Journal of Biotechnology, 24 (1992) 129-139 © 1992 Elsevier Science Publishers B.V. All rights reserved 0168-1656/92/$05.00
129
BIOTEC 00757
Control of enzyme properties in supramolecular systems F. Alfani, M. Cantarella, N. Cutarella and N. Spreti Department of Chemistry, Chemical Engineering and Materials, University of L'Aquila, Italy
R. Germani and G. Savelli Department of Chemistry, Uniuersityof Perugia, 06100 Perugia, Italy
(Received 21 January 1991; revision accepted 28 April 1991)
Summary The study deals with stability and activity of enzymes in supramolecular systems. Acid phosphatase (EC 3.1.3.2) has been studied as model enzyme. The organic phase is rich in C 2 - C 4 acetates. Didodecyldimethylammonium chloride (DDDACI) has been mainly used as ionic surfactants. The rate of enzyme inactivation is smaller than in buffer and is less dependent on storage temperature. Specific activity of the enzyme is lowered because of a less affinity towards the substrate and of reduction of maximal velocity. Acid phosphatase; Stability; Activity; Supramolecular system
Introduction The biocatalysis in organic solvents recently received great attention because of many potential advantages as regards bioreactions in aqueous media. Enzyme Correspondence to: Francesco Alfani, Dipartimento di Chimica, Ingegneria Chimica e Materiali, Universit~ di L'Aquila, Monteluco di Roio, 67040 L'Aquila, Italia.
130 recovery at the end of the process is easier and immobilization could become useless, since positive cooperations, which improve biocatalyst life time, could also occur. The conditions exist for lack of microbial contamination and facilitated down-stream processing. Finally, the increased substrate solubility and the decreased inhibition of substrate a n d / o r product together with more favorable biochemical equilibria, may determine higher reaction yields (Tramper et al., 1985; Dordick, 1989; Laane et al., 1987). On the other hand, the presence of very small amounts of water is always necessary in enzymatic reactions in order to preserve the active structure of the protein (Zaks and Klibanov, 1985). Therefore, two different media have been mainly investigated and results of bioconversions either in biphasic systems (water and immiscible organic solvents; Brink and Tramper, 1985; Semenov et al., 1987) or in supramolecular organized systems, as micelles and vesicles (Luisi et al., 1988), are reported in the literature. Solutions of surfactants in apolar organic solvents contain assemblies which are described as reverse or inverse micelles (Menger et al., 1973; Frendler, 1982; Flechter and Robinson, 1984; Luisi, 1985). These solutions may tolerate appreciable amounts of water and the assemblies can be regarded as 'water pools', i.e. microdroplets of water stabilized by surfactants with the polar or ionic residues in contact with the water and the apolar residues in contact with the organic solvent (Menger et al., 1973). The properties of these water pools depend upon the water-surfactants ratio: at low values, most of the water is interacting with the surfactant ions, while at high values, water has in the interior of the pool properties similar to those of bulk water (Kondo et al., 1982). The ionic and polar residues can enter these water droplets, which concentrate them, and bimolecular reactions of hydrophilic reagents are often very fast in solutions of reverse micelle (Frendler, 1982). The" rate effects on spor;taneous unimolecular reactions are due to changes in the submicroscopic reaction medium and can provide considerable informations on the surface structures of micro-assemblies (Romsted, 1984; Bunton and Savelli, 1986). These 'water pools' can be regarded as formally homogeneous micro-reactors and the use of surfactants in organic solvents for stabilizing the activity of soluble enzymes has been studied (Menger and Yamada, 1979; Martinek et al., 1981). In many reacting bio-systems the water necessitates as cosubstrate and a high water to organic chemical ratio facilitates the formation of a microenvironment similar to the one existing in vivo. This study deals with stability and activity of enzymes in solutions of surfactants in organic solvent-water media, i.e. in the presence of supramolecular organized systems (reverse micelles). In the literature many data which deal with the effects of surfactants on enzymic properties are available but little attention has been devoted to the effects caused by surfactants and supramolecular systems on biochemical reaction rates. Most of the studies are done with the anionic AOT surfactant and, in very few cases, in the presence of the cationic surfactant, cetyltrimethylammonium bromide (CTABr). The investigations mainly aim to understand the relationships between structure and property of supramolecular systems and chemistry and biochemistry of bound molecules.
131 The surfactants may control both the amount of solubilized water and the properties of organized systems. In physical, chemical and biological systems, organization can develop new functions (Ringsdorf et al., 1988). Since the micellization is strongly controlled by the length of the hydrophobic chains, by the nature of the head groups and of the counterions, various ionic and non ionic surfactants, with different structural features, have been synthesized and some of them have been tested in this work. Hydrolysis of p-nitrophenyl phosphate is studied in the presence of acetates. The activity and stability of acid phosphatase in different media are well known and this enzyme is largely adopted as model system (Cantarella et al., 1991). The experiments have been mainly carried out with didodecyldimethylammonium chloride (DDDACI) which is a good stabilizer of biocatalyst activity. The solubilization of large water volumes in the organic phase is allowed and the conditions of the enzymatic assay are not altered too much.
Experimental Materials The acid phosphatase (EC 3.1.3.2) used in this study is from Boehringer Biochemia (Italy). The following companies supplied the listed reagents and the substrate: Ega-Chemie (FRG), p-nitrophenyl phosphate (sodium salt); Baker Chemicals (Holland), ethyl acetate and i-amyl acetate; Aldrich Chemic (Germany), propyl acetate and n-butyl acetate. The solvents have been used without further purification. All other chemicals were pure grade reagents commercially available.
Surfactant preparation The preparation and the purification of single chain surfactants, cetyltripropylammonium bromide (CTPABr), cetyltributylammonium bromide (CTBABr), twin chain surfactants, didodecyldimethylammonium chloride (DDDACI) and bromide (DDDABr) and trioctylethylammonium bromide (TOEABr) have been previously described (Cipiciani et al., 1987; Bonan et al., 1990; Germani et al., 1990).
Assay of acid phosphatase activity The activity of acid phosphatase is determined in 50 mM sodium citrate/citric .acid buffer, pH 5.6, and 2 mM p-nitrophenyl phosphate, unless otherwise specified. The p-nitrophenol liberated under the experimental conditions is measured spectrophotometrically at 405 nm. The alkalinization of reaction samples is carried out with an equal volume of NaOH solution (1 N). A molar absorbance coefficient of 18 500 l mol-1 cm-1 is used to evaluate product concentration.
132
Tests of water solubilization. The water solubilization in the acetates has been initially tested. Surfactant concentration is always 0.1 M, the volume of organic phase is 2 ml and the temperature is kept constant at 30°C. Small volumes of water are progressively added under stirring. This is stopped at regular intervals and the presence of phase separation is checked. Stability of acid phosphatase.
Acid phosphatase is stored at 30°C under stirred conditions; 50 ~1 of enzymatic solution (2.5 mg m1-1) are added to 1.95 ml of acetate and DDDACI. Surfactant concentration in the organic solvent ranges from 0.5 to 0.15 M. Temperature is varied between 30 and 45°C. At regular time intervals samples (80 /zl) are withdrawn from the vessel and assay of residual activity is carried out at the following conditions: T = 30°C, 50 mM Na-citrate buffer pH 5.6, 2 mM substrate, 15 min of incubation.
Activity of acid phosphatase in supramolecular systems A part of this study has been devoted to investigate the effect of media composition on the activity of acid phosphatase. All the experiments have been carried out in a stirred and thermostated batch reactor. Temperature is kept constant at 30°C and enzyme concentration is 5 /xg m1-1. Conditions of either differential or integral reactor have been explored. The p-nitrophenyl phosphate and p-nitrophenol typify problems posed by partitioning into the two phases. The substrate is retained in the aqueous phase while all the product moves in the organic one (Cantarella et al., 1991).
Results and Discussion
Water solubilization As previously pointed out and as recently shown (Germani et al., 1990) water tends to organize the surfactant in order to minimize its contact with the organic solvent. The addition of small amount of water causes hydration of head groups and of counterions in monomeric forms. If the amount of water is increased, the surfactants are forced to cooperatively organize themselves in new supramolecular structures which can accommodate more water molecule per surfactant molecule. Obviously this possibility strongly depends on the hydrophobic and hydrophilic residues peculiar of each surfactant. Phase separation should be observed if favourable conditions for the reorganization process do not occur. In Table 1 values are reported which refer to the system butyl acetate and water. The amount of dissolved water depends on the structure of surfactants and the phase separation is observed above the critical value. Surfactants with single (CTPABr and CTBABr), twin (DDDACI and D D D A B r ) and triple chains (TOEABr) of hydrophobic residues have been studied. Methyl, butyl and propyl radicals have been investigated as head groups. Bromide and chloride are the
133 TABLE 1 Water solubilityin 2 ml of butyl acetate Surfactant Cetyltripropylammoniumbromide Cetyltributylammoniumbromide Didodecyldimethylammoniumchloride Didodecyldimethylammoniumbromide Trioctylethylammoniumbromide
CTPABr CTBABr DDDACI DDDABr TOEABr
Water (~1) 70 30 80 40 40
Surfactant concentration 0.1 M, temperature 30°C. counterions. Since the organic phase retains the highest quantity of water in the presence of DDDAC1, this surfactant has been used in this preliminary investigation. The possibility of varying composition of media during storage and assay of the enzyme activity in a wide spectrum is important for the understanding of enzyme behaviour.
Stability of acid phosphatase Since the lack of enzyme stability is one of the present limits to industrial application of bioreactors the first part of the kinetic characterization of the enzyme in supramolecular systems has been devoted to determine the deactivation rate. On the basis of previous studies (Menger and Yamada, 1979; Martinek et al., 1981; Bunton and Savelli, 1986; Germani et al., 1990), the organization of surfactants may be regarded as 'water pools' micelles and the enzyme should be confined into the reverse micelle or close to the boundary surface. The results achieved in the presence of ethyl, propyl, butyl and i-amyl acetate are plotted in a semilog diagram (Fig. 1) and compared with data of enzyme stability in aqueous buffer and in distilled water. This representation of experimentally determined enzyme deactivation is usually adopted in the literature since it allows a rapid identification of a first-order kinetics (Greco et al., 1979). The results clarify that such a deactivation mechanism holds for acid phosphatase under all the operational conditions investigated in this study. Comparison with stability in distilled water is also necessary since the organic solutions are always prepared with distilled water because of the instability of the supramolecular organization when buffer ions are present in the media. All the lines show two different slopes which indicate two different values of the deactivation rate constants, kal and ka2. The rate transition occurs at storage periods and extent of inactivation which are characteristics of each system. The rapid initial inactivation is less important using propyl, butyl and ethyl acetate and justifies the observed higher stability of acid phosphatase in these solvents. In Figs. 2 and 3 the dependence of enzyme inactivation on molar concentration of the surfactant and temperature is reported. Results show that the lower
134 lOO -_
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.
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.
.
.
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.
.
.
. 300
'
400
f, m i n Fig. 1. Thermal stability of acid phosphatase at 30°C. Enzyme concentration 62.5 ~g ml-~. ~,, ethyl acetate; e, propyl acetate; , , butyl acetate; O, i-amyl acetate; 6 #, 5o mM Na-citrate buffer, pH 5.6; • , distilled water.
DDDACI molarity the higher initial rate of deactivation. On the other hand, kd2 value is almost unaffected by variations of surfactant concentration. Both rate constants of first and second deactivation depend on storage temperature. Activation energies of the two decay mechanisms were measured in experiments per-
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13
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•
•
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300
f, rain Fig. 2. Thermal stability of acid phosphatase as function of DDDACI molarity in butyl acetate at 30°C and 62.5/~g ml -~ of e n z y m e . . , 0.05 M; ~,, 0.075 M; O, 0.10 M; e, 0.15 M.
135
~1
0
0
~
"~ 0 0 0
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100
~,mln
200
300
Fig. 3. Thermal stability of acid phosphatase as function of temperature at 0.1 M DDDACI in butyl acetate and 62.5/zg ml- 1 of enzyme. @, 3 0 ° C ; . , 35°C; Q, 40°C; &, 45°C.
formed with a constant concentration of acid phosphatase stored in butyl acetate and 0.1 M DDDAC1 between 30 and 45°C and they were Eal = 20230 cal tool -1 and Ea2 = 14 460 cal mol-1. These values are much lower than those measured for the initial rate of acid phosphatase deactivation in buffered media, Eab = 34 700 cal m o l - 1 (Alfani et al., 1989). The following conclusions arise from this part of the investigation. Inactivation of acid phosphatase can be lowered using solutions of C 2 - C 4 acetates rich in 0.1 M D D D A C I . E n h a n c e m e n t of stability can be further increased using higher surfactant concentration (0.15 M) but the achievement is not enough to justify the higher consumption of surfactant and the decrease of specific activity, as afterwards discussed. The resort to organized supramolecular systems gives larger benefits at high t e m p e r a t u r e because of the lower activation energy than that in buffered media.
Enzyme activity O t h e r experiments have been set up to study the dependence of acid phosphatase kinetics on the composition of reaction media. The rate of hydrolysis obeys a M i c h a e l i s - M e n t e n equation. The values of Michaelis constant g m and maxim u m velocity Vm have been evaluated according to the L i n e w e a v e r - B u r k analysis from data of reaction rate at different substrate concentrations. In aqueous buffer, K m is 0.597 m M and Vm is 2.73 /xmol min -1 per mg E. The addition of 0.1 M D D D A C I in the aqueous buffer causes the increase of Michaelis constant, K m = 1.48 mM, while it does not affect Vm. Similar results are achieved in organic phases differently rich in surfactant. Experiments m a d e with butyl and propyl acetate at
136 1
:~0.8
0.6
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~0.2
o
o
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80 lOO 12o 14o 16o 18o f, h Fig. 4. Product concentration vs. process time at 30°C and 5/zg m1-1 of enzyme. , , butyl acetate and 0.04 M DDDACI; &, butyl acetate and 0.1 M DDDACI; Q, propyl acetate and 0.04 M DDDACI; O, propyl acetate and 0.1 M DDDACI.
two DDDACI concentrations, 0.04 and 0.1 M, and 3% v / v of aqueous phase show that both K m and Vm depend on media composition but exhibit opposite trends. The increase of the DDDAC1 concentration from 0.04 to 0.1 M in butyl acetate causes K m variation from 1.54 to 0.98 mM and Vm reduction from 1.914 to 0.68 /zmol min -~ per mg E. Moreover, K m is the same in butyl and propyl acetate but maximum velocity is 18% lower in propyl acetate. The surfactant seems to affect enzyme affinity towards the substrate while the organic chemicals decrease its specific activity. Therefore, the observed enhancement of enzyme stability and the measured reduction of biocatalyst activity typify problems of optimization of reaction conditions. Temperature, surfactant concentration and organic phase should be selected in order to assure a prolonged reuse of the enzyme, which could balance the loss of specific reaction rate. In such a way, productions per unit weight of protein higher than that in aqueous environments are possible. A first attempt is shown in Fig. 4 which refers to experiments carried out at 30°C with both propyl and butyl acetate and at two concentrations of DDDACI, 0.04 and 0.1 M. Enzyme and substrate concentrations are 5 / x g ml -~ and 2 mM respectively. The reactor is stirred and total volume is 25 ml. The aqueous phase is 3% by volume. Product concentration is plotted vs. process time and the slopes of the curves indicate that the enzyme is still active after 120-140 h of reaction. At lower DDDACI concentration, 0.04 M, p-nitrophenol formation is higher and propyl acetate creates a better environment to preserve the native activity of the enzyme. Biocatalysis in supramolecular systems is a complex phenomenon. The prediction of reaction yield can not be exclusively formulated on the basis of this
137
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Fig. 5. Specific activity of acid phosphatase vs. water to surfactant ratio at 30°C and 5 /zg ml - I of enzyme in the presence of 3% v / v water, m , propyl acetate; • , butyl acetate.
preliminary investigation and deserves analysis of further experiments. These have shown that specific activity of the enzyme also depends on the molar ratio of water to surfactant (R) and the total volume of aqueous phase. Results are plotted in
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Fig. 6. Specific activity of acid phosphatase vs. water volume at 30°C, 5 / z g m l - 1 of enzyme, 2 ml total volume, m , propyl acetate, R = 5 0 ; - ~ , propyl acetate, R = 20.15; • , butyl acetate, R = 50; ~,, butyl acetate, R = 20.15.
138 Figs. 5 and 6. Experiments have been carried out at 30°C in stirred batch reactors, 2 ml, constant volume of aqueous phase, 60/zl, saturating substrate concentration, 2 mM, and with 5/xg m1-1 of acid phosphatase. Product formation during 20 min of incubation was measured under differential reaction conditions. At first, different quantities of surfactant have been used in order to investigate a range of R between 17.2 and 52.4. Surfactant concentration and water quantity have been successively varied keeping constant the value of R. Data of Fig. 5 show that the specific activity of acid phosphatase is an increasing function of R and is roughly the same in butyl and propyl acetate with the exception of media very rich in DDDAC1. It also appears in Fig. 6 that the curves of specific activity vs. water volume present maxima which depend on the value of R and the organic chemicals. The highest specific activity of the enzyme is always observed at R = 50 and 2% v / v of water in the system. If the amount of surfactant is increased, up to R = 20.15, the enzyme activity decreases and the maximum occurs at 3.5% v / v of aqueous phase in propyl acetate and at roughly 2.5% v / v in butyl acetate. Finally, activity of acid phosphatase in propyl acetate is more dependent than in butyl acetate on composition.
Conclusions Cationic surfactants have been prepared which allow a supramolecular organization in apolar organic solvents and good retention of water quantities, up to 4 - 5 % v/v. Stability of acid phosphatase in these media is improved especially above room temperature. Specific activity of the enzyme depends on the organic solvent, the water to surfactant ratio and the total volume of aqueous phase. In general, affinity towards the substrate and maximum velocity are worse than the ones in buffer. However, operational conditions can be selected in order to assure prolonged reuse of the biocatalyst and to achieve high yield of overall production per unit weight of enzyme. This behaviour has been observed working with a hydrolytic enzyme and a substrate totally confined in the aqueous environment. Better results could be expected with other enzymes and water insoluble substrates.
Acknowledgment This study was supported by Italian Ministry of Public Education (MURST) and by National Research Council (CNR).
References Alfani, F., Cantarella, L., Cantarella, M., Gallifuoco,A. and Scardi, V. (1989) Thermodynamicanalysis of the stability enhancement of acid phosphatase by gel immobilizationin proteins. Lat. Am. Appl. J. 20, 47-51.
139 Bonan, C., Germani, R., Ponti, P.P., Savelli, G., Cerichelli, G. and Bunton, C.A. (1990) Micellar headgroup size and anion nucleophilicity in SN2 reactions. J. Phys. Chem. 94, 5331-5335. Brink, L.E.S. and Tramper, J. (1985) Optimization of organic solvent in multiphase biocatalysis. Biotechnol. Bioeng. 27, 1258-1269. Bunton, C.A. and Savelli, G. (1986) Organic reactivity in aqueous micelles and similar assemblies. Adv. Phys. Org. Chem. 22, 213-309. Cantarella, M., Cantarella, L. and Alfani, F. (1991) Hydrolytic reactions in two phase systems. Effect of water-immiscible organic solvents on stability and activity of acid phosphatase, /3-glucosidase and /3-fructofuranosidase. Enzyme Microb. Technol. 13, 547-553. Cipiciani, A., Germani, R., Savelli, G., Bunton, C.A., Mhala, M. and Moffatt, J.R. (1987) The effects of single- and twin-tailed ionic surfactants upon aromatic nucleophilic substitution. J. Chem. Soc. Perkin II, 541-546. Dordick, J.S. (1989) Enzymatic catalysis in monophasic organic solvents. Enzyme Microb. Technol. 11, 194-211. Fletcher, P.D.I. and Robinson, B.H. (1984) Effect of organized surfactant system on the kinetics of metal ligand complex formation and dissociation. J. Chem. Soc. Faraday Trans. I, 80, 2417-2437. Frendler, J.H. (1982) Membrane Mimetic Chemistry. Wiley-Interscience, New York. Germani, R., Ponti, P.P., Spreti, N., Savelli, G., Cipiciani, A., Cerichelli, G., Bunton, C.A. and Si, V. (1990) Decarboxylation of e-nitrobenzisoxazole-3-carboxylate ion in dichloromethane: the possible role of reverse micelles. J. Colloid Interface Sci. 138, 443-450. Greco, G., Albanesi, D., Cantarella, M., Gianfreda, L., Palescandolo, R. and Scardi, V. (1979) Enzyme inactivation and stabilization studies in an ultrafiltration reactor. Eur. J. Appl. Microbiol. Biotechnol. 8, 249-261. Kondo, H., Miwa, I. and Sunamoto, J. (1982) Biphasic structure model for reversed micelles. Depressed acid dissociation of excited-state pyranine in the restricted reaction field. J. Phys. Chem. 86, 4826-4831. Laane, C., Boeren, S., Vos, K. and Veerger, C. (1987) Rules for optimization of biocatalysis in organic solvents. Biotechnol. Bioeng. 30, 81-87. Luisi, P.L. (1985) Enzymes hosted in reverse micelles in hydrocarbon solution. Angew. Chem. Int. Ed. Eng. 24, 439-450. Luisi, P.L., Giomini, M., Pileni, M.P. and Robinson, B.H. (1988) Reverse micelles as hosts for proteins and small molecules. Biochim. Biophys. Acta 947, 209-246. Martinek, K., Levashov, A.V., Klyachko, N.L., Pantin, V.I. and Berezin I.V. (1981) The principles of enzyme stabilization. VI. Catalysis by water-soluble enzymes entrapped into reversed micelles of surfactants in organic solvents. Biochim. Biophys. Acta 657, 277-294. Menger, F.M., Donohue, J.A. and Williams, R.F. (1973) C~talysis in water pools. J. Am. Chem. Soc. 95, 286-288. Menger, F.M. and Yamada, K. (1979) Enzyme catalysis in water pools. J. Am. Chem. Soc. 101, 6731-6734. Ringsdorf, H., Schlarb, B. and Venzmer, J. (1988) Molecular architecture and function of polymeric oriented systems: models for the study of organization, surface recognition, and dynamic of biomembranes. Angew. Chem. Int. Ed. Engl. 27, 113-158. Romsted, L.S. (1984) Micellar effects on reaction rates and equilibria. In: Mittal, K.L. and Lindman, B. (Eds.), Surfactants in solution, Plenum, New York, Vol. 2, pp. 1015-1068. Semenov, A.N, Klmelnitski, Y.L., Berezin, I.V. and Martinek, K. (1987) Water-organic solvent two-phase systems as media for biocatalytic reactions: the potential for shifting chemical equilibria towards higher yield of end products. Biocatalysis i, 3-8. Tramper, J., Van der Plas, H.C. and Linko, P. (Eds.) (1985) Biocatalysts in Organic Synthesis, Elsevier, Amsterdam/New York. Zaks, A. and Klibanov, A. (1985) Enzyme-catalyzed processes in organic solvents. Proc. Nat. Acad. Sci. USA, 82, 3192-3196.
129
BIOTEC 00757
Control of enzyme properties in supramolecular systems F. Alfani, M. Cantarella, N. Cutarella and N. Spreti Department of Chemistry, Chemical Engineering and Materials, University of L'Aquila, Italy
R. Germani and G. Savelli Department of Chemistry, Uniuersityof Perugia, 06100 Perugia, Italy
(Received 21 January 1991; revision accepted 28 April 1991)
Summary The study deals with stability and activity of enzymes in supramolecular systems. Acid phosphatase (EC 3.1.3.2) has been studied as model enzyme. The organic phase is rich in C 2 - C 4 acetates. Didodecyldimethylammonium chloride (DDDACI) has been mainly used as ionic surfactants. The rate of enzyme inactivation is smaller than in buffer and is less dependent on storage temperature. Specific activity of the enzyme is lowered because of a less affinity towards the substrate and of reduction of maximal velocity. Acid phosphatase; Stability; Activity; Supramolecular system
Introduction The biocatalysis in organic solvents recently received great attention because of many potential advantages as regards bioreactions in aqueous media. Enzyme Correspondence to: Francesco Alfani, Dipartimento di Chimica, Ingegneria Chimica e Materiali, Universit~ di L'Aquila, Monteluco di Roio, 67040 L'Aquila, Italia.
130 recovery at the end of the process is easier and immobilization could become useless, since positive cooperations, which improve biocatalyst life time, could also occur. The conditions exist for lack of microbial contamination and facilitated down-stream processing. Finally, the increased substrate solubility and the decreased inhibition of substrate a n d / o r product together with more favorable biochemical equilibria, may determine higher reaction yields (Tramper et al., 1985; Dordick, 1989; Laane et al., 1987). On the other hand, the presence of very small amounts of water is always necessary in enzymatic reactions in order to preserve the active structure of the protein (Zaks and Klibanov, 1985). Therefore, two different media have been mainly investigated and results of bioconversions either in biphasic systems (water and immiscible organic solvents; Brink and Tramper, 1985; Semenov et al., 1987) or in supramolecular organized systems, as micelles and vesicles (Luisi et al., 1988), are reported in the literature. Solutions of surfactants in apolar organic solvents contain assemblies which are described as reverse or inverse micelles (Menger et al., 1973; Frendler, 1982; Flechter and Robinson, 1984; Luisi, 1985). These solutions may tolerate appreciable amounts of water and the assemblies can be regarded as 'water pools', i.e. microdroplets of water stabilized by surfactants with the polar or ionic residues in contact with the water and the apolar residues in contact with the organic solvent (Menger et al., 1973). The properties of these water pools depend upon the water-surfactants ratio: at low values, most of the water is interacting with the surfactant ions, while at high values, water has in the interior of the pool properties similar to those of bulk water (Kondo et al., 1982). The ionic and polar residues can enter these water droplets, which concentrate them, and bimolecular reactions of hydrophilic reagents are often very fast in solutions of reverse micelle (Frendler, 1982). The" rate effects on spor;taneous unimolecular reactions are due to changes in the submicroscopic reaction medium and can provide considerable informations on the surface structures of micro-assemblies (Romsted, 1984; Bunton and Savelli, 1986). These 'water pools' can be regarded as formally homogeneous micro-reactors and the use of surfactants in organic solvents for stabilizing the activity of soluble enzymes has been studied (Menger and Yamada, 1979; Martinek et al., 1981). In many reacting bio-systems the water necessitates as cosubstrate and a high water to organic chemical ratio facilitates the formation of a microenvironment similar to the one existing in vivo. This study deals with stability and activity of enzymes in solutions of surfactants in organic solvent-water media, i.e. in the presence of supramolecular organized systems (reverse micelles). In the literature many data which deal with the effects of surfactants on enzymic properties are available but little attention has been devoted to the effects caused by surfactants and supramolecular systems on biochemical reaction rates. Most of the studies are done with the anionic AOT surfactant and, in very few cases, in the presence of the cationic surfactant, cetyltrimethylammonium bromide (CTABr). The investigations mainly aim to understand the relationships between structure and property of supramolecular systems and chemistry and biochemistry of bound molecules.
131 The surfactants may control both the amount of solubilized water and the properties of organized systems. In physical, chemical and biological systems, organization can develop new functions (Ringsdorf et al., 1988). Since the micellization is strongly controlled by the length of the hydrophobic chains, by the nature of the head groups and of the counterions, various ionic and non ionic surfactants, with different structural features, have been synthesized and some of them have been tested in this work. Hydrolysis of p-nitrophenyl phosphate is studied in the presence of acetates. The activity and stability of acid phosphatase in different media are well known and this enzyme is largely adopted as model system (Cantarella et al., 1991). The experiments have been mainly carried out with didodecyldimethylammonium chloride (DDDACI) which is a good stabilizer of biocatalyst activity. The solubilization of large water volumes in the organic phase is allowed and the conditions of the enzymatic assay are not altered too much.
Experimental Materials The acid phosphatase (EC 3.1.3.2) used in this study is from Boehringer Biochemia (Italy). The following companies supplied the listed reagents and the substrate: Ega-Chemie (FRG), p-nitrophenyl phosphate (sodium salt); Baker Chemicals (Holland), ethyl acetate and i-amyl acetate; Aldrich Chemic (Germany), propyl acetate and n-butyl acetate. The solvents have been used without further purification. All other chemicals were pure grade reagents commercially available.
Surfactant preparation The preparation and the purification of single chain surfactants, cetyltripropylammonium bromide (CTPABr), cetyltributylammonium bromide (CTBABr), twin chain surfactants, didodecyldimethylammonium chloride (DDDACI) and bromide (DDDABr) and trioctylethylammonium bromide (TOEABr) have been previously described (Cipiciani et al., 1987; Bonan et al., 1990; Germani et al., 1990).
Assay of acid phosphatase activity The activity of acid phosphatase is determined in 50 mM sodium citrate/citric .acid buffer, pH 5.6, and 2 mM p-nitrophenyl phosphate, unless otherwise specified. The p-nitrophenol liberated under the experimental conditions is measured spectrophotometrically at 405 nm. The alkalinization of reaction samples is carried out with an equal volume of NaOH solution (1 N). A molar absorbance coefficient of 18 500 l mol-1 cm-1 is used to evaluate product concentration.
132
Tests of water solubilization. The water solubilization in the acetates has been initially tested. Surfactant concentration is always 0.1 M, the volume of organic phase is 2 ml and the temperature is kept constant at 30°C. Small volumes of water are progressively added under stirring. This is stopped at regular intervals and the presence of phase separation is checked. Stability of acid phosphatase.
Acid phosphatase is stored at 30°C under stirred conditions; 50 ~1 of enzymatic solution (2.5 mg m1-1) are added to 1.95 ml of acetate and DDDACI. Surfactant concentration in the organic solvent ranges from 0.5 to 0.15 M. Temperature is varied between 30 and 45°C. At regular time intervals samples (80 /zl) are withdrawn from the vessel and assay of residual activity is carried out at the following conditions: T = 30°C, 50 mM Na-citrate buffer pH 5.6, 2 mM substrate, 15 min of incubation.
Activity of acid phosphatase in supramolecular systems A part of this study has been devoted to investigate the effect of media composition on the activity of acid phosphatase. All the experiments have been carried out in a stirred and thermostated batch reactor. Temperature is kept constant at 30°C and enzyme concentration is 5 /xg m1-1. Conditions of either differential or integral reactor have been explored. The p-nitrophenyl phosphate and p-nitrophenol typify problems posed by partitioning into the two phases. The substrate is retained in the aqueous phase while all the product moves in the organic one (Cantarella et al., 1991).
Results and Discussion
Water solubilization As previously pointed out and as recently shown (Germani et al., 1990) water tends to organize the surfactant in order to minimize its contact with the organic solvent. The addition of small amount of water causes hydration of head groups and of counterions in monomeric forms. If the amount of water is increased, the surfactants are forced to cooperatively organize themselves in new supramolecular structures which can accommodate more water molecule per surfactant molecule. Obviously this possibility strongly depends on the hydrophobic and hydrophilic residues peculiar of each surfactant. Phase separation should be observed if favourable conditions for the reorganization process do not occur. In Table 1 values are reported which refer to the system butyl acetate and water. The amount of dissolved water depends on the structure of surfactants and the phase separation is observed above the critical value. Surfactants with single (CTPABr and CTBABr), twin (DDDACI and D D D A B r ) and triple chains (TOEABr) of hydrophobic residues have been studied. Methyl, butyl and propyl radicals have been investigated as head groups. Bromide and chloride are the
133 TABLE 1 Water solubilityin 2 ml of butyl acetate Surfactant Cetyltripropylammoniumbromide Cetyltributylammoniumbromide Didodecyldimethylammoniumchloride Didodecyldimethylammoniumbromide Trioctylethylammoniumbromide
CTPABr CTBABr DDDACI DDDABr TOEABr
Water (~1) 70 30 80 40 40
Surfactant concentration 0.1 M, temperature 30°C. counterions. Since the organic phase retains the highest quantity of water in the presence of DDDAC1, this surfactant has been used in this preliminary investigation. The possibility of varying composition of media during storage and assay of the enzyme activity in a wide spectrum is important for the understanding of enzyme behaviour.
Stability of acid phosphatase Since the lack of enzyme stability is one of the present limits to industrial application of bioreactors the first part of the kinetic characterization of the enzyme in supramolecular systems has been devoted to determine the deactivation rate. On the basis of previous studies (Menger and Yamada, 1979; Martinek et al., 1981; Bunton and Savelli, 1986; Germani et al., 1990), the organization of surfactants may be regarded as 'water pools' micelles and the enzyme should be confined into the reverse micelle or close to the boundary surface. The results achieved in the presence of ethyl, propyl, butyl and i-amyl acetate are plotted in a semilog diagram (Fig. 1) and compared with data of enzyme stability in aqueous buffer and in distilled water. This representation of experimentally determined enzyme deactivation is usually adopted in the literature since it allows a rapid identification of a first-order kinetics (Greco et al., 1979). The results clarify that such a deactivation mechanism holds for acid phosphatase under all the operational conditions investigated in this study. Comparison with stability in distilled water is also necessary since the organic solutions are always prepared with distilled water because of the instability of the supramolecular organization when buffer ions are present in the media. All the lines show two different slopes which indicate two different values of the deactivation rate constants, kal and ka2. The rate transition occurs at storage periods and extent of inactivation which are characteristics of each system. The rapid initial inactivation is less important using propyl, butyl and ethyl acetate and justifies the observed higher stability of acid phosphatase in these solvents. In Figs. 2 and 3 the dependence of enzyme inactivation on molar concentration of the surfactant and temperature is reported. Results show that the lower
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.
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f, m i n Fig. 1. Thermal stability of acid phosphatase at 30°C. Enzyme concentration 62.5 ~g ml-~. ~,, ethyl acetate; e, propyl acetate; , , butyl acetate; O, i-amyl acetate; 6 #, 5o mM Na-citrate buffer, pH 5.6; • , distilled water.
DDDACI molarity the higher initial rate of deactivation. On the other hand, kd2 value is almost unaffected by variations of surfactant concentration. Both rate constants of first and second deactivation depend on storage temperature. Activation energies of the two decay mechanisms were measured in experiments per-
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f, rain Fig. 2. Thermal stability of acid phosphatase as function of DDDACI molarity in butyl acetate at 30°C and 62.5/~g ml -~ of e n z y m e . . , 0.05 M; ~,, 0.075 M; O, 0.10 M; e, 0.15 M.
135
~1
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Fig. 3. Thermal stability of acid phosphatase as function of temperature at 0.1 M DDDACI in butyl acetate and 62.5/zg ml- 1 of enzyme. @, 3 0 ° C ; . , 35°C; Q, 40°C; &, 45°C.
formed with a constant concentration of acid phosphatase stored in butyl acetate and 0.1 M DDDAC1 between 30 and 45°C and they were Eal = 20230 cal tool -1 and Ea2 = 14 460 cal mol-1. These values are much lower than those measured for the initial rate of acid phosphatase deactivation in buffered media, Eab = 34 700 cal m o l - 1 (Alfani et al., 1989). The following conclusions arise from this part of the investigation. Inactivation of acid phosphatase can be lowered using solutions of C 2 - C 4 acetates rich in 0.1 M D D D A C I . E n h a n c e m e n t of stability can be further increased using higher surfactant concentration (0.15 M) but the achievement is not enough to justify the higher consumption of surfactant and the decrease of specific activity, as afterwards discussed. The resort to organized supramolecular systems gives larger benefits at high t e m p e r a t u r e because of the lower activation energy than that in buffered media.
Enzyme activity O t h e r experiments have been set up to study the dependence of acid phosphatase kinetics on the composition of reaction media. The rate of hydrolysis obeys a M i c h a e l i s - M e n t e n equation. The values of Michaelis constant g m and maxim u m velocity Vm have been evaluated according to the L i n e w e a v e r - B u r k analysis from data of reaction rate at different substrate concentrations. In aqueous buffer, K m is 0.597 m M and Vm is 2.73 /xmol min -1 per mg E. The addition of 0.1 M D D D A C I in the aqueous buffer causes the increase of Michaelis constant, K m = 1.48 mM, while it does not affect Vm. Similar results are achieved in organic phases differently rich in surfactant. Experiments m a d e with butyl and propyl acetate at
136 1
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~0.2
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80 lOO 12o 14o 16o 18o f, h Fig. 4. Product concentration vs. process time at 30°C and 5/zg m1-1 of enzyme. , , butyl acetate and 0.04 M DDDACI; &, butyl acetate and 0.1 M DDDACI; Q, propyl acetate and 0.04 M DDDACI; O, propyl acetate and 0.1 M DDDACI.
two DDDACI concentrations, 0.04 and 0.1 M, and 3% v / v of aqueous phase show that both K m and Vm depend on media composition but exhibit opposite trends. The increase of the DDDAC1 concentration from 0.04 to 0.1 M in butyl acetate causes K m variation from 1.54 to 0.98 mM and Vm reduction from 1.914 to 0.68 /zmol min -~ per mg E. Moreover, K m is the same in butyl and propyl acetate but maximum velocity is 18% lower in propyl acetate. The surfactant seems to affect enzyme affinity towards the substrate while the organic chemicals decrease its specific activity. Therefore, the observed enhancement of enzyme stability and the measured reduction of biocatalyst activity typify problems of optimization of reaction conditions. Temperature, surfactant concentration and organic phase should be selected in order to assure a prolonged reuse of the enzyme, which could balance the loss of specific reaction rate. In such a way, productions per unit weight of protein higher than that in aqueous environments are possible. A first attempt is shown in Fig. 4 which refers to experiments carried out at 30°C with both propyl and butyl acetate and at two concentrations of DDDACI, 0.04 and 0.1 M. Enzyme and substrate concentrations are 5 / x g ml -~ and 2 mM respectively. The reactor is stirred and total volume is 25 ml. The aqueous phase is 3% by volume. Product concentration is plotted vs. process time and the slopes of the curves indicate that the enzyme is still active after 120-140 h of reaction. At lower DDDACI concentration, 0.04 M, p-nitrophenol formation is higher and propyl acetate creates a better environment to preserve the native activity of the enzyme. Biocatalysis in supramolecular systems is a complex phenomenon. The prediction of reaction yield can not be exclusively formulated on the basis of this
137
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Fig. 5. Specific activity of acid phosphatase vs. water to surfactant ratio at 30°C and 5 /zg ml - I of enzyme in the presence of 3% v / v water, m , propyl acetate; • , butyl acetate.
preliminary investigation and deserves analysis of further experiments. These have shown that specific activity of the enzyme also depends on the molar ratio of water to surfactant (R) and the total volume of aqueous phase. Results are plotted in
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Fig. 6. Specific activity of acid phosphatase vs. water volume at 30°C, 5 / z g m l - 1 of enzyme, 2 ml total volume, m , propyl acetate, R = 5 0 ; - ~ , propyl acetate, R = 20.15; • , butyl acetate, R = 50; ~,, butyl acetate, R = 20.15.
138 Figs. 5 and 6. Experiments have been carried out at 30°C in stirred batch reactors, 2 ml, constant volume of aqueous phase, 60/zl, saturating substrate concentration, 2 mM, and with 5/xg m1-1 of acid phosphatase. Product formation during 20 min of incubation was measured under differential reaction conditions. At first, different quantities of surfactant have been used in order to investigate a range of R between 17.2 and 52.4. Surfactant concentration and water quantity have been successively varied keeping constant the value of R. Data of Fig. 5 show that the specific activity of acid phosphatase is an increasing function of R and is roughly the same in butyl and propyl acetate with the exception of media very rich in DDDAC1. It also appears in Fig. 6 that the curves of specific activity vs. water volume present maxima which depend on the value of R and the organic chemicals. The highest specific activity of the enzyme is always observed at R = 50 and 2% v / v of water in the system. If the amount of surfactant is increased, up to R = 20.15, the enzyme activity decreases and the maximum occurs at 3.5% v / v of aqueous phase in propyl acetate and at roughly 2.5% v / v in butyl acetate. Finally, activity of acid phosphatase in propyl acetate is more dependent than in butyl acetate on composition.
Conclusions Cationic surfactants have been prepared which allow a supramolecular organization in apolar organic solvents and good retention of water quantities, up to 4 - 5 % v/v. Stability of acid phosphatase in these media is improved especially above room temperature. Specific activity of the enzyme depends on the organic solvent, the water to surfactant ratio and the total volume of aqueous phase. In general, affinity towards the substrate and maximum velocity are worse than the ones in buffer. However, operational conditions can be selected in order to assure prolonged reuse of the biocatalyst and to achieve high yield of overall production per unit weight of enzyme. This behaviour has been observed working with a hydrolytic enzyme and a substrate totally confined in the aqueous environment. Better results could be expected with other enzymes and water insoluble substrates.
Acknowledgment This study was supported by Italian Ministry of Public Education (MURST) and by National Research Council (CNR).
References Alfani, F., Cantarella, L., Cantarella, M., Gallifuoco,A. and Scardi, V. (1989) Thermodynamicanalysis of the stability enhancement of acid phosphatase by gel immobilizationin proteins. Lat. Am. Appl. J. 20, 47-51.
139 Bonan, C., Germani, R., Ponti, P.P., Savelli, G., Cerichelli, G. and Bunton, C.A. (1990) Micellar headgroup size and anion nucleophilicity in SN2 reactions. J. Phys. Chem. 94, 5331-5335. Brink, L.E.S. and Tramper, J. (1985) Optimization of organic solvent in multiphase biocatalysis. Biotechnol. Bioeng. 27, 1258-1269. Bunton, C.A. and Savelli, G. (1986) Organic reactivity in aqueous micelles and similar assemblies. Adv. Phys. Org. Chem. 22, 213-309. Cantarella, M., Cantarella, L. and Alfani, F. (1991) Hydrolytic reactions in two phase systems. Effect of water-immiscible organic solvents on stability and activity of acid phosphatase, /3-glucosidase and /3-fructofuranosidase. Enzyme Microb. Technol. 13, 547-553. Cipiciani, A., Germani, R., Savelli, G., Bunton, C.A., Mhala, M. and Moffatt, J.R. (1987) The effects of single- and twin-tailed ionic surfactants upon aromatic nucleophilic substitution. J. Chem. Soc. Perkin II, 541-546. Dordick, J.S. (1989) Enzymatic catalysis in monophasic organic solvents. Enzyme Microb. Technol. 11, 194-211. Fletcher, P.D.I. and Robinson, B.H. (1984) Effect of organized surfactant system on the kinetics of metal ligand complex formation and dissociation. J. Chem. Soc. Faraday Trans. I, 80, 2417-2437. Frendler, J.H. (1982) Membrane Mimetic Chemistry. Wiley-Interscience, New York. Germani, R., Ponti, P.P., Spreti, N., Savelli, G., Cipiciani, A., Cerichelli, G., Bunton, C.A. and Si, V. (1990) Decarboxylation of e-nitrobenzisoxazole-3-carboxylate ion in dichloromethane: the possible role of reverse micelles. J. Colloid Interface Sci. 138, 443-450. Greco, G., Albanesi, D., Cantarella, M., Gianfreda, L., Palescandolo, R. and Scardi, V. (1979) Enzyme inactivation and stabilization studies in an ultrafiltration reactor. Eur. J. Appl. Microbiol. Biotechnol. 8, 249-261. Kondo, H., Miwa, I. and Sunamoto, J. (1982) Biphasic structure model for reversed micelles. Depressed acid dissociation of excited-state pyranine in the restricted reaction field. J. Phys. Chem. 86, 4826-4831. Laane, C., Boeren, S., Vos, K. and Veerger, C. (1987) Rules for optimization of biocatalysis in organic solvents. Biotechnol. Bioeng. 30, 81-87. Luisi, P.L. (1985) Enzymes hosted in reverse micelles in hydrocarbon solution. Angew. Chem. Int. Ed. Eng. 24, 439-450. Luisi, P.L., Giomini, M., Pileni, M.P. and Robinson, B.H. (1988) Reverse micelles as hosts for proteins and small molecules. Biochim. Biophys. Acta 947, 209-246. Martinek, K., Levashov, A.V., Klyachko, N.L., Pantin, V.I. and Berezin I.V. (1981) The principles of enzyme stabilization. VI. Catalysis by water-soluble enzymes entrapped into reversed micelles of surfactants in organic solvents. Biochim. Biophys. Acta 657, 277-294. Menger, F.M., Donohue, J.A. and Williams, R.F. (1973) C~talysis in water pools. J. Am. Chem. Soc. 95, 286-288. Menger, F.M. and Yamada, K. (1979) Enzyme catalysis in water pools. J. Am. Chem. Soc. 101, 6731-6734. Ringsdorf, H., Schlarb, B. and Venzmer, J. (1988) Molecular architecture and function of polymeric oriented systems: models for the study of organization, surface recognition, and dynamic of biomembranes. Angew. Chem. Int. Ed. Engl. 27, 113-158. Romsted, L.S. (1984) Micellar effects on reaction rates and equilibria. In: Mittal, K.L. and Lindman, B. (Eds.), Surfactants in solution, Plenum, New York, Vol. 2, pp. 1015-1068. Semenov, A.N, Klmelnitski, Y.L., Berezin, I.V. and Martinek, K. (1987) Water-organic solvent two-phase systems as media for biocatalytic reactions: the potential for shifting chemical equilibria towards higher yield of end products. Biocatalysis i, 3-8. Tramper, J., Van der Plas, H.C. and Linko, P. (Eds.) (1985) Biocatalysts in Organic Synthesis, Elsevier, Amsterdam/New York. Zaks, A. and Klibanov, A. (1985) Enzyme-catalyzed processes in organic solvents. Proc. Nat. Acad. Sci. USA, 82, 3192-3196.
