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Relating the microstructure of enzyme dispersions in organic solvents to their kinetic behavior
ELSEVIER
Relating the microstructure of enzyme dispersions in organic solvents to their kinetic behavior Emma Bracey,* Robert A. Stenning,* and Brian E. Brooker* *Institute
qf Food Research, Reading, United Kingdom
The effect qf ultrasound irradiation on activit?, and particle size of a subtilisin-catalyzed interesterifcation reaction in an organic solvent was studied. Across a range of levels of water addition and irradiation amplitude. sonication failed to increuse reaction rate compared with stirred systems. Sonieation was shown to be effective in reducing particle size, but the effect of any reduction in diffisional limitations on reaction rate was exceeded by an inhibitory effect of the treatment. This inhibition was found to be irreversible upon cessation of sonication 0 1998 Elsevier and aqueous assay qf activity, or recycling of the enzyme into a stirred reaction system. Sciellce Inc. Keywords:
Subtilisin: ultrasound irradiation; organic solvent; electron microscopy; particle size
Introduction The use of enzyme-catalyzed reactions in nearly anhydrous organic solvents for biotransformations has been widely a small amount of reported. ’ -’ Under these conditions, water (< 2%) is required in order to maintain the enzyme in its catalytically active conformation. Since enzymes are insoluble in almost all organic solvents, molecular contact with their hydrophobic substrates can be achieved using an enzyme dispersion. Although this means they are easily recovered for reuse, this may also present problems due to diffusional limitations of the substrate to the enzyme surface. This may be overcome by adsorption of the enzyme onto an inert support to give better distribution of the biocatalyst and thus significantly enhance reaction rates. Alternatively, ultrasound irradiation of the enzyme suspension to reduce such mass transfer limitations has also been reported.6 It has been shown6 that there is a significant enhancement of the reaction rate of subtilisin-catalyzed interesterification of amino acid esters in organic solvents by exposure of subtilisin powder to ultrasound. One explanation for this enhancement was an increase in the amount of available surface area of the enzyme by fragmentation, but the structural basis for this rate enhancement was not
Address reprint requests to Dr. Robert A. Stenning, Reading Laboratory. Institute of Food Research, Earley Gate, Whiteknights Road, Reading RG6 6BZ, U.K.
Received 9 January
1997; revised 23 June 1997; accepted
1 July 1997
Enzyme and Microbial Technology 22:147-151, 1998 0 1998 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
clarified. Apparently in contradiction to these observations, Zaks and Klibanov7 reported that ultrasonication of a suspension of chymotrypsin in octane (resulting in a reduction of an average enzyme particle from 270 to 5 p.m, as revealed by direct microscopic examination) had no appreciable effect on the enzyme transesterification rate, thus suggesting that there were no limitations of internal diffusion. Previous reports 8.9 about the ultrasonication of enzyme reactions in aqueous media have shown both enhanced activity and inactivation of the enzyme depending on the intensity of the irradiation. Ishimori et aL8 reported acceleration of both free and immobilized a-chymotrypsin activity with ultrasound which decreased with increasing sonication power. This effect in aqueous systems probably is due to partial denaturation of the enzyme by cavitation. Sakakibara ef al9 found that at high ultrasonic intensities, inactivation of the enzyme occurred after 4 h; however, at reduced intensities, a significant enhancement of reaction rate was reported probably due to a reduction in substrate inhibition and aggregation based on hydrogen bonding of molecules. This report seems to suggest that differences in response to sonication are the result of differences in energy input, and emphasizes the difficulties of comparing ultrasonic work of different researchers due to problems with estimation of the ultrasonic power applied. The present work therefore set out to relate the enhancement of enzyme activity by continuous sonication to enzyme particle size as determined by laser light scattering. Enzyme particles dispersed in organic solvent were exam-
0141-0229/98/$19.00 PII SOl41-0229(97)00138-5
Papers ined by electron microscopy before and during sonication to determine changes in structure produced by this treatment. Preliminary comparisons of stirred and sonicated systems, however, showed no enhancement in the reaction rate of sonicated enzyme as previously reported.” This unexpected result led to further investigations of the effect of sonication on subtilisin-catalyzed interesterification reactions.
Materials and methods Materials Subtilisin [bacterial protease Type XXIV (EC 3.4.21.62); activity 12.9 U mgg’], N-acetyl-phenylalanine ethyl ester (APEE), and I-hexanol were all obtained from Sigma Chemicals Ltd. (Poole, England). The hexanol was the highest available purity and was stored over a 3 A molecular sieve. Subtilisin was dissolved in 20 mM potassium phosphate buffer pH 7.8 at a concentration of 25 mg ml- ‘. The enzyme solution was divided into 10 ml vacuum ampules, shell frozen in liquid nitrogen, and freeze-dried overnight using an Edwards high vacuum freezedryer (EF03). Secondary drying after constriction of the ampules was performed over phosphorous pentoxide. These were then sealed under vacuum. A new sealed ampule was opened and used for each new experiment. Preliminary electron microscopic studies used enzyme stored in a desiccator until it was found to give inconsistent activities. For laser particle sizing, technical grade propan-2-01 from BDH Chemicals (St. Albans, UK) was used as the bulk solvent.
General methods Stirring and sonication. All reactions to compare the catalytic activity of sonicated and stirred enzyme systems used the method described by Vulfson et aZ.h as follows. The lyophilized enzyme at a concentration of 1.5 mg ml-’ was added to 12 ml of 10 mM APEE in hexanol containing 1% (v/v) 100 mrvr potassium phosphate buffer pH 7.8. The reaction was performed in a thermostatically controlled jacketed vessel and the temperature maintained at 38°C. Sonication with continuous stirring was applied at a constant amplitude of 6 pm using an MSE Soniprep 150 fitted with a 9.5 mm diameter probe. All stirring was performed with a small magnetic stirrer bar; the tip of the sonicator probe was placed in a central position in the reaction vessel. At various intervals, 0.1 ml samples were removed and placed in Eppendorf tubes along with 1.0 ml of the mobile phase [containing 0.1% (v/v) perchloric acid to stop the reaction]. These were then spun in a Heraeus Biofuge at 11,000 g for 5 min to sediment the enzyme. The supernatant was then further diluted prior to HPLC analysis. HPLC analysis. HPLC analysis was performed on a Waters 600 system fitted with a UV detector (wavelength = 214 nm) and a 250 mm ODS2 5 pm Spherisorb reverse-phase column maintained at a temperature of 45°C. Methanol and water in the volume ratio 80:20 [containing 0.1% (v/v) perchloric acid] was used as the isocratic eluent at a flow rate of 1 ml mini ‘. The rate of growth of the AP-hexyl ester peak was monitored. Particle sizing. The determined by laser reaction mixture was or stirring and added presentation unit of
148
size distribution of enzyme dispersions was light scattering. A small volume of the removed after different periods of sonication to propan-2-o) in the small volume sample a Malvern Mastersizer X. The following
Enzyme Microb. Technol.,
1998, vol. 22, February
parameters were used in the processing algorithm: particle refractive index of 1.54 and propanol refractive index of I .383. The presentation 2NHE, previously calculated from these values. was used to predict scatter angles. The refractive index of the enzyme was determined with an Abbe refractometer. Electron microscopy. To study the structure of lyophilized enzyme by scanning electron microscopy (SEM), freeze-dried enzyme was sprinkled onto a specimen stub coated with conductive cement and sputter coated with gold before examination in an Hitachi S570 SEM. To examine the morphology of enzyme particles in sonicated and stirred reaction mixtures by transmission electron microscopy (TEM), a drop of the enzyme dispersion was placed on a carbon-coated electron microscope grid at various intervals and rapidly frozen by plunging into liquid propane. It was then transferred to a Bal-Tee 400T freeze-etching unit and freeze-dried at a stage temperature of -50°C for 8 h according to a modified version of the technique described by Willison and Rowe.‘” The dried grid was examined in an Hitachi H600 TEM operating at 75kV.
Experiments The effect of dispersing subtilisin by stirring or sonication on the rate of reaction was investigated using the method discussed above. Possible adverse effects of sonication and stirring on enzyme activity were examined in the following experiment. In separate experiments, enzyme reaction systems were sonicated or stirred for 4 h. The reaction mixtures were then sedimented in an MSE centrifuge at 600 g for 15 min and the supernatant removed and drained from the enzyme pellet. Water was added and the enzyme dissolved to give a concentration of 25 mg rnl~ ‘. This solution was freeze-dried in the normal manner. Both treated enzyme samples were then used in separate stirred experiments and their reaction rates compared. In addition, specific enzyme activity (U rng- ‘) before and after periods of stirring or sonication was determined by aqueous assay using benzyloxycarbonyl-glycl-glcyl-L-leucine p-nitroanilide (ZGGLNA) as the chromogenic substrate.” The concentration of protein in the enzyme samples was determined by the method of Lowry rt 01.” using bovine serum albumin as the standard. Reaction systems were either stirred or sonicated for 2 and 4 h. The enzyme suspension was centrifuged as described previously and the resulting enzyme pellet dissolved in water at a concentration of 25 mg ml-’ prior to freeze-drying and subsequent determination of activity. To investigate the possibility that contaminants in the enzyme preparations were affecting the catalytic activity in stirred and sonicated systems, the activity of the commercial enzyme was compared with the same enzyme that had been purified by the following method. Contaminating materials such as bulking agents in the Sigma preparation of subtilisin were removed by dialysis. Subtilisin was dissolved in 20 mM phosphate buffer pH 7.8 at a concentration of 27 mg ml-’ and dialyzed against water for 4 h (2 X 1 1) at 4°C. This was followed by dialysis against 2 1 of phosphate buffer pH 7.8 for 3 h. A sample was retained for aqueous assays and the remainder was freeze-dried in the normal manner. A 1 mg ml-’ solution of Sigma subtilisin in its original state was prepared and analyzed along with the dialyzed subtilisin (27 mg ml-‘) prior to freeze-drying. Any autoproteolysis of the subtilisin during dialysis was analyzed for by SDS-polyacrylamide gel electrophoresis of the two subtilisin samples. A Pharmacia PhastGel system was used with precast homogeneous 20% acrylamide gels. Electrophoresis was performed as recommended by Pharmacia (Leicester, UK) using limiting values of 250V. IOmA. and 3W for 95Vh.13
15
Microstructure Table 1 different media
of enzyme
1.5
3.0
4.5
Activity (enzyme
0 120 240
6.0
6
0
1.5
3.0
4.5
6.0
Time (hours)
Figure 1 Evolution of N-acetyl phenylalanine hexyl ester as a function of time: in stirred (0) and sonicated (0) media catalyzed by subtilisin (a); in stirred (0) and sonicated (0) media using subtilisin purified by dialysis (b); and in stirred media using subtilisin recovered from stirred (0) or sonicated (0) systems for 4 h (cl
The specific activity of these two subtilisin samples was determined to establish whether the dialysis process had caused any significant change in enzyme activity. The activity of the subtilisin before and after dialysis was monitored using ZGGLNA as the chromogenic substrate using a method described previously.” Protein concentrations of the two samples were again estimated by the method of Lowry et al.”
Results Kinetic behavior
of subtilisin
The data obtained for the subtilisin-catalyzed interesterification of APEE in hexanol shows that, contrary to previous reports,6 no enhancement in the reaction rate was obtained when the enzyme suspension was sonicated (Figure la). Indeed, in the first 2 h of the reaction, the enzyme activity in the sonicated system was the same as that produced by stirring but after 6 h, the stirred system contained the greater accumulation of reaction product. During sonication, the rate of reaction was seen to fall before complete conversion of the substrate. Electrophoresis and aqueous assay of dialyzed subtilisin indicated that its integrity and activity were preserved
E. Bracey et al.
Activity of subtilisin determined by aqueous assay at intervals during stirring and sonication in reaction
Length of treatment (min)
0
dispersions:
U mg -‘)
Stirred
Sonicated
5.11 ? 0.26 4.82 2 0.39 4.44 t 0.49
5.11 + 0.26 4.41 k 0.61 3.76 -t 0.13
following purification. When this enzyme was stirred or sonicated, similar results were obtained (Figure lb) to those with the unpurified enzyme. The reaction rate of sonicated subtilisin was found to decline with time, thereby indicating that there was some loss of activity associated with this treatment. To determine whether this was an irreversible effect, enzyme was recovered after 4 h of stirring or sonication in the reaction medium, freeze-dried, and reused in a stirred system (Figure lc). If sonication had caused reversible damage to the subtilisin, then with subsequent stirring, it would show similar behavior to controls; however, reaction profiles showed that this was not the case because the ultrasound-treated enzyme had less activity than that produced by stirring. In addition to determining loss of activity in an interesterification reaction. inactivation of the enzyme was determined in aqueous solution. Results for enzyme activities (enzyme Unit mg-‘) after periods of treatment of stirring or sonication also showed a greater loss of subtilisin activity after 2 and 4 h of sonication treatment than with stirring (Table I). Microstructure
of subtilisin
Examination of freeze-dried subtilisin by SEM revealed a cellular structure (representing the eutectic phase of the frozen enzyme solution) consisting of thin plates and rods whose dimensions varied slightly from one preparation to another (Figure 2). The dispersed enzyme consisted of plate and rod-shaped fragments when examined by TEM. Figure 3 shows their appearance after 30 min of sonication. This method of dispersion produced smaller particles than did stirring for 4 h. The effect of sonication or stirring on mean enzyme particle diameters obtained by laser light scattering is shown in Table 2. The results are consistent with those obtained by microscopy and show that the greatest change in particle size occurred during the first 30 min of sonication. Further sonication (up to 6 h) had relatively little effect on particle size. In preliminary studies, dispersed enzyme that had been stored in a desiccator was examined by TEM and found to consist of rounded particles. Plate-like particles were only observed with material that was maintained under vacuum after freeze-drying.
Discussion The work described here shows not only that continuous sonication of a subtilisin-catalyzed interesterification reac-
Enzyme Microb.
Technol.,
1998, vol. 22, February
15
149
Papers
Figure2 showing
Scanning electron micrograph lamellar structure
of lyophilized subtilisin
tion produces no enhancement of reaction rate compared to stirring but also that it may progressively retard the reaction rate if prolonged. A previous report6 has suggested that fragmentation of the enzyme by sonication may enhance reaction rate by increasing the surface area. In our work, the structure of the freeze-dried enzyme powder was observed by SEM as an open honeycomb structure which had a large amount of available surface area and consisted of flat plates and rods. Disintegration of this structure into its component rods and plates occurred during stirring and sonication but both laser light scattering and TEM confirmed that considerably greater particle size reduction was achieved by sonication especially in the first 30 min; however, the plate-like nature of particles means that only a relatively small increase in surface area accompanies this size reduction during fragmentation. This may explain the lack of enhancement in reaction rate with sonication observed both in the present study and by Zaks and Klibanov.’ In this work, continuous sonication appears to progressively and irreversibly retard reaction rate. Two authors have proposed explanations for declining enzyme activity during catalysis in near-anhydrous organic media. Goldberg et a1.‘4 reported an esterification reaction using a lipase catalyst in media containing only the substrates and the powdered enzyme. It was found that higher shaking rates produced decreased reaction velocities. They hypothesized that enzyme particles have to share water molecules in order 150
Enzyme Microb.
Technol.,
1998, vol. 22, February
Figure 3 Transmission electron micrograph of subtilisin particles after 30 min sonication in reaction media
to be active and therefore vigorous dispersion reduces activity. Increased dispersion alone cannot be responsible for reduced catalytic activity in the present work since activity did not recover upon cessation of sonication. The hydration state of the enzyme in this study may also explain the similarity of activities observed in the initial stages of stirred and sonicated reactions. An Environmental SEM studyi of the morphology of lyophilized subtilisin with increasing humidity reported that the flaky powder swells to form a branchlike structure. From this observation, Roziewski and Russell” proposed that hydration of a subtilisin particle in hydrophobic solvent causes it to swell. Table 2 Mean diameter of subtilisin particles after differing periods of sonication or stirring in reaction medium as determined by laser light scattering
Length of treatment (min) la 30 90 360
Mean particle diameter Stirred system
Sonicated
51.19 37.89 35.90 31.35
“This value was measured after 1 min of stirring disperse and wet the aggregated enzyme mass
15
(km) system
51.19 1.93 1.62 1.32 required
to
Microstructure increasing the length of the diffusional path to its center. In addition, saturation of pores with water rather than organic solvent further impedes reactant diffusion. In the present work, the plate and rod-shaped fragments observed by TEM resemble the nonhydrated flakes of Roziewski and Russell. This would suggest that, in the present experiments, water adsorption by the enzyme had not taken place; however, preliminary TEM observations of the enzyme before storage in evacuated ampules showed that the subtilisin was in the form of rounded particles, perhaps corresponding to the swollen and hydrated enzyme previously reported.” If adsorption of atmospheric moisture prior to addition of enzyme to the reaction system increases mass transport limitations, then the strong agitation of the enzyme produced by sonication might be expected to promote activity by reducing these limitations. This could explain the observed benefit of sonication reported by Vulfson et ~1.~ using enzyme that had been stored over phosphorous pentoxide in a desiccator, and the lack of enhancement in the present study where the experimental protocol ensured a new ampule of enzyme was used for each experiment and where the problem of diffusional limitations caused by water adsorption would be minimal. It can be argued that if the degree of hydration is so critical to the effect of sonication on activity, then a beneficial effect of sonication should have been observed in the present work when higher levels of water were added to the medium in order to identify an optimum level. No difference in response to sonication was observed with differing levels of water in the reaction medium, supporting the premise that it is exposure to water prior to immersion that effects the response of the enzyme to sonication. It may be that the small levels of water dispersed in the solvent prior to enzyme addition are not available for enzyme modification of the type observed when it is exposed to atmospheric moisture. The reason for the disparity between this observation and results of other workers may be that enzyme used in this work was dryer than that used by others and therefore that there was no mass transport inhibition to be overcome by sonication. After prolonged sonication, an irreversible progressive deactivation of the enzyme was observed that appears to be the result of direct enzyme degradation by the sonication. The fact that this observation is not common to
of enzyme
dispersions:
E. Bracey et al.
all such work may be indicative of the difficulties of irradiating the reaction systems with a reproducible pattern and level of energy.
Acknowledgments This work was supported by a grant from the Biotechnology and Biological Sciences Research Council.
References I. 2. 3. 4. 5. 6.
I. 8.
9.
IO.
I I.
12.
13. 14.
15.
Klibanov, A. M. Enzymes that work in organic solvents. Chrm. Tech. 1986, 6, 354-359 Zaks. A. and Klibanov. A. M. Enzyme-catalyzed processes in organic solvents. Proc. Nat/. Acud. Sci. USA 1985. 82, 3192-3 196 Zaks, A. and Klibanov, A. M. Enzymatic catalysis in nonaqueous solvents. J. Bid. Chem. 1988. 263, 3194-3201 Klibanov, A. M. Enzymatic catalysis in anhydrous organic solvents. Trends Biochem. Sci. 1989. 14, 141-144 Klibanov, A. M. Why are enzymes less active in organic solvents than in water? Trends Biotechrd. 1997. 15, 97-101 Vulfson, E. N., Sarney. D. B.. and Law. B. A. Enhancement of subtilisin-catalyzed interesterification in organic solvents by ultrasound irradiation. Enzyme Microb. Technol. I99 I. 13, 123-l 26 Zaks, A. and Klibanov, A. M. Enzymatic catalysis in nonaqueous solvents. J. Bid. Chem. 1988, 263, 3 194-3201 Ishimori, Y., Karube, I.. and Suzuki. S. Acceleration of immobilised o-chymotrypsin activity with ultrasonic irradiation. J. Mol. Curd. 1981. 12, 253-259 Sakakibara. M., Wang, D.. Takahashi, K., and Mori. S. Influence of ultrasound irradiation on hydrolysis of sucrose catalyzed by invertase. Enzyme Micmb. Technd. 1096. 18, 444-448 Willison. J. H. M. and Rowe. A. J. Replica techniques in the biological sciences. In: Rr@icrr. Shadowing. trnd Free:c-etching Techniques (Glauert, A. M.. Ed.). Elsevier/North Holland Biomedical Press, 1980. 153 Lyublinskaya. L. A., Belyaev. S. V.. Strongin. Y. A., Matyash, L. F.. Levi% E. D.. and Stepanov, V. M. A new chromogenic substrate for subtilisin. Ad. Biochem. 1974. 62, 37 l-376 Lowry, 0. H.. Rosebrough, N. J.. Farr, A. L.. and Randall. R. J. Protem measurement with the folin phenol reagent. J. Riol. Chem. I95 I. 193,265-275 Pharmacia PhastSystem separation technique File No. I I I Goldberg. M., Thomas, D.. and Legoy, M. Water activity as a key parameter of synthesis reactions: The example of lipase in biphasic tliquid/solid) media. .!?I,-,vme Micmh. Tdmol. 1990. 12, 976-981 Roziewski. K. and Russell, A. J. Effect of hydration on the morphology of enzyme powder. Biotechnol. Bioen,~. 1992, 39, 1171-1175
Enzyme Microb.
Technol.,
1998, vol. 22, February
15
151
Relating the microstructure of enzyme dispersions in organic solvents to their kinetic behavior Emma Bracey,* Robert A. Stenning,* and Brian E. Brooker* *Institute
qf Food Research, Reading, United Kingdom
The effect qf ultrasound irradiation on activit?, and particle size of a subtilisin-catalyzed interesterifcation reaction in an organic solvent was studied. Across a range of levels of water addition and irradiation amplitude. sonication failed to increuse reaction rate compared with stirred systems. Sonieation was shown to be effective in reducing particle size, but the effect of any reduction in diffisional limitations on reaction rate was exceeded by an inhibitory effect of the treatment. This inhibition was found to be irreversible upon cessation of sonication 0 1998 Elsevier and aqueous assay qf activity, or recycling of the enzyme into a stirred reaction system. Sciellce Inc. Keywords:
Subtilisin: ultrasound irradiation; organic solvent; electron microscopy; particle size
Introduction The use of enzyme-catalyzed reactions in nearly anhydrous organic solvents for biotransformations has been widely a small amount of reported. ’ -’ Under these conditions, water (< 2%) is required in order to maintain the enzyme in its catalytically active conformation. Since enzymes are insoluble in almost all organic solvents, molecular contact with their hydrophobic substrates can be achieved using an enzyme dispersion. Although this means they are easily recovered for reuse, this may also present problems due to diffusional limitations of the substrate to the enzyme surface. This may be overcome by adsorption of the enzyme onto an inert support to give better distribution of the biocatalyst and thus significantly enhance reaction rates. Alternatively, ultrasound irradiation of the enzyme suspension to reduce such mass transfer limitations has also been reported.6 It has been shown6 that there is a significant enhancement of the reaction rate of subtilisin-catalyzed interesterification of amino acid esters in organic solvents by exposure of subtilisin powder to ultrasound. One explanation for this enhancement was an increase in the amount of available surface area of the enzyme by fragmentation, but the structural basis for this rate enhancement was not
Address reprint requests to Dr. Robert A. Stenning, Reading Laboratory. Institute of Food Research, Earley Gate, Whiteknights Road, Reading RG6 6BZ, U.K.
Received 9 January
1997; revised 23 June 1997; accepted
1 July 1997
Enzyme and Microbial Technology 22:147-151, 1998 0 1998 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
clarified. Apparently in contradiction to these observations, Zaks and Klibanov7 reported that ultrasonication of a suspension of chymotrypsin in octane (resulting in a reduction of an average enzyme particle from 270 to 5 p.m, as revealed by direct microscopic examination) had no appreciable effect on the enzyme transesterification rate, thus suggesting that there were no limitations of internal diffusion. Previous reports 8.9 about the ultrasonication of enzyme reactions in aqueous media have shown both enhanced activity and inactivation of the enzyme depending on the intensity of the irradiation. Ishimori et aL8 reported acceleration of both free and immobilized a-chymotrypsin activity with ultrasound which decreased with increasing sonication power. This effect in aqueous systems probably is due to partial denaturation of the enzyme by cavitation. Sakakibara ef al9 found that at high ultrasonic intensities, inactivation of the enzyme occurred after 4 h; however, at reduced intensities, a significant enhancement of reaction rate was reported probably due to a reduction in substrate inhibition and aggregation based on hydrogen bonding of molecules. This report seems to suggest that differences in response to sonication are the result of differences in energy input, and emphasizes the difficulties of comparing ultrasonic work of different researchers due to problems with estimation of the ultrasonic power applied. The present work therefore set out to relate the enhancement of enzyme activity by continuous sonication to enzyme particle size as determined by laser light scattering. Enzyme particles dispersed in organic solvent were exam-
0141-0229/98/$19.00 PII SOl41-0229(97)00138-5
Papers ined by electron microscopy before and during sonication to determine changes in structure produced by this treatment. Preliminary comparisons of stirred and sonicated systems, however, showed no enhancement in the reaction rate of sonicated enzyme as previously reported.” This unexpected result led to further investigations of the effect of sonication on subtilisin-catalyzed interesterification reactions.
Materials and methods Materials Subtilisin [bacterial protease Type XXIV (EC 3.4.21.62); activity 12.9 U mgg’], N-acetyl-phenylalanine ethyl ester (APEE), and I-hexanol were all obtained from Sigma Chemicals Ltd. (Poole, England). The hexanol was the highest available purity and was stored over a 3 A molecular sieve. Subtilisin was dissolved in 20 mM potassium phosphate buffer pH 7.8 at a concentration of 25 mg ml- ‘. The enzyme solution was divided into 10 ml vacuum ampules, shell frozen in liquid nitrogen, and freeze-dried overnight using an Edwards high vacuum freezedryer (EF03). Secondary drying after constriction of the ampules was performed over phosphorous pentoxide. These were then sealed under vacuum. A new sealed ampule was opened and used for each new experiment. Preliminary electron microscopic studies used enzyme stored in a desiccator until it was found to give inconsistent activities. For laser particle sizing, technical grade propan-2-01 from BDH Chemicals (St. Albans, UK) was used as the bulk solvent.
General methods Stirring and sonication. All reactions to compare the catalytic activity of sonicated and stirred enzyme systems used the method described by Vulfson et aZ.h as follows. The lyophilized enzyme at a concentration of 1.5 mg ml-’ was added to 12 ml of 10 mM APEE in hexanol containing 1% (v/v) 100 mrvr potassium phosphate buffer pH 7.8. The reaction was performed in a thermostatically controlled jacketed vessel and the temperature maintained at 38°C. Sonication with continuous stirring was applied at a constant amplitude of 6 pm using an MSE Soniprep 150 fitted with a 9.5 mm diameter probe. All stirring was performed with a small magnetic stirrer bar; the tip of the sonicator probe was placed in a central position in the reaction vessel. At various intervals, 0.1 ml samples were removed and placed in Eppendorf tubes along with 1.0 ml of the mobile phase [containing 0.1% (v/v) perchloric acid to stop the reaction]. These were then spun in a Heraeus Biofuge at 11,000 g for 5 min to sediment the enzyme. The supernatant was then further diluted prior to HPLC analysis. HPLC analysis. HPLC analysis was performed on a Waters 600 system fitted with a UV detector (wavelength = 214 nm) and a 250 mm ODS2 5 pm Spherisorb reverse-phase column maintained at a temperature of 45°C. Methanol and water in the volume ratio 80:20 [containing 0.1% (v/v) perchloric acid] was used as the isocratic eluent at a flow rate of 1 ml mini ‘. The rate of growth of the AP-hexyl ester peak was monitored. Particle sizing. The determined by laser reaction mixture was or stirring and added presentation unit of
148
size distribution of enzyme dispersions was light scattering. A small volume of the removed after different periods of sonication to propan-2-o) in the small volume sample a Malvern Mastersizer X. The following
Enzyme Microb. Technol.,
1998, vol. 22, February
parameters were used in the processing algorithm: particle refractive index of 1.54 and propanol refractive index of I .383. The presentation 2NHE, previously calculated from these values. was used to predict scatter angles. The refractive index of the enzyme was determined with an Abbe refractometer. Electron microscopy. To study the structure of lyophilized enzyme by scanning electron microscopy (SEM), freeze-dried enzyme was sprinkled onto a specimen stub coated with conductive cement and sputter coated with gold before examination in an Hitachi S570 SEM. To examine the morphology of enzyme particles in sonicated and stirred reaction mixtures by transmission electron microscopy (TEM), a drop of the enzyme dispersion was placed on a carbon-coated electron microscope grid at various intervals and rapidly frozen by plunging into liquid propane. It was then transferred to a Bal-Tee 400T freeze-etching unit and freeze-dried at a stage temperature of -50°C for 8 h according to a modified version of the technique described by Willison and Rowe.‘” The dried grid was examined in an Hitachi H600 TEM operating at 75kV.
Experiments The effect of dispersing subtilisin by stirring or sonication on the rate of reaction was investigated using the method discussed above. Possible adverse effects of sonication and stirring on enzyme activity were examined in the following experiment. In separate experiments, enzyme reaction systems were sonicated or stirred for 4 h. The reaction mixtures were then sedimented in an MSE centrifuge at 600 g for 15 min and the supernatant removed and drained from the enzyme pellet. Water was added and the enzyme dissolved to give a concentration of 25 mg rnl~ ‘. This solution was freeze-dried in the normal manner. Both treated enzyme samples were then used in separate stirred experiments and their reaction rates compared. In addition, specific enzyme activity (U rng- ‘) before and after periods of stirring or sonication was determined by aqueous assay using benzyloxycarbonyl-glycl-glcyl-L-leucine p-nitroanilide (ZGGLNA) as the chromogenic substrate.” The concentration of protein in the enzyme samples was determined by the method of Lowry rt 01.” using bovine serum albumin as the standard. Reaction systems were either stirred or sonicated for 2 and 4 h. The enzyme suspension was centrifuged as described previously and the resulting enzyme pellet dissolved in water at a concentration of 25 mg ml-’ prior to freeze-drying and subsequent determination of activity. To investigate the possibility that contaminants in the enzyme preparations were affecting the catalytic activity in stirred and sonicated systems, the activity of the commercial enzyme was compared with the same enzyme that had been purified by the following method. Contaminating materials such as bulking agents in the Sigma preparation of subtilisin were removed by dialysis. Subtilisin was dissolved in 20 mM phosphate buffer pH 7.8 at a concentration of 27 mg ml-’ and dialyzed against water for 4 h (2 X 1 1) at 4°C. This was followed by dialysis against 2 1 of phosphate buffer pH 7.8 for 3 h. A sample was retained for aqueous assays and the remainder was freeze-dried in the normal manner. A 1 mg ml-’ solution of Sigma subtilisin in its original state was prepared and analyzed along with the dialyzed subtilisin (27 mg ml-‘) prior to freeze-drying. Any autoproteolysis of the subtilisin during dialysis was analyzed for by SDS-polyacrylamide gel electrophoresis of the two subtilisin samples. A Pharmacia PhastGel system was used with precast homogeneous 20% acrylamide gels. Electrophoresis was performed as recommended by Pharmacia (Leicester, UK) using limiting values of 250V. IOmA. and 3W for 95Vh.13
15
Microstructure Table 1 different media
of enzyme
1.5
3.0
4.5
Activity (enzyme
0 120 240
6.0
6
0
1.5
3.0
4.5
6.0
Time (hours)
Figure 1 Evolution of N-acetyl phenylalanine hexyl ester as a function of time: in stirred (0) and sonicated (0) media catalyzed by subtilisin (a); in stirred (0) and sonicated (0) media using subtilisin purified by dialysis (b); and in stirred media using subtilisin recovered from stirred (0) or sonicated (0) systems for 4 h (cl
The specific activity of these two subtilisin samples was determined to establish whether the dialysis process had caused any significant change in enzyme activity. The activity of the subtilisin before and after dialysis was monitored using ZGGLNA as the chromogenic substrate using a method described previously.” Protein concentrations of the two samples were again estimated by the method of Lowry et al.”
Results Kinetic behavior
of subtilisin
The data obtained for the subtilisin-catalyzed interesterification of APEE in hexanol shows that, contrary to previous reports,6 no enhancement in the reaction rate was obtained when the enzyme suspension was sonicated (Figure la). Indeed, in the first 2 h of the reaction, the enzyme activity in the sonicated system was the same as that produced by stirring but after 6 h, the stirred system contained the greater accumulation of reaction product. During sonication, the rate of reaction was seen to fall before complete conversion of the substrate. Electrophoresis and aqueous assay of dialyzed subtilisin indicated that its integrity and activity were preserved
E. Bracey et al.
Activity of subtilisin determined by aqueous assay at intervals during stirring and sonication in reaction
Length of treatment (min)
0
dispersions:
U mg -‘)
Stirred
Sonicated
5.11 ? 0.26 4.82 2 0.39 4.44 t 0.49
5.11 + 0.26 4.41 k 0.61 3.76 -t 0.13
following purification. When this enzyme was stirred or sonicated, similar results were obtained (Figure lb) to those with the unpurified enzyme. The reaction rate of sonicated subtilisin was found to decline with time, thereby indicating that there was some loss of activity associated with this treatment. To determine whether this was an irreversible effect, enzyme was recovered after 4 h of stirring or sonication in the reaction medium, freeze-dried, and reused in a stirred system (Figure lc). If sonication had caused reversible damage to the subtilisin, then with subsequent stirring, it would show similar behavior to controls; however, reaction profiles showed that this was not the case because the ultrasound-treated enzyme had less activity than that produced by stirring. In addition to determining loss of activity in an interesterification reaction. inactivation of the enzyme was determined in aqueous solution. Results for enzyme activities (enzyme Unit mg-‘) after periods of treatment of stirring or sonication also showed a greater loss of subtilisin activity after 2 and 4 h of sonication treatment than with stirring (Table I). Microstructure
of subtilisin
Examination of freeze-dried subtilisin by SEM revealed a cellular structure (representing the eutectic phase of the frozen enzyme solution) consisting of thin plates and rods whose dimensions varied slightly from one preparation to another (Figure 2). The dispersed enzyme consisted of plate and rod-shaped fragments when examined by TEM. Figure 3 shows their appearance after 30 min of sonication. This method of dispersion produced smaller particles than did stirring for 4 h. The effect of sonication or stirring on mean enzyme particle diameters obtained by laser light scattering is shown in Table 2. The results are consistent with those obtained by microscopy and show that the greatest change in particle size occurred during the first 30 min of sonication. Further sonication (up to 6 h) had relatively little effect on particle size. In preliminary studies, dispersed enzyme that had been stored in a desiccator was examined by TEM and found to consist of rounded particles. Plate-like particles were only observed with material that was maintained under vacuum after freeze-drying.
Discussion The work described here shows not only that continuous sonication of a subtilisin-catalyzed interesterification reac-
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Papers
Figure2 showing
Scanning electron micrograph lamellar structure
of lyophilized subtilisin
tion produces no enhancement of reaction rate compared to stirring but also that it may progressively retard the reaction rate if prolonged. A previous report6 has suggested that fragmentation of the enzyme by sonication may enhance reaction rate by increasing the surface area. In our work, the structure of the freeze-dried enzyme powder was observed by SEM as an open honeycomb structure which had a large amount of available surface area and consisted of flat plates and rods. Disintegration of this structure into its component rods and plates occurred during stirring and sonication but both laser light scattering and TEM confirmed that considerably greater particle size reduction was achieved by sonication especially in the first 30 min; however, the plate-like nature of particles means that only a relatively small increase in surface area accompanies this size reduction during fragmentation. This may explain the lack of enhancement in reaction rate with sonication observed both in the present study and by Zaks and Klibanov.’ In this work, continuous sonication appears to progressively and irreversibly retard reaction rate. Two authors have proposed explanations for declining enzyme activity during catalysis in near-anhydrous organic media. Goldberg et a1.‘4 reported an esterification reaction using a lipase catalyst in media containing only the substrates and the powdered enzyme. It was found that higher shaking rates produced decreased reaction velocities. They hypothesized that enzyme particles have to share water molecules in order 150
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1998, vol. 22, February
Figure 3 Transmission electron micrograph of subtilisin particles after 30 min sonication in reaction media
to be active and therefore vigorous dispersion reduces activity. Increased dispersion alone cannot be responsible for reduced catalytic activity in the present work since activity did not recover upon cessation of sonication. The hydration state of the enzyme in this study may also explain the similarity of activities observed in the initial stages of stirred and sonicated reactions. An Environmental SEM studyi of the morphology of lyophilized subtilisin with increasing humidity reported that the flaky powder swells to form a branchlike structure. From this observation, Roziewski and Russell” proposed that hydration of a subtilisin particle in hydrophobic solvent causes it to swell. Table 2 Mean diameter of subtilisin particles after differing periods of sonication or stirring in reaction medium as determined by laser light scattering
Length of treatment (min) la 30 90 360
Mean particle diameter Stirred system
Sonicated
51.19 37.89 35.90 31.35
“This value was measured after 1 min of stirring disperse and wet the aggregated enzyme mass
15
(km) system
51.19 1.93 1.62 1.32 required
to
Microstructure increasing the length of the diffusional path to its center. In addition, saturation of pores with water rather than organic solvent further impedes reactant diffusion. In the present work, the plate and rod-shaped fragments observed by TEM resemble the nonhydrated flakes of Roziewski and Russell. This would suggest that, in the present experiments, water adsorption by the enzyme had not taken place; however, preliminary TEM observations of the enzyme before storage in evacuated ampules showed that the subtilisin was in the form of rounded particles, perhaps corresponding to the swollen and hydrated enzyme previously reported.” If adsorption of atmospheric moisture prior to addition of enzyme to the reaction system increases mass transport limitations, then the strong agitation of the enzyme produced by sonication might be expected to promote activity by reducing these limitations. This could explain the observed benefit of sonication reported by Vulfson et ~1.~ using enzyme that had been stored over phosphorous pentoxide in a desiccator, and the lack of enhancement in the present study where the experimental protocol ensured a new ampule of enzyme was used for each experiment and where the problem of diffusional limitations caused by water adsorption would be minimal. It can be argued that if the degree of hydration is so critical to the effect of sonication on activity, then a beneficial effect of sonication should have been observed in the present work when higher levels of water were added to the medium in order to identify an optimum level. No difference in response to sonication was observed with differing levels of water in the reaction medium, supporting the premise that it is exposure to water prior to immersion that effects the response of the enzyme to sonication. It may be that the small levels of water dispersed in the solvent prior to enzyme addition are not available for enzyme modification of the type observed when it is exposed to atmospheric moisture. The reason for the disparity between this observation and results of other workers may be that enzyme used in this work was dryer than that used by others and therefore that there was no mass transport inhibition to be overcome by sonication. After prolonged sonication, an irreversible progressive deactivation of the enzyme was observed that appears to be the result of direct enzyme degradation by the sonication. The fact that this observation is not common to
of enzyme
dispersions:
E. Bracey et al.
all such work may be indicative of the difficulties of irradiating the reaction systems with a reproducible pattern and level of energy.
Acknowledgments This work was supported by a grant from the Biotechnology and Biological Sciences Research Council.
References I. 2. 3. 4. 5. 6.
I. 8.
9.
IO.
I I.
12.
13. 14.
15.
Klibanov, A. M. Enzymes that work in organic solvents. Chrm. Tech. 1986, 6, 354-359 Zaks. A. and Klibanov. A. M. Enzyme-catalyzed processes in organic solvents. Proc. Nat/. Acud. Sci. USA 1985. 82, 3192-3 196 Zaks, A. and Klibanov, A. M. Enzymatic catalysis in nonaqueous solvents. J. Bid. Chem. 1988. 263, 3194-3201 Klibanov, A. M. Enzymatic catalysis in anhydrous organic solvents. Trends Biochem. Sci. 1989. 14, 141-144 Klibanov, A. M. Why are enzymes less active in organic solvents than in water? Trends Biotechrd. 1997. 15, 97-101 Vulfson, E. N., Sarney. D. B.. and Law. B. A. Enhancement of subtilisin-catalyzed interesterification in organic solvents by ultrasound irradiation. Enzyme Microb. Technol. I99 I. 13, 123-l 26 Zaks, A. and Klibanov, A. M. Enzymatic catalysis in nonaqueous solvents. J. Bid. Chem. 1988, 263, 3 194-3201 Ishimori, Y., Karube, I.. and Suzuki. S. Acceleration of immobilised o-chymotrypsin activity with ultrasonic irradiation. J. Mol. Curd. 1981. 12, 253-259 Sakakibara. M., Wang, D.. Takahashi, K., and Mori. S. Influence of ultrasound irradiation on hydrolysis of sucrose catalyzed by invertase. Enzyme Micmb. Technd. 1096. 18, 444-448 Willison. J. H. M. and Rowe. A. J. Replica techniques in the biological sciences. In: Rr@icrr. Shadowing. trnd Free:c-etching Techniques (Glauert, A. M.. Ed.). Elsevier/North Holland Biomedical Press, 1980. 153 Lyublinskaya. L. A., Belyaev. S. V.. Strongin. Y. A., Matyash, L. F.. Levi% E. D.. and Stepanov, V. M. A new chromogenic substrate for subtilisin. Ad. Biochem. 1974. 62, 37 l-376 Lowry, 0. H.. Rosebrough, N. J.. Farr, A. L.. and Randall. R. J. Protem measurement with the folin phenol reagent. J. Riol. Chem. I95 I. 193,265-275 Pharmacia PhastSystem separation technique File No. I I I Goldberg. M., Thomas, D.. and Legoy, M. Water activity as a key parameter of synthesis reactions: The example of lipase in biphasic tliquid/solid) media. .!?I,-,vme Micmh. Tdmol. 1990. 12, 976-981 Roziewski. K. and Russell, A. J. Effect of hydration on the morphology of enzyme powder. Biotechnol. Bioen,~. 1992, 39, 1171-1175
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