abstract of DOI:10.1023/A:1013954219627

In this work it is shown that the bienzyme lipase/lipoxygenase system can function in reversed micelles of bis(2-ethyl)hexyl sulfosuccinate (AOT) in octane. As a lipase substrate, a fish fat preparation (fat of sea mammals) with a high content of polyunsa

Full text of DOI:10.1023/A:1013954219627

Russian Journal of Bioorganic Chemistry, Vol. 28, No. 1, 2002, pp. 44–49. Translated from Bioorganicheskaya Khimiya, Vol. 28, No. 1, 2002, pp. 50–55.
Original Russian Text Copyright © 2002 by Pavlenko, Kuptsova, Klyachko, Levashov.
The Lipase/Lipoxygenase Bienzyme System
in AOT Reversed Micelles in Octane
1
I. M. Pavlenko* , O. S. Kuptsova, N. L. Klyachko, and A. V. Levashov
Moscow State University, Chemical Faculty, Vorob’evy gory, GSP-3 Moscow, 119899 Russia
Received January 17, 2001; in final form, March 30, 2001
Abstract—In this work it is shown that the bienzyme lipase/lipoxygenase system can function in reversed
micelles of bis(2-ethyl)hexyl sulfosuccinate (AOT) in octane. As a lipase substrate, a fish fat preparation (fat of
sea mammals) with a high content of polyunsaturated fatty acids was used. It was demonstrated that the bien-
zyme reaction proceeded in a stationary mode and had a rate-limiting step catalyzed by lipase. Under optimal
conditions, the efficacy of functioning of the bienzyme system was by an order of magnitude higher than that
in water. The lipase/lipoxygenase bienzyme system can be used as a new method of spectrophotometrical deter-
mination of lipase activity.
Key words: bienzyme system, fatty acids, lipase, lipoxygenase, reversed micelles, triglycerides
INTRODUCTION
than the aforementioned methods. However, its appli-
cation is limited, because phenyl esters of short-chain
fatty acids used as lipase substrates cannot be regarded
as suitable, specific lipase substrates. The methods in
which the amount of enzyme-released fatty acids is
measured are most applicable for the determination of
lipase activity [16, 17]. Colorimetric methods can con-
siderably enhance the sensitivity of the determination
of fatty acids [18, 19]. The pH-stating method allows
Lipase (triacylglyceride hydrolase, CE 3.1.1.3), an
enzyme hydrolyzing esters of glycerol and fatty acids,
widely occurs in animals [1], plants [2], and microor-
ganisms [3–5]. It plays a key role in lipid metabolism of
all organisms and contributes to deposition and mobili-
zation of fat, which serves as a source of cell energy.
Lipase, a biocatalyst with diverse substrate specificity,
is widely used nowadays in organic synthesis [6, 7], the determination of initial rates of lipase reactions, but
medicine [8–10], and food [11] and paper [12] indus- is relatively low sensitive and is applicable only at basic
pH values [20]. A highly sensitive fluorimetric method
using synthetic fluorogenic acyl glycerides should be
mentioned [21]. However, the preparation of triglycer-
ides containing a fluorophore at the end of the aliphatic
chain is not facile and makes this method expensive.
This work describes the study of the lipase/lipoxy-
genase bienzyme system in the AOT–water–octane
reversed micelles (Fig. 1). As the second enzyme,
lipoxygenase was chosen due to its ability to oxidize
polyunsaturated fatty acids resulted from the triglycer-
ide hydrolysis catalyzed by lipase (the first enzyme)
tries. In general, the determination of lipolytic activity
is not simple; the methods currently known are as a rule
rather complicated, not convenient in practice, and not
highly sensitive. For example, turbidimetric [13] and
stalagmometric methods [14], based on the measure-
ment of a difference in physical properties of the reac-
tion system are not reliable, since these properties
depend on many factors including pH and the presence
of metal ions, and are therefore not widely used. Colo-
rimetric and fluorometric methods based on the mea-
surement of the amount of released alcohols, for exam-
ple, the Fletcher method [15], ensure higher accuracy (Scheme).
lipase
lipoxygenase
fat/oil
unsaturated
fatty
Hydroperoxide of
fatty
acid
acid
Scheme
RESULTS AND DISCUSSION
(NPL) and linoleic acid, respectively, were used as sub-
strates. The kcat values for lipase in water exceeded
those in reversed micelles (Table). However, as is seen
in Fig. 2, the inhibitory effect observed in water was not
found in micelles. The value of kcat for lipoxygenase in
reversed micelles with a high water content virtually
To obtain kinetic characteristics of separately func-
tioning lipase and lipoxygenase, p-nitrophenyl laurate
1
To whom correspondence should be addressed; fax: +7 (095) 939-
3429; phone +7 (095) 939-3429; e-mail: ivankap@aport.ru.
1068-1620/02/2801-0044$27.00 © 2002 MAIK “Nauka /Interperiodica”

THE LIPASE/LIPOXYGENASE BIENZYME SYSTEM
Äéí
45
Octane
Lipase
Fatty acid
Lipoxygenase
Water
Triglyceride
Fig. 1. A scheme of the lipase/lipoxygenase bienzyme system in reversed micelles.
coincided with the kcat value in water, whereas Km con- matrix when the amount of water in the micelle was
small. When analyzing pH dependences of both
enzymes, one can conclude that pH 8.0 is optimal for
functioning of the bienzyme system lipase/lipoxygen-
ase in reversed micelles. To study the reaction kinetics
in the lipase/lipoxygenase bienzyme system, fish fat
(fat of sea mammals) was used as a lipase substrate,
which is characterized by a high content of polyunsat-
urated fatty acids in triglycerides. At its bienzyme
transformation both in water and in micelles, a lag
period was observed. According to the kinetics of bien-
zyme reactions [22], the lag period occurs provided that
the rate of the first step is much lower than that of the
second.
siderably increased. This may result from the fact that
the substrate concentrates in the organic phase (octane).
It was found in an independent experiment that lipase
in the linoleic acid/lipoxygenase system and lipoxyge-
nase in the NPL/lipase system within the working range
of concentrations did not affect each other’s activity
and, what is most important, did not display inhibitory
effects.
The pH dependences of initial rates of NPL hydrol-
ysis and linoleic acid oxidation catalyzed by lipase and
lipoxygenase, respectively, are given in Fig. 3. The pH
values for the reversed micelles corresponded to pH of
the buffer solution that was used for the preparation of
reversed micelles of the required hydration degree
(w0 = [H2O]/[AOT]). It was found that the optimum of
catalytic activity of lipase in reversed micelles at w0 25
and 15 is at pH 8.5, which corresponds to its pH opti-
mum in water. Lipoxygenase was most active in water
and reversed micelles at w0 25 at pH 7.0. In reversed
For the determination of the limiting stage of suc-
cessive transformations of fish fat, dependences of the
initial rates of the bienzyme reaction on the concentra-
tions of each enzyme were found. As is seen in Fig. 4,
the initial rate of the bienzyme process little depends on
the concentration of the other enzyme (lipoxygenase),
micelles with lower water content, the pH optimum of whereas for lipase (the first enzyme) this dependence is
lipoxygenase activity shifted to 8.0. This result may be linear. These dependences imply that the lipase-cata-
accounted for by an alteration of enzyme properties lyzed reaction was in this case the limiting stage of the
upon the induced contact with the AOT polyelectrolyte overall process. It is worth mentioning the lack of the
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 1 2002

46
PAVLENKO et al.
v× 108, å s–1
v × 109, å s–1
20
16
12
8
16
1
12
2
8
4
3
4
4
1
2
0
4
8
12
16
20
[S], mM
0
4
5
6
7
8
9
10
pH
Fig. 2. The dependence of the initial rate of NPL hydrolysis
catalyzed by lipase on the substrate concentration (1) in
water (0.1 M Tris, pH 8.0) and (2) in reversed micelles
(w 25, 0.05 M AOT in octane, 0.1 M Tris, pH 8.0); [E]
Fig. 3. The pH-effect on the catalytic activities of lipase
(1, 2) and lipoxygenase (3, 4) in reversed micelles; w 25
0
0
0
–6
–7
1.36 × 10 M.
(1, 3) and 15 (3, 4); [E] 4.5 × 10 M.
0
background hydrolysis of fish fat and the inability of
lipoxygenase to oxidize it. Thus, it is lipase activity that
is characterized by the rate observed.
responded to functioning of various protein forms
including its subunits. It is known that porcine pancre-
atic lipase is a monomeric protein with a molecular
mass of 50 kDa [25]. According to the principle of cor-
relation between the protein size and micelle internal
cavity at optimal w0, this protein would display the
maximum catalytic activity at w0 15. The presence of
another maximum at w0 25 may imply the ability of
lipase to form molecular aggregates, in this case,
according to calculation, a tetramer. It is known that the
dependence of the catalytic activity of soybean lipoxy-
genase and w0 does not have a clear maximum [26]; the
enzyme is most active at hydration degree w0 > 20. For
the lipase/lipoxygenase bienzyme system, the optimal
catalytic activity was observed at w0 25 (Fig. 7). A com-
parison of curves presented in Figs. 6 and 7 allows the
conclusion that the optimum for the bienzyme system
at w0 25 corresponds to the function of lipase tetramer.
The shoulder at the curve of dependence (Fig. 7) at
great w0 values can be related to the formation of more
complex bienzyme associates.
As is seen in Fig. 5, unlike the bienzymic transfor-
mation of fish fat in water solution, in the AOT–octane
reversed micelles the substrate did not show inhibitory
properties. The maximum rate of the reaction in
reversed micelles was by an order of magnitude higher
than in water.
It is known that the catalytic activity of many
enzymes depends on the hydration degree of detergent
molecules (a parameter characterizing the size of the
micelle internal cavity) [23]. This dependence is
described by a curve with a maximum. The optimal cat-
alytic activity of enzymes solubilized in reversed
micelles is as a rule observed when the sizes of the pro-
tein and micellar matrix correlate [24].
Figure 6 presents the dependence of the lipase cata-
lytic activity on the hydration degree, which is a curve
with two maximums at w0 15 and 25. Similar dependen-
ces with several maximums are observed for enzymes
that can function as associates of various oligomeric
compositions. In this case optimums on the curve cor-
The observed maximum rate of this bienzyme reac-
tion in micelles at varied w0 exceeds the maximum rate
The enzyme kinetic characteristics in water and in AOT–octane reversed micelles
Lipase
Lipoxygenase
Km × 103, M
System
kcat × 103, s–1
Km × 103, M
kcat, s–1
0.1 M Tris
22.3
1.5
3.7
3.9
1.4
9.4
5.9
5.1
2.1
0.4
1.9
2.0
4.4
29.5
26.1
20.8
0.05 M AOT, w0 10
0.05 M AOT, w0 15
0.05 M AOT, w0 25
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 1 2002

THE LIPASE/LIPOXYGENASE BIENZYME SYSTEM
47
v× 103, rel. unit
v× 103, rel. unit
5
40
30
20
10
1
2
4
3
2
1
6
4
2
1
1
0
0.05
0.10
[S], mg/ml
2
0
2
4
6
8
10
0
0.5
1.0
1.5
2.0
[S], mg/ml
[E] × 106, å
Fig. 5. The dependence of the initial rate of fish fat bien-
zyme transformation on the substrate concentration (1) in
Fig. 4. The dependence of the initial rate of the bienzyme
transformation of fish fat (S) in reversed micelles (w 25) on
0
water and (2) in reversed micelles (w 25). [Lipase] 4.5 ×
0
0
(1) the lipase and (2) lipoxygenase concentrations. [S]
1.136 mg/ml.
0
−6
–7
10 M; [lipoxygenase] 9.1 × 10 M.
0
max × 108, å s–1
V
max × 102, rel. unit
V
8
30
25
20
15
10
5
6
4
2
0
1
2
0
10
15 20 25 30 35 40 45
w0
8
12
16
20
24
28
32
w0
Fig. 7. The dependence of the maximum rate of fish fat
hydrolysis catalyzed by lipase/lipoxygenase system on the
Fig. 6. The dependence of the maximum rate of NPL
–6
AOT hydration degree. [E] 9.1 × 10 M (1). For compari-
0
hydrolysis catalyzed by lipase in reversed micelles on the
son, the level of maximum rate of this process in water is
given (2).
–6
AOT hydration degree. [E] 9.1 × 10 M.
0
in water by an order of magnitude. These data imply the
lack of diffusion hindrances in the process of redistribu-
tion of lipase and lipoxygenase substrates in the organic
phase, which emphasizes once again the advantage of
reversed micelles over water as a reaction medium.
unsaturated fatty acids, since the utilization of these
acids in human organism decreases, in particular, the
risk of cardiovascular diseases. In addition, the final
products of metabolism of unsaturated fatty acids that
contain a 1,4-cis,cis-pentadienyl fragment and are nat-
To conclude, we showed a possibility of the express ural substrates of lipoxygenases, represent biologically
and continuous determination of lipase activity using
the lipase/lipoxygenase bienzyme system in reversed
micelles. The system is facile in preparation and easy in
operation.
important compounds, such as leucotrienes, trombox-
anes, and prostaglandins implementing regulatory
functions in human organism [27, 28]. Therefore, the
lipase/lipoxygenase bienzyme system proposed by us
may be a convenient test system for the determination
One of the currently important problems in food
industry is the preparation of triglycerides containing of fatty acids in the oil and fat triglycerides, which
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 1 2002

48
PAVLENKO et al.
enables the assessment of their nutrient value. Appar- of the buffer solution in accordance with the required
ently, the replacement of lipase by phospholipase in the hydration degree and the lipoxygenase solution (10 µl,
system described will enable the determination of 2 mg/ml) were added. The resulting solution was ther-
phospholipase activity. The conjugated lipase/lipoxy- mostated for 2 min at 30°ë, and the lipase solution
genase system can be also convenient for studying the (10 µl, 50 mg/ml) was added. The reaction mixture was
lipase regioselectivity when triglycerides containing intensely shaken, and the accumulation of fatty acid
unsaturated fatty acids in various positions were used as hydroxyperoxide was registered at λ 245 nm at 30°ë.
substrates.
The reaction rate was expressed in optical absorption
units per minute (relative units). The background
hydrolysis of the substrate did not occur under experi-
mental conditions. It was found that lipoxygenase did
not oxidize fatty acids comprising triglycerides.
EXPERIMENTAL
Porcine pancreatic lipase (EC 3.1.1.3), soybean
lipoxygenase 1 (EC 1.13.11.12), p-nitrophenyl laurate,
linoleic acid, and sodium di(2-ethyl)hexyl sulfosucci-
nate (aerosol OT, AOT) were purchased from Sigma
(United States); tris(hydroxymethyl)aminomethane
(Tris) was from Fluka (Switzerland); sodium glycocho-
late was from Ferak (Germany); sodium hydrophos-
phate dodecahydrate, sodium dihydrophosphate, and
distilled octane were from Reakhim (Russia); the fish
fat preparation was purchased from a pharmacy.
ACKNOWLEDGMENTS
The work was supported by the state program
Advances in Bioengineering, Biocatalytic Technolo-
gies; NWO, project 047-007-005 (The Netherlands);
and NATO, project LST.CLG.974984.
REFERENCES
Kinetic parameters were measured at 30°ë on a Shi-
madzu UV-265 FW spectrophotometer (Japan) supple-
mented with a thermostatic cell. For experiments in
water, the substrate and enzyme solutions in 0.1 M Tris
(pH 8.0) containing 10 mM NaCl, 5 mM CaCl2, and
1 wt % sodium glycocholate were prepared directly
before the experiment. In reversed micelles, 0.05 M
AOT in octane and 0.1 M Tris (pH 8.0) were used.
The lipase activity was determined according to the
Fletcher method [15] under continual spectrophotomet-
ric registration at λ 400 nm on the bases of the initial
accumulation rate of the product (p-nitrophenol) in the
reaction mixture in the course of the enzymic hydroly-
sis of the substrate (NPL). The background hydrolysis
of the substrate did not occur under experimental con-
ditions.
Lipoxygenase activity was determined by the ini-
tial accumulation rate of the product (linoleic acid
hydroxyperoxide) in the reaction mixture in the course
of the enzymic oxidation of linoleic acid as in [26]. The
measurements were made using continuous spectro-
photometrical registration of the reaction kinetics at λ
234 nm in water and 245 nm in AOT–octane reversed
micelles.
To determine the maximum rate of the bienzyme
reaction in water, the reaction mixture containing
buffer solution (2.2 ml), the corresponding amount of
aqueous emulsion of fish fat at a concentration of
1. Carriere, F., Bezzine, S., and Verger, R., J. Mol. Catal. B,
1997, vol. 3, pp. 55–56.
2. Borgstrom, B. and Brockman, H.L., Lipases, Amster-
dam: Elsevier, 1984.
3. Jaeger, K.E., Ransas, S., Dijkstra, B.W., Colson, C.,
van Heuvel, M., and Misset, O., FEMS, Microbiol. Rev.,
1994, vol. 15, pp. 29–63.
4. Gilbert, E.J., Enzyme Microb. Technol., 1993, vol. 15,
pp. 634–645.
5. Wohlfahrt, S. and Jaeger, K.E., BioEngineering, 1993,
vol. 9, pp. 39–46.
6. Gutman, A.L. and Shapira, M., Adv. Biochem. Eng. Bio-
technol., 1995, vol. 52, pp. 87–128.
7. Chopineau, J., Kieboom, P.G., Klibanov, A.M., and
Riva, S., J. Am. Chem. Soc., 1998, vol. 110, pp. 584–589.
8. Wicke-Planquart, C., Canaan, S., Riviere, M., Dupuis, L.,
and Verger, R., Protein Eng., 1996, vol. 9, pp. 1225–
1232.
9. Carriere, F., Moreau, H., Raphel, V., Laugier, R., Beni-
court, C., Junien, J.L., and Verger, R., Eur. J. Biochem.,
1991, vol. 202, pp. 75–83.
10. Suzuki, A., Mizumoto, A., Sarr, M.J., and Dimagno, E.P.,
Gastroenterology, 1997, vol. 112, pp. 2048–2055.
11. Gudmundsson, B.O., Almarsson, O., and Haralds-
son, G.G., Tetrahedron. Lett., 1993, vol. 34, pp. 5791–
5794.
12. Fujita, Y., Matsukura, M., Hata, K., Shimoto, H.,
Sharyo, M., Skaguchi, H., and Gibson, K., Tappi J.,
1992, vol. 75, pp. 117–122.
2 mg/ml, and lipoxygenase solution (10 µl, 2 mg/ml) 13. Vogel, W.C. and Zieve, L., Clin. Chem. (Washington, D.C.),
1963, vol. 9, pp. 168–172.
was thermostated for 2 min at 30°ë followed by addi-
tion of the lipase solution (10 µl, 50 mg/ml). The kinet-
ics of accumulation of hydroxyperoxide of the fatty
acid was monitored at λ 234 nm. In reversed micelles,
the reaction mixture was prepared directly in the spec-
trophotometrical cuvette. To this end, fish fat was solu-
bilized in 0.05 M AOT solution in octane (2.2 ml) at a
concentration of 50 mg/ml, and the necessary amount
14. Rona, P. and Michaelis, L., Biochemistry, 1911, vol. 31,
pp. 345–351.
15. Fletcher, P.D.I. and Robinson, B.H., J. Chem. Soc.,
Faraday Trans. 1, 1985, vol. 81, pp. 2667–2679.
16. Dole, V.P., J. Clin. Invest., 1956, vol. 35, pp. 150–156.
17. Dole, V.P. and Meinertz, H., J. Biol. Chem., 1960,
vol. 235, pp. 2595–2601.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 1 2002

THE LIPASE/LIPOXYGENASE BIENZYME SYSTEM
49
18. Han, D., Kwon, D.Y., and Rhee, J.S., Agric. Biol. Chem., 24. Martinek, K., Levashov, A.V., Klyachko, N.L., Pan-
1987, vol. 51, pp. 615–618.
tin, V.I., and Berezin, I.V., Biochim. Biophys. Acta, 1981,
19. Walde, P., J. Amer. Oil. Chem. Soc., 1990, vol. 67,
vol. 657, pp. 277–294.
pp. 110–115.
25. Schmid, R.D. and Verger, R., Angew. Chem. Int. Ed.,
20. Mattson, F.H. and Volpenhein, R.A., J. Amer. Oil. Chem.
Soc., 1966, vol. 43, pp. 286–291.
1998, vol. 37, pp. 1608–1633.
21. Zandonella, G., Haalck, L., Spener, F., Paltauf, F., and
Hermetter, A., J. Mol. Catal., B: Enzym., 1997, vol. 3,
p. 127–130.
26. Kurganov, B.I., Shkarina, T.N., Malalhova, E.A., Davy-
dov, D.R., and Chebotareva, N.A., Biochimie, 1989,
vol. 71, pp. 573–578.
22. Varfolomeev, S.D. and Gurevich, K.G., Biokinetika
(Biokinetics), Moscow: Fair Press, 1999, pp. 280–289.
27. Marx, J.L., Science, 1982, vol. 215, pp. 1380–1382.
23. Martinek, K., Levashov, A.V., Klyachko, N.L.,
Khmel’nitskii, Yu.L., and Berezin, I.V., Biol. Membr.,
1985, vol. 2, pp. 669–696.
28. Lagaede, M., Gualde, N., and Rigaud, M., Biochemistry,
1989, vol. 257, pp. 313–320.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 1 2002