ELSEVIER

The effects of ultrasound on the activities of some glycosidase enzymes of industrial importance
Stephen Barton, Clive Bullock, and Deborah Weir
School of Life Sciences, Roehampton Institute, West Hill, London SW15 3SN, United Kingdom

The effects of ultrasound on the activities of alpha-amylase and amyloglucosidase toward starch and glycogen hydrolysis, and of invertase toward sucrose hydrolysis, were investigated using an ultrasound bath. A fixed-time assay was employed with product formation determined by reducing equivalents formed per unit time using the 3,S-dinitrosalicylate reagent (DNS). Reaction rates in the presence and absence of ultrasound were compared. Increases in rates were observed in the presence of ultrasound at most concentrations with a marked increase in invertase activity towards sucrose (37% at 0.9 M) and a reduction in the inhibition of sucrose hydrolysis observed at high substrate concentrations. Improvements in the efficiency of mixing and disruption of intra- and intermolecular substrate molecule interactions at high concentrations are possible explanations for the changes observed.
Keywords:

Ultrasound; alpha-amylase; glucoamylase; invertase; enzyme activity

Introduction
Enzyme-catalyzed hydrolyses are commonly employed in the production of sugar syrups in the food industry. The rates of these reactions show a tendency to slow at high substrate concentrations particularly in the case of invertase at sucrose concentrations greater than 5% w/w. The nature of invertase inhibition is complex due partly to product inhibition by o-fructose (competitive) and o-glucose (partial noncompetitive) as well as substrate inhibition attributed to a modification of the intra- and intermolecular hydrogen bonds between sucrose molecules at increasing sucrose concentrations.3 Reports that the use of ultrasound may enhance the activity of certain enzymes4 have led us to investigate its effects on the enzymatic hydrolysis of starch, glycogen, and sucrose, and in particular, its potential for modifying substrate/product inhibition of these reactions at high substrate concentrations. The use of an ultrasonic bath in this context has the advantages of simplicity, low cost, and modest power outputs (l-2 W cm- for each transducer) reducing the likeli-

hood of localized heating effects associated with ultrasound probe and cuphorn systems.5 The possibility of direct (nonenzymatic) substrate degradation which has been reported for some polymers including dextrins,5.6 though not for starch, is also minimized. The most obvious limitations are that the operating frequency is normally fixed and power densities vary within the bath, consequently standardization of sample locations is essential for comparative purposes. The effects of ultrasound on the action of alpha-amylase (EC 3.2.1.1) and glucoamylase (amyloglucosidase; EC 3.2.1.3) on starch and/or glycogen, and of invertase (p-ofructofuranoside fructohydrolase; EC 3.2.1.26) on sucrose were the subject of this investigation. The progress of substrate hydrolysis was measured by determination of the reducing sugar equivalents released using the standard 3,5dinitrosalicylate (DNS) assay following a 10 min fixedtime incubation period with the enzyme in the presence or absence of ultrasound in each case.

Materials and methods

Materials Soluble (potato) starch, (oyster) glycogen, sucrose, alpha-amylase (Type Xl-A; Bacillus spp.; 90 U mg-I), and glucoamylase (Rhizopus spp.; 8.4 U mg-) were obtained from Sigma Chemicals. An invertase extract was obtained from Bakers yeast (4400 U ml-) according to the method described by Clark et a1.9

Address reprint requests to Dr. Clive Bullock, Department of Biological and Chemical Sciences, Roehampton Institute, West Hill, London SW15 3SN, United Kingdom Received 9 December 1994: revised 26 April 1995; accepted 4 May 1995

Enzyme and Microbial Technology 18:190-194, 1996 0 1996 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

0141-0229/96/$15.00 SSDI 0141-0229(95)00092-J

Effects
DNS reagent was prepared from 100 ml 5% (w/v) 3,5dinitrosalicylic acid in 2 M sodium hydroxide, mixed with 60% (w/v) aqueous sodium tartrate, and made up to 500 ml with water. Aqueous enzyme solutions were the following concentrations: alpha-amylase (1 .O g 1-l); 0.1% (v/v) glucoamylase; and 0.1% (v/v) invertase. The substrate solutions were: 1% or 5% (w/v) starch in 0.02 M phosphate buffer pH 6.9 containing 0.05 M sodium chloride: 5% or 10% (w/v) glycogen in phosphate buffer pH 6.9 containing 0.05 M sodium chloride; and 1.8 M sucrose in 0.05 M acetate buffer pH 4.7. Ultrasound experiments were carried out using a Kerry Pulsatron 60W ultrasonic bath with a capacity of 1.75 1 and internal dimensions of 176 x 162 x 250 mm. Round-bottomed Pyrex sample tubes (internal diameter, 15 mm; thickness, 1.0 mm) were

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Invertuse-catalyzed

hydrolysis of sucrose

An optimum substrate concentration of 0.7 M was recorded at 40C without sonication with a significant reduction in the rate at increased concentrations. An unexplained but consistent increase in rate at the highest sucrose concentration studied (2.0 M) was also observed (Figure I). Combes and Monsan3 have recorded a rate reduction above a lower optimum of 0.15 M sucrose. The application of ultrasound gave an optimum at 1.0 M sucrose with a substantially enhanced rate of 0.3 pmol min- compared to 0.2 pmol min- for unsonicated samples. Alpha-amylase catalyzed hydrolysis of starch

employed. Incubation and assay procedure

Substrate (2.0 ml) and buffer ( 1.O ml) solutions were equilibrated at 40C for 5 min. Following addition of 0.05 ml enzyme, the reaction was incubated for 10 min at 40C. All samples were mechanically shaken at 100 rpm throughout the procedure. DNS reagent (2 ml) was then added and the sample heated to 100C for 5 min. Following the addition of 20 ml water, the absorbance of the sample was determined at 540 nm against a blank where 0.05 ml water was substituted for enzyme. Experiments were performed in triplicate. Reducing equivalents were calculated from calibration graphs obtained using absorbance data for standard solutions of n-glucose reacted with DNS as above.

At lower substrate concentrations, increased reaction rates were observed with sonication (Figure 2); rates were approximately 50% greater at 8 g I-. At higher starch concentrations (15-50 g l-l), both sonicated and unsonicated samples showed similar patterns, reaching a maximum rate of around 7.0 pmol min- beyond 15 g 1-l. Alpha-amylase catalyzed hydrolysis of glycogen

Determination of power density at sample sites in the ultrasound bath

For each site, 3 ml water was placed in the reaction tube and samples were equilibrated at 40C for 10 min. The thermostat was then removed and the temperature increase recorded during a 1 min period of exposure to ultrasound. Power density was estimated from the relationship: dT power density = c,,m z

A major drawback of the use of starch as substrate in kinetic studies is its limited solubility. The use of glycogen should eliminate any influence of insoluble material on the rate of reaction. A decrease in the maximum rate was recorded at glycogen concentrations above 130 g 1-l with no significant difference in the pattern or rate of reaction between sonicated and unsonicated samples (Figure 3). Glucoamylase-catalyzed hydrolysis of starch

The use of ultrasound consistently enhanced the rate of reaction over the O-50 g 1-l substrate concentration range tested with a 20% increase recorded at 50 g 1-l. There was
0.4

(I)

where c,, = heat capacity of the solvent (J g-

K-l)

li .g Z E i g

0.3

M =

mass of solvent

(g) (C)

T = temperature f = time (s)

increase

0.2

This provides a measurement of power entering the system (3 ml) and thus can be expressed as watts per ml sample (W ml-).

Results and discussion

Determination of optimum power density
0.1

Maximum power density (0.35 W ml-) was obtained in the center of the bath; this location was used for all enzyme measurements. Values as low as 0.25 W ml- were recorded in other locations, thereby demonstrating the need to standardize sample location within the bath.

[Sucrose]

Figure 1 Invertase-catalyzed hydrolysis of sucrose in the presence and absence of ultrasound. Error bars indicate 95% confidence limits

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1.5

7 .i a E x g

1.0

;-

2.5 -

-J

2.0 -

0.5

0.0 2 [Starch] 4 g L
-1

10

20

30

40
-1

50

60

[Starch]

gL

Figure 2 Alpha-amylase-catalyzed hydrolysis of starch in the presence and absence of ultrasound. Error bars indicate 95% confidence limits

Figure 4 Glucoamylase-catalyzed hydrolysis of starch in the presence and absence of ultrasound. Error bars indicate 95% confidence limits

no evidence for inhibition even at the highest substrate concentrations (Figure 4). A marked difference in the shape of the rate curves is observed when starch was used as substrate (a slow initial increase in reaction velocity occurs) compared to the use of sucrose or glycogen which was characterized by a rapid initial increase. The decreased solubility of starch compared

to the other substrates mentioned tion for this difference.

may provide an explana-

The effects of ultrasound on enzymatic activity

Ultrasound has the potential to influence the reaction rate in several ways. A localized increase in temperature and enhanced mixing of substrate, enzyme, and products would be expected, Reaction temperatures were maintained at 40 + 2C so that any temperature effect would make only a marginal contribution to an increase in reaction rate. Sucrose solutions at concentrations greater than 1 M become visibly more viscous making it difficult to achieve efficient mixing using conventional mechanical methods. Particular attention was paid to ensure efficient mixing using vigorous and continuous mechanical shaking in the experiments described. This may account for the absence of significant invertase inhibition at substrate concentrations below 0.7 M. The application of ultrasound would undoubtedly achieve a more homogeneous reaction mixture and facilitate diffusion to and from the active sites of the enzyme. Similar improvements in mixing would be expected with the glycogen and starch substrate solutions employed with alpha-amylase and glucoamylase. A number of factors have been proposed to account for the depression of invertase activity at high sucrose concentrations3,. I; primarily high substrate viscosities, low water activities, and direct substrate and/or product inhibition of the reaction are considered. Combes and Manson demonstrated that invertase activity was unaffected when the viscosity of the reaction medium was increased using carboxymethyl cellulose, but was reduced by the decrease in free water concentration resulting from the addition of reagents such as magnesium chloride,9 an effect reported earlier using ethanol. I.* They concluded that the reduction in 15

,
6 CI 5

*El

50

100

150

[Glycogen]

g L

-i

Figure 3 Alpha-amylase-catalyzed hydrolysis of glycogen in the presence and absence of ultrasound. Error bars indicate 95% confidence limits

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Effects activity at sucrose concentrations in excess of 0.4 M was attributable to substrate inhibition resulting from hydrogen bonding interactions leading to the folding and clustering of sucrose molecules. These clusters may be resistant to invertase hydrolysis and form inactive enzyme-substrate complexes. It is possible that the application of ultrasound may lead to a disruption of water-sucrose and sucrose-sucrose hydrogen-bonding interactions, reducing the occurrence of inactive substrate forms. Substrate degradation via a nonenzymatic route is also possible. As early as 1933, Flosdorf and Chambers used an intense audible sound source to hydrolyze both starch and sucrose; similar effects have been demonstrated with ultrasound at 723 kHz.l Recent studies on potato starch suggested that although ultrasound may degrade starch granules leading to a decrease in viscosity, there was no degradation of the starch molecules themselves. Some degradation of dextrans in aqueous solution has been reported,6 but in the present study nonenzyme controls showed no evidence of significant substrate hydrolysis with ultrasound under the prevailing conditions. One further possibility is enzyme activation with ultrasound. Only a limited number of reports have recorded an increase in activity in the presence of ultrasound for free enzymes in vitro. ls Azhar and Hamdy observed that ultrasound had no effect on the activity of beta-amylase from sweet potato. Studies of alpha-amylase and glucoamylase immobilized on porous polystyrene beads demonstrated an increase in starch and maltose hydrolysis in the presence of sonic radiation..lh This increase was proportional for O-5 kW cm- sound intensity and was more pronounced for larger substrate molecules and larger carrier beads; however. the effect was attributed to an increase in substrate/ product diffusion rates at the carrier surface rather than a change in enzyme activity. There have been numerous reports that ultrasound decreases enzyme activity13,17 and in some cases, an increase in rate at lower intensities followed by a decrease in rate at higher intensities has been recorded, most notably for the activity of alpha-chymotrypsin on casein.lK

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equally expensive and pose potential contamination problems. Large sonic probes are a cheaper alternative providing an efficient circulation of the reaction mixture through the zones of high ultrasound activity (maximum 120 W cmP2).18 In view of this, the effect of ultrasound intensity on the rate of the reactions described would also benefit from further investigation.

Conclusions
The experiments described demonstrate the potential role of ultrasound in enhancing the rate of the enzyme-catalyzed hydrolysis of starch and sucrose over a wide range of substrate concentrations. The most likely mechanisms are increased efficiency of mixing and diffusion of reaction components, although direct enzyme activation is also feasible at the low power densities employed. In the case of invertase, ultrasound may also have an effect on intermolecular interactions involving substrate and water molecules. thereby relieving substrate inhibition.

Acknowledgments
We thank Dr. Juliusz Chrzastowski for his advice and support on all matters related to ultrasound. This research was supported by the Roehampton Institute Central Research Fund.

References
I. 2. 3. Godfrey, T. and Reichelt. J. Indusfrial Microbioln,g~ 2nd ed., Macmillan. New York, 1989. pp. 375-396 Dixon, M. and Webb, E. C. En:~mes 3rd ed., Longman, London, 1979 Combes, D. and Monsan. P. Sucrose hydrolysis by invertase: characterisation of products and substrate inhibition. Curbohydr. Rex 1983, 117.215-228 Mason, T. J. Sonochemistry: Current Trends and Future Prospects. In: Currenr Trends in Sonochemistyv Price, G. J. (Ed.). Royal Society of Chemistry. Cambridge, 1992, 168-178 Mason. T. J. Pructical Sonochemistry. Ellis Horwood. New York. 1991 Basedow. A. M. and Ebert, K. H. Zum mechanismus des abbaus von polymeren in losung durch ultraschall. A4ukmmc1l. Chem. 1975, 176, 745-157 Azhar, A. and Hamdy, M. K. Sonication effect on potato starch and sweet potato powder. J. Food Sci. 1979, 44, 801-804 Sumner, J. B. and Howell, S. F. A method for determination saccharase activity. J. Biol. Chem. 1935, 108, 51-54 Clark, J. M. (ed.) Experimenral cisco, 1964, pp. 25-27
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applicability

6.

For the ultrasound-enhanced activity of enzymes such as invertase or glucoamylase to be of commercial use, the feasibility of performing the reaction directly in an ultrasound bath and ultimately in a large-scale sonoreactor would require investigation. The problems associated with the scaleup of ultrasound processes for industrial use have been discussed by Mason5 The use of 5 m3 capacity cleaning bath reactors is feasible and has the advantage that the chance of reaction contamination by erosion of the ultrasound source is not present. A nonuniform field is one disadvantage of all large-scale ultrasound systems; efficient stirring and cooling systems would be required for the reactions investigated here. The chief disadvantage is cost; to achieve even 10% coverage of the vessel walls, an estimate of approximately 570 transducers would be required to give a system based on 9 1 medium for each transducer.19 Immersible transducers are a more flexible alternative but are

7. 8. 9. 10.

of

Freeman. San Fran-

Besserdich, E., Kahrig, E., Krenz, R.. and Kirstein, D. Kinetische untersuchungen zur substratuberschusshemmung geloster und tragerfixierter invertase. J. Mol. Catal. 1977, 2, 361-367 Mathlouthi, M. X-ray diffraction study of the molecular association in aqueous solutions of D-fructose. o-glucose and sucrose. Curhohydr. Rex 1981. 91, 113-123 Nelson, J. M. and Schubert, M. P. Water concentration and the rate of hydrolysis of sucrose by invertase. J. Am. Chem. SK. 1928, 50, 2188 Flosdorf. E. W. and Chambers, L. A. The chemical action of audible sound. J. Am. Chem. Sot. 1933. 55, 3051-3052 Szent-Gyorgi, A. Chemical and biological effects of ultrasonic radiation. Nature 1933, 131, 278

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15. Schmidt, P., Rosenfeld, E., Millner, R., and Schellenberger, A. Effects of ultrasound on the catalytic activity of matrix-bound glucoamylase. Ultrasonics 1987, 25, 295-299 Czemer, R., Millner, R., Roenfeld, E., Schellenberger, A., and Schmidt, P. Theoretical and experimental studies on the influence of ultrasound on immobilised enzymes. Biotechnol Bioeng. 1987, 30. 928-935 Macleod, R. M. and Dunn, F. Effects of intense non-cavitating ultrasound on selected enzymes. J. Acousr. Sock. Am. 1968. 44. 932940 Ishimori, Y., Karube, I.. and Suzuki. S. Acceleration of immohilised alpha-chymotrypsin activity with ultrasonic irradiation. ./. Mol. Caral. 198 1. 12, 253-259 Goodwin. T. .I. Scale-up considerations in sonochemistry. In: Sonochemistry: The Uses of Ultrasound in Chemistry (Mason, T. J., Ed.). Royal Society of Chemistry. Cambridge, 1990, 138-151

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