Evangelopoulos, A.E.

Papadimitriou, Vassiliki

Xenakis, Aristotelis

HELIOS Repository

ACTIVITY STUDIES OF CHYMOTRYPSIN 
IN MICROEMULSIONS 
V. Papadimitriou, A. Xenakis and A.E. Evangelopoulos 
Inst of Biological Research & Biotechnology, 
The National Hellenic Research Foundation, 
48, Vus. Constantinou Ave., Athens 
The interest of using water in oil microemulsions as a medium 
for enzymatic studies has been growing up, since it was shown 
that, generally, enzymes keep their biocatalytic ability which hosted 
in such microheterogeneous systems. In this case the enzyme 
molecules are not handled in classical aqueous buffer solutions, 
but in reverse micellar systems of water in organic solvents 
(microemulsions) (1,2). In these systems, which are thermodynami-
cally stable and optically isotropic solutions (3), the dispersed 
water phase acts like a microreactor where the enzyme transforms 
the substrates under restricted conditions, miming the in vivo con­
ditions. 
The composition of the microemulsions seems to play an im­
portant role on the enzyme behavior. By changing the nature of the 
amphiphile molecules, used for the formulation of these systems, 
the stability and the activity of the enzyme may be altered. 
Proteases have been considered as good model enzyme systems 
that can function in microemulsions (4,5,6). We have studied in a 
comparative basis the catalytic behavior of a-chymotrypsin in vari­
ous types of microemulsion systems formulated either with anionic 
(AOT) or cationic (CTAB) surfactants. We have examined the effect 
of many parameters related to the enzyme properties (pH, T) and 
to the microemulsion characteristics (water content, presence and 
nature of cosurfactant). 
In the anionic AOT/isooctane/water systems, the hydrolysis of 
Ν - glutaryl - L - phenylalanine - ρ - nitroanilide (GPNA) followed 
a Michaelis - Menten pattern, with a K
m
 value of 0.14 ± 0.01 mM, 
84 
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0 
11.5 
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7.5 
ί 
ι 
Ο 
Ο 
\ 
ο 
1
 ' ι 
5 10 14 18 
Wo 
• 
, φ φ ! 
• 
Ι 
• 
• 
ι 
• 
ο 10 15 20 25 
00 
U1 
Wo = [Η 2 0]/[Α0Τ] 
Fig. 1. Activity of a - chymotrypsin in AOT (50mM)Iisooctane microemulsions as a function of the hydration ratio 
which is similar to the value found in water (Km=0.1 mM). The 
catalysis strongly depended on the hydration ratio w 0 * 
(Η2θ]/[ΑΟΤ] of the reverse micelles and on the initial pH of the 
aqueous dispersed phase. A typical example is showed in Fig. 1 
where the enzyme activity is plotted vs. w 0 at pH » 8.6. The opti­
mum of the bell - shaped curve shifts towards lower values when 
the initial pH of the enzyme is increased (Fig. 1., inset.). 
The effect of the nature of the surfactant used was also tested 
by using the cationic amphiphile cetyltrimethylamrnonium bromide 
(CTAB) in the presence of a series of short - chained aliphatic 
alcohols as cosurfactants. The rate of hydrolysis of GPNA was con­
siderably slower in this case L :an in the anionic systems. When 
the hydrolysis of Ν - acetyl - tryptophan - ethyl ester (ATEE) was 
considered, it was found that the chain length of the cosurfactant 
used affected the kinetics, expressed in terms of the ratio Kcat/Km. 
Namely, this ratio varied from 4.5 10 M s for butanol, to 2.3 
103M _ 1 s"1 for pentanol and to 1.1 103M _ 1 s"1 for hexanol. These 
results are related to the variation of the polarity of the dispersed 
phase induced by the nature of the amphiphiles. 
REFERENCES 
1. Martinek, K., Levashov, A.V., Klyachko, N., Khmelnitski, Y. and 
Berezin, I.V. (1986) Eur. J. BiocherrL 155, 453 - 468. 
2. Luisi, P.L., Giomini, M., Pileni, M.P. and Robinson, B.H. (1988) 
Biochim. Biophys. Acta 947, 209 - 246. 
3. Danielsson, I. & Lindman, B. (1982) Colloids Surf. 3, 81 . 
4. Barbaric, S. and Luisi, P.L. (1981) J. Am. Chem. Soc. 103, 4239 
- 44. 
5. Martinek, K., Levashov, A.V., Klyachko, N., Pantin, V.l. and 
Berezin, I.V., (1981) Biochim. Biophys. Acta. 657, 277 - 294. 
6. Fletcher, P.D.I., Rees, G.D., Robinson, B.H. and Freedman, R.B. 
(1985) Biochim. Biophys. Acta. 832, 204 - 214. 
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Activity studies of chymotrypsin in microemulsions

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