glycosidase and malate

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The effects of ultrasound on theactivities of some glycosidase enzymesof industrial importanceStephen Barton, Clive Bullock, and Deborah WeirSchool 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 glycogenhydrolysis, and of invertase toward sucrose hydrolysis, were investigated using an ultrasound bath. A fixed-timeassay was employed with product formation determined by reducing equivalents formed per unit time using the3,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 increasein invertase activity towards sucrose (37% at 0.9 M) and a reduction in the inhibition of sucrose hydrolysisobserved at high substrate concentrations. Improvements in the efficiency of mixing and disruption of intra- andintermolecular substrate molecule interactions at high concentrations are possible explanations for the changesobserved.

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

IntroductionEnzyme-catalyzed hydrolyses are commonly employed inthe production of sugar syrups in the food industry. Therates of these reactions show a tendency to slow at highsubstrate concentrations particularly in the case of inver-tase at sucrose concentrations greater than 5% w/w. Thenature of invertase inhibition is complex due partly to prod-uct inhibition by o-fructose (competitive) and o-glucose(partial noncompetitive) as well as substrate inhibition at-tributed to a modification of the intra- and intermolecularhydrogen bonds between sucrose molecules at increasingsucrose concentrations.3 Reports that the use of ultrasoundmay enhance the activity of certain enzymes4 have led us toinvestigate its effects on the enzymatic hydrolysis of starch,glycogen, and sucrose, and in particular, its potential formodifying substrate/product inhibition of these reactions athigh substrate concentrations.

The use of an ultrasonic bath in this context has theadvantages of simplicity, low cost, and modest power out-puts (l-2 W cm- for each transducer) reducing the likeli-

Address reprint requests to Dr. Clive Bullock, Department of Biologicaland Chemical Sciences, Roehampton Institute, West Hill, London SW153SN, United KingdomReceived 9 December 1994: revised 26 April 1995; accepted 4 May 1995

hood of localized heating effects associated with ultrasoundprobe and cuphorn systems.5 The possibility of direct (non-enzymatic) substrate degradation which has been reportedfor some polymers including dextrins,5.6 though not forstarch, is also minimized. The most obvious limitations arethat the operating frequency is normally fixed and powerdensities vary within the bath, consequently standardizationof 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; EC3.2.1.3) on starch and/or glycogen, and of invertase (p-o-fructofuranoside fructohydrolase; EC 3.2.1.26) on sucrosewere the subject of this investigation. The progress of sub-strate hydrolysis was measured by determination of the re-ducing sugar equivalents released using the standard 3,5-dinitrosalicylate (DNS) assay following a 10 min fixed-time incubation period with the enzyme in the presence orabsence of ultrasound in each case.

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

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Effects of ultrasound on glycosidase enzymes: S. Barton et al.DNS reagent was prepared from 100 ml 5% (w/v) 3,5-

dinitrosalicylic 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: al-pha-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 in0.02 M phosphate buffer pH 6.9 containing 0.05 M sodium chlo-ride: 5% or 10% (w/v) glycogen in phosphate buffer pH 6.9 con-taining 0.05 M sodium chloride; and 1.8 M sucrose in 0.05 Macetate buffer pH 4.7.

Inv ertuse-cataly zed hydroly sis of sucrose

Ultrasound experiments were carried out using a Kerry Pulsa-tron 60W ultrasonic bath with a capacity of 1.75 1 and internaldimensions of 176 x 162 x 250 mm. Round-bottomed Pyrex sam-ple tubes (internal diameter, 15 mm; thickness, 1.0 mm) wereemployed.

An optimum substrate concentration of 0.7 M was recordedat 40C without sonication with a significant reduction inthe rate at increased concentrations. An unexplained butconsistent increase in rate at the highest sucrose concentra-tion studied (2.0 M) was also observed (Figure I). Combesand Monsan3 have recorded a rate reduction above a loweroptimum of 0.15 M sucrose. The application of ultrasoundgave an optimum at 1.0 M sucrose with a substantially en-hanced rate of 0.3 pmol min- compared to 0.2 pmol min-for unsonicated samples.

Incubation and assay procedureSubstrate (2.0 ml) and buffer (1 O ml) solutions were equilibratedat 40C for 5 min. Following addition of 0.05 ml enzyme, thereaction was incubated for 10 min at 40C. All samples weremechanically shaken at 100 rpm throughout the procedure. DNSreagent (2 ml) was then added and the sample heated to 100C for5 min. Following the addition of 20 ml water, the absorbance ofthe sample was determined at 540 nm against a blank where 0.05ml water was substituted for enzyme. Experiments were performedin triplicate. Reducing equivalents were calculated from calibra-tion graphs obtained using absorbance data for standard solutionsof n-glucose reacted with DNS as above.

Alpha-amy lase catalyzed hy drolysis of starchAt lower substrate concentrations, increased reaction rateswere observed with sonication (Figure 2); rates were ap-proximately 50% greater at 8 g I-. At higher starch con-centrations (15-50 g l-l), both sonicated and unsonicatedsamples showed similar patterns, reaching a maximum rateof around 7.0 pmol min- beyond 15 g 1-l.

Determinat ion of power density at sample sites inthe ultrasound bath

Alpha-amy lase catalyzed hy drolysis of glycogenA major drawback of the use of starch as substrate in kineticstudies is its limited solubility. The use of glycogen shouldeliminate any influence of insoluble material on the rate ofreaction. A decrease in the maximum rate was recorded atglycogen concentrations above 130 g 1-l with no significantdifference in the pattern or rate of reaction between soni-cated and unsonicated samples (Figure 3).

For each site, 3 ml water was placed in the reaction tube andsamples were equilibrated at 40C for 10 min. The thermostat wasthen removed and the temperature increase recorded during a 1min period of exposure to ultrasound. Power density was estimatedfrom the relationship:

Glucoamy lase-catalyzed hydrolysis of starchThe use of ultrasound consistently enhanced the rate ofreaction over the O-50 g 1-l substrate concentration rangetested with a 20% increase recorded at 50 g 1-l. There was

dTpower density = c,,m z

where

(I)

c,, = heat capacity of the solvent (J g- K-l)M = mass of solvent (g)T = temperature increase (C)f = time (s)

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 discussionDeterminat ion of optim um power density

0.4

li 0.3.gZEi

- 0.2g

0.1

Maximum power density (0.35 W ml-) was obtained in thecenter of the bath; this location was used for all enzymemeasurements. Values as low as 0.25 W ml- were recordedin other locations, thereby demonstrating the need to stan-dardize sample location within the bath.

[Sucrose] M

Figure 1 Invertase-catalyzed hydrolysis of sucrose in the pres-ence and absence of ultrasound. Error bars indicate 95% confi-dence limits

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Papers1.5

- 1.07.iaEx- 0.5g

0.02 4

[Starch] g L-16 a 0 10 20 30 40 50 60

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

no evidence for inhibition even at the highest substrate con-centrations (Figure 4).

A marked difference in the shape of the rate curves isobserved when starch was used as substrate (a slow initialincrease in reaction velocity occurs) compared to the use ofsucrose or glycogen which was characterized by a rapidinitial increase. The decreased solubility of starch compared

,6

5CI7*El 4

1

0 I I I I0 50 100 150[Glycogen] g L-i

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

-; 2.5 -

-J 2.0 -

[Starch] g L 1

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

to the other substrates mentioned may provide an explana-tion for this difference.The effects of ultrasound on enzymatic activityUltrasound has the potential to influence the reaction rate inseveral ways. A localized increase in temperature and en-hanced mixing of substrate, enzyme, and products would beexpected, Reaction temperatures were maintained at 40 +2C so that any temperature effect would make only a mar-ginal contribution to an increase in reaction rate. Sucrosesolutions at concentrations greater than 1 M become visiblymore viscous making it difficult to achieve efficient mixingusing conventional mechanical methods. Particular atten-tion was paid to ensure efficient mixing using vigorous andcontinuous mechanical shaking in the experiments de-scribed. This may account for the absence of significantinvertase inhibition at substrate concentrations below 0.7 M.The application of ultrasound would undoubtedly achieve amore homogeneous reaction mixture and facilitate diffusionto and from the active sites of the enzyme. Similar improve-ments in mixing would be expected with the glycogen andstarch substrate solutions employed with alpha-amylase andglucoamylase.

A number of factors have been proposed to account forthe depression of invertase activity at high sucrose concen-trations3,. I; primarily high substrate viscosities, low wa-ter activities, and direct substrate and/or product inhibitionof the reaction are considered. Combes and Manson dem-onstrated that invertase activity was unaffected when theviscosity of the reaction medium was increased using car-boxymethyl cellulose, but was reduced by the decrease infree water concentration resulting from the addition of re-agents such as magnesium chloride,9 an effect reported ear-lier using ethanol. I.* They concluded that the reduction in

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Effects of ultrasound on glycosidase enzymes: S. Barton et al.activity at sucrose concentrations in excess of 0.4 M wasattributable to substrate inhibition resulting from hydrogenbonding interactions leading to the folding and clustering ofsucrose molecules. These clusters may be resistant to inver-tase hydrolysis and form inactive enzyme-substrate com-plexes. It is possible that the application of ultrasound maylead to a disruption of water-sucrose and sucrose-sucrosehydrogen-bonding interactions, reducing the occurrence ofinactive substrate forms.

Substrate degradation via a nonenzymatic route is alsopossible. As early as 1933, Flosdorf and Chambers usedan intense audible sound source to hydrolyze both starchand sucrose; similar effects have been demonstrated withultrasound at 723 kHz.l Recent studies on potato starchsuggested that although ultrasound may degrade starchgranules leading to a decrease in viscosity, there was nodegradation of the starch molecules themselves. Some deg-radation of dextrans in aqueous solution has been reported,6but in the present study nonenzyme controls showed noevidence of significant substrate hydrolysis with ultrasoundunder the prevailing conditions.

One further possibility is enzyme activation with ultra-sound. Only a limited number of reports have recorded anincrease in activity in the presence of ultrasound for freeenzymes in vitro. ls Azhar and Hamdy observed that ultra-sound had no effect on the activity of beta-amylase fromsweet potato. Studies of alpha-amylase and glucoamylaseimmobilized on porous polystyrene beads demonstrated anincrease in starch and maltose hydrolysis in the presence ofsonic radiation..lh This increase was proportional for O-5kW cm- sound intensity and was more pronounced forlarger substrate molecules and larger carrier beads; how-ever. the effect was attributed to an increase in substrate/product diffusion rates at the carrier surface rather than achange in enzyme activity. There have been numerous re-ports that ultrasound decreases enzyme activity13,17 and insome cases, an increase in rate at lower intensities followedby a decrease in rate at higher intensities has been recorded,most notably for the activity of alpha-chymotrypsin oncasein.lK

Commercial applicabilityFor the ultrasound-enhanced activity of enzymes such asinvertase or glucoamylase to be of commercial use, thefeasibility of performing the reaction directly in an ultra-sound bath and ultimately in a large-scale sonoreactorwould require investigation. The problems associated withthe scaleup of ultrasound processes for industrial use havebeen discussed by Mason5 The use of 5 m3 capacity clean-ing bath reactors is feasible and has the advantage that thechance of reaction contamination by erosion of the ultra-sound source is not present. A nonuniform field is one dis-advantage of all large-scale ultrasound systems; efficientstirring and cooling systems would be required for the re-actions investigated here. The chief disadvantage is cost; toachieve even 10% coverage of the vessel walls, an estimateof approximately 570 transducers would be required to givea system based on 9 1 medium for each transducer.19 Im-mersible transducers are a more flexible alternative but are

equally expensive and pose potential contamination prob-lems. Large sonic probes are a cheaper alternative providingan efficient circulation of the reaction mixture throughthe zones of high ultrasound activity (maximum 120 WcmP2).18 In view of this, the effect of ultrasound intensity onthe rate of the reactions described would also benefit fromfurther investigation.

ConclusionsThe experiments described demonstrate the potential role ofultrasound in enhancing the rate of the enzyme-catalyzedhydrolysis of starch and sucrose over a wide range of sub-strate concentrations. The most likely mechanisms are in-creased efficiency of mixing and diffusion of reaction com-ponents, although direct enzyme activation is also feasibleat the low power densities employed. In the case of inver-tase, ultrasound may also have an effect on intermolecularinteractions involving substrate and water molecules.thereby relieving substrate inhibition.

AcknowledgmentsWe thank Dr. Juliusz Chrzastowski for his advice and sup-port on all matters related to ultrasound. This research wassupported by the Roehampton Institute Central ResearchFund.

ReferencesI.

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fects of ultrasound on the catalytic activity of matrix-bound glu-coamylase. Ultrasonics 1987, 25, 295-299

16. Czemer, R., Millner, R., Roenfeld, E., Schellenberger, A., andSchmidt, P. Theoretical and experimental studies on the influence ofultrasound on immobilised enzymes. Biotechnol Bioeng. 1987, 30.928-935

17. Macleod, R. M. and Dunn, F. Effects of intense non-cavitating ul-

trasound on selected enzymes. J. Acousr. Sock. Am. 1968. 44. 932-940

18. Ishimori, Y., Karube, I.. and Suzuki. S. Acceleration of immohilisedalpha-chymotrypsin activity with ultrasonic irradiation. ./. Mol.Caral. 198 1. 12, 253-259

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glycosidase and malate

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