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ENZYMATIC HYDROLYSIS OF STARCH BYUSING A SONIFIERDilek Kiliç Apar a , Meltem Turhan a & Belma Özbek aa Department of Chemical Engineering , Yιldιz Technical University ,Davutpaşa Campus, Esenler/Istanbul, TurkeyPublished online: 30 Aug 2006.

To cite this article: Dilek Kiliç Apar , Meltem Turhan & Belma Özbek (2006) ENZYMATIC HYDROLYSISOF STARCH BY USING A SONIFIER, Chemical Engineering Communications, 193:9, 1117-1126, DOI:10.1080/00986440500354424

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Enzymatic Hydrolysis of Starch by Using a Sonifier

DILEK KILIC APAR, MELTEM TURHAN, ANDBELMA OZBEK

Department of Chemical Engineering, Yildiz Technical University,Davutpasa Campus, Esenler=Istanbul, Turkey

In the present study, the ultrasonication method was used to investigate the effect ofultrasonic energy on the hydrolysis of corn, rice, and wheat starch by using thealpha-amylase enzymes produced by Bacillus species and Bacillus licheniformis.The effects of sonifier operation variables such as duty cycle and acoustic power rateon the stability of alpha-amylase enzymes and hydrolysis degrees of three types ofstarches were investigated at a temperature of 40�C and pH 6.5. To determine theeffect of temperature with sonication on the hydrolysis process, wheat starchhydrolysis experiments were also carried out at a temperature of 50�C. Then, therelation between duty cycle and enzyme stability during hydrolysis for each enzymeat 50�C was expressed by a zero-order inactivation model.

Keywords Alpha-amylase; Enzyme stability; Starch hydrolysis; Sonicationprocess; Duty cycle; Acoustic power

Introduction

Starch is a major storage product of many economically important crops such aswheat, rice, corn, maize tapioca, and potatoes. A large-scale starch processing indus-try has emerged in the past century. In recent decades, there has been a shift from theacid hydrolysis of starch to the use of starch-converting enzymes in the production ofmaltodextrin-modified starches or glucose and fructose syrups. Besides their use instarch hydrolysis, starch-converting enzymes are also used in a number of otherindustrial applications, such as laundry and porcelain detergents or as anti-stalingagents in baking (van der Maarel et al., 2002; Colonna et al., 1998; Textor et al.,1998; Hill et al., 1997).

Amylases, which are starch-hydrolyzing enzymes, are the most important indus-trial enzymes. The enzymes belonging to the amylase class represent catalysts thatcan hydrolytically break the glucosidic bonds on the starch molecule and its deriva-tives, by adding water molecules to them and by transforming the polymers to lowermolecular weight molecules (oligomers and=or monomers). In the food industrythese enzymes are employed especially for the production of oven food products,glucose syrup, food-stuff products, and also alcohols and sugars; moreover, amylo-lytic enzymes are used in paper and drapery manufacture, in malt saccharificationfor brewing beer, and in the detergents industry (Salieri et al., 1995; Komolprasertand Ofoli, 1991).

Address correspondence to Belma Kin Ozbek, Department of Chemical Engineering,Yildiz Technical University, Davutpasa Campus, 34210, Esenler=Istanbul, Turkey. E-mail:bozbek@yildiz.edu.tr

Chem. Eng. Comm., 193:1117–1126, 2006Copyright # Taylor & Francis Group, LLCISSN: 0098-6445 print/1563-5201 onlineDOI: 10.1080/00986440500354424

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In biotechnological processes, the ultrasonication method (Feliu et al., 1998;Middelberg, 1996) is widely used at the laboratory scale, and it does not requiresophisticated equipment or extensive technical training. The structure and functionof biological molecules can be changed by ultrasound irradiation. The most commoninteraction mechanisms involved in this case are heat or chemical effects and acous-tically induced cavitational activity. In addition, inactivation of released products byultrasonication can also be caused by mechanical effects, i.e., shear stress developedby eddies arising from shock waves. The level of ultrasound intensity plays the majorpart in the activity of many enzymes. On the other hand, some researchers reportedthat the activity of free enzymes increased under mild ultrasound irradiation(Sakakibara et al., 1996; Choi and Kim, 1994; Ozbek and Ulgen, 2000).

For this study, the ultrasonication method was chosen to investigate the effect ofultrasonic energy on the hydrolysis of corn, rice, and wheat starch with the alpha-amylases produced by different Bacillus strains. Enzyme inactivations are one ofthe constraints in the rapid development of the biotechnological processes. There-fore, before designing a successful hydrolysis system, information is required todescribe phenomena that affect the kinetics of starch hydrolysis.

Materials and Methods

Experimental Apparatus

The experimental apparatus used for this work is shown in Figure 1. All enzymatichydrolysis reactions were carried out in a 500 mL cylindrical glass reactor (8 cm indiameter, 10 cm in height). The ultrasonication experiments were carried out at20 kHz on a Bandelin Sonopuls HD 2200 equipped with a horn. The tip of the horn(KE 76 titanium tapered tip of 6 mm diameter) was immersed about 2 cm into

Figure 1. Experimental apparatus used for enzymatic hydrolysis of starch.

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250 mL solution to be processed in the vessel. The temperature and pH inside thesolutions were intermittently checked and kept constant by the use of an outer jacketconnected to a temperature-controlled water bath and pH controller module. Thetemperature in the reactor was also measured continuously by a digital thermometer.The acoustic power was controlled manually between 20 and 100 W. The ultrasonicenergy was pulsed using the Duty Cycle Control of the instrument in order to reducethe formation of free radicals. In pulsed mode, ultrasonic vibrations are transmittedto the solution at a rate of one pulse per second.

Enzymatic Hydrolysis of Starch

The effects of sonifier duty cycle (from 10 to 80%; inreased by increments of 10) andacoustic power (20 and 100 W) were investigated by checking the stability of thealpha-amylases and hydrolysis degrees of corn, rice, and wheat starch at 40�C andpH 6.5. Wheat starch hydrolysis experiments were also performed at a temperatureof 50�C for determination of the effect of temperature with the sonication on thehydrolysis process. The reactions were carried out in 250 mL of aqueous solutionscontaining 10 g=L starch and approximately 90000 units enzyme per liter, with pro-cessing time of 30 minutes for each experiment. Enzyme activities prior to ultrasoni-cation process were determined as the initial activities. In calculations, the activitieswere determined as 100% activity. Activities at any operational conditions (A) werethen obtained as the percentage values of the initial activities.

Determination of Hydrolysis Degree of Starch

The hydrolysis degrees of starch were calculated using the residual starch concentra-tions. For determination of the residual starch concentrations (Astolfi-Filfo et al.,1986) in the reaction solution, the samples were taken at the begining and at theend of the process. A 5 mL amount of iodine solution (0.5% KI and 0.15% I2)and known volumes of the samples were mixed. The final volume was completedto 15 mL by addition of distilled water. The absorbencies were measured at550 nm against a blank containing 5 mL of iodine solution and 10 mL of distilledwater. Then, absorbencies were converted to starch concentration using the cali-bration chart. At least five measurements were made for each condition and the datawere averaged.

Determination of Alpha-Amylase Activity

Two types of alpha-amylases from different Bacillus strains were employed in thepresent work, those produced from Bacillus species and from Bacillus licheniformis(obtained from Sigma Chemical Company, Product Code: A6814 and Product Code:A3403, respectively). The determination of alpha-amylase activity was done accord-ing to De Moraes et al. (1995) as follows: 0.2 g of soluble starch was dissolved in100 mL boiling 50 mM sodium acetate buffer (pH 5.9). The solution was cooled to40�C, and 200 mL of the appropriately diluted reaction solution was added to 1 mLof starch solution and the mixture was incubated at 40�C in a water bath for 10 min-utes. The iodine reagent was prepared by addition of 1 mL stock solution (0.5% I2 in5% KI) and 5 mL 5 M HCl to 0.5 L of distilled water. Then 200 mL incubated reactionmixture was added to 5 mL iodine solution to stop the reaction. The degradation of

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starch by the enzyme was measured at 620 nm against 200 mL water in 5 mL of iodinesolution as a blank. One unit of alpha-amylase activity was defined as the quantity ofenzyme required to hydrolyse 0.1 mg starch in 10 minutes at 40�C when 2 mg starchwas present at the start of the reaction. At least five measurements were made for eachcondition, and the data given are an average of these results.

Results and Discussion

Effect of Duty Cycle and Acoustic Power on Alpha-Amylase Stability andCorn Starch Hydrolysis

In this work, the effects of sonifier operational parameters on corn starch hydrolysisby using alpha-amylase enzymes produced by Bacillus species and Bacillus lichenifor-mis were investigated at temperature of 40�C. Figures 2 and 3 show the residual alpha-amylase enzyme activities (%) and hydrolysis degrees of corn starch (%) depending onthe duty cycle rates at acoustic powers 20 and 100 W.

According to the data obtained from the experiments by using alpha-amylaseproduced by Bacillus species (see Figure 2) and Bacillus licheniformis (see Figure 3),no significant change was observed for the hydrolysis degrees of corn starch(approximately constant at 5% and 16%, respectively) and the enzyme activities(approximately constant at 94% and 92% of original activity, respectively) byincreasing the duty cycle rates from 10% to 80% at acoustic powers 20 and 100 W.

Effect of Duty Cycle and Acoustic Power on Alpha-Amylase Stability and RiceStarch Hydrolysis

In order to determine the effects of sonifier operational parameters on rice starchhydrolysis by using alpha-amylase enzymes, the experiments were carried out atthe same conditions as mentioned above for corn starch. Figures 4 and 5 show the

Figure 2. Residual enzyme activity (%) and hydrolysis degree (%) for corn starch hydrolyzedby B. species at a temperature of 40�C and acoustic powers 20 and 100 W.

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residual alpha-amylase enzyme activities (%) and hydrolysis degrees of rice starch(%) depending on the duty cycle rates at acoustic powers 20 and 100 W.

As seen from Figure 4, showing the data obtained by using alpha-amylase pro-duced by Bacillus species, no significant change was observed for the hydrolysisdegrees of rice starch (approximately constant at 11%) and the enzyme activities(approximately 98%), and again from the data obtained by using alpha-amylase pro-duced by Bacillus licheniformis (see Figure 5), no significant change was observed forhydrolysis degrees (approximately constant at 30%) and enzyme activities (approxi-mately 95%) by increasing the duty cycle rates from 10% to 80% at acoustic powers20 and 100 W.

Figure 3. Residual enzyme activity (%) and hydrolysis degree (%) for corn starch hydrolyzedby B. licheniformis at a temperature of 40�C and acoustic powers 20 and 100 W.

Figure 4. Residual enzyme activity (%) and hydrolysis degree (%) for rice starch hydrolyzedby B. species at a temperature of 40�C and acoustic powers 20 and 100 W.

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Effect of Duty Cycle and Acoustic Power on Alpha-Amylase Stability and WheatStarch Hydrolysis

To investigate the effect of temperature with the sonication on the hydrolysis processand enzyme stability, wheat starch hydrolysis experiments were also carried out at atemperature of 50�C. The residual alpha-amylase enzyme activities (%) and hydroly-sis degrees of wheat starch (%) depending on the duty cycle rates at acoustic powers20 and 100 W are given in Figures 6–9.

According to the data obtained from these experiments performed at a tempera-ture of 40�C by using alpha-amylase produced by Bacillus species (see Figures 6

Figure 5. Residual enzyme activity (%) and hydrolysis degree (%) for rice starch hydrolyzedby B. licheniformis at a temperature of 40�C and acoustic powers 20 and 100 W.

Figure 6. Residual enzyme activity (%) and hydrolysis degree (%) for wheat starch hydro-lyzed by B. species at temperatures of 40� and 50�C and acoustic power 20 W.

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and 7), no significant change was observed for the hydrolysis degrees of wheat starch(approximately constant at 18%) and the enzyme activities (approximately constantat 92%), and again from the data obtained by using alpha-amylase produced byBacillus licheniformis (see Figures 8 and 9), no significant change was observed forhydrolysis degrees (approximately constant at 32%) and enzyme activities (approxi-mately constant at 92%) by increasing the duty cycle rates from 10% to 80% atacoustic powers 20 and 100 W, respectively.

At the temperature of 50�C by using alpha-amylase produced by Bacillus species(see Figures 6 and 7) and Bacillus licheniformis (see Figures 8 and 9), no significant

Figure 7. Residual enzyme activity (%) and hydrolysis degree (%) for wheat starch hydro-lyzed by B. species at temperatures of 40� and 50�C and acoustic power 100 W.

Figure 8. Residual enzyme activity (%) and hydrolysis degree (%) for wheat starch hydro-lyzed by B. licheniformis at temperatures of 40� and 50�C and acoustic power 20 W.

Enzymatic Hydrolysis of Starch 1123

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change was observed for the hydrolysis degrees of wheat starch (approximately con-stant at 61% and 76%, respectively) by increasing the duty cycle rates from 10% to80% at acoustic powers 20 and 100 W, respectively. By increasing the temperaturefrom 40� to 50�C, the hydrolysis degrees of wheat starch obtained by using alpha-amylase enzymes produced by Bacillus species and Bacillus licheniformis increasedsignificantly for all duty cycle rates and acoustic powers. These increases in thehydrolysis degrees of wheat starch can be explained by the temperature increaseand were not related to the sonication itself.

On the other hand, at the temperature of 50�C, by increasing the duty cycle ratesfrom 10% to 80% at acoustic powers 20 and 100 W, the activities of the enzyme pro-duced by Bacillus species (see Figures 6 and 7) decreased from 91% to 77% and from89% to 75%, respectively, and the activities of the enzyme produced by Bacilluslicheniformis (see Figures 8 and 9) decreased from 89% to 81% and from 89% to80%, respectively. Decreases in activities of both enzymes could be explained by thecombined effects of the temperature and sonifier duty cycle. At the acoustic powerrange of 20–100 W, no change in enzymatic stability was observed. Sonication powersused in this work may not be high enough to make any changes in the stabilityof enzymes.

To predict the effect of the duty cycle on enzyme stability, data of residuala-amylase enzyme activity versus duty cycle were evaluated for each enzymeused at acoustic powers 20 and 100 W at a temperature of 50�C. A zero-orderinactivation model (Equation (1)), which effectively simulated the experimental data,was used.

A ¼ Amax � kDðDCÞ ð1Þ

where Amax is enzyme activity without ultrasonication, and kD is the zero-orderinactivation coefficient dependent on duty cycle (DC). The estimated para-meters and statistical values for each acoustic power and for each enzyme are givenin Table I.

Figure 9. Residual enzyme activity (%) and hydrolysis degree (%) for wheat starch hydro-lyzed by B. licheniformis at temperatures of 40� and 50�C and acoustic power 100 W.

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Enzymatic Starch Hydrolysis Experiments without Sonication

The experiments of starch hydrolysis without sonication with alpha-amylase fromtwo bacterial sources at temperatures of 40� and 50�C were conducted in a stirredbatch reator at an impeller speed of 300 rpm and pH 6.5 to illustrate the effects ofsonication. The experimental results are given in Table II. Lower starch hydrolysisdegrees and higher residual enzyme activity values with sonication were obtained.These results show that sonication was not beneficial for starch hydrolysis at thegiven experimental conditions. The lower hydrolysis degrees could be explained bythe accumulation that sonication could not help to disturb of the structure of anytype of starch used for this study or by the mass transfer being limited by the oscillat-ing frequency of the sonifier. In addition to this, the forces involved in the sonifiedfluid may play a significant role in the reaction mechanism that reduces hydrolysisdegree, although enzymes remained stable.

Conclusion

In this study, for corn starch, no sufficient hydrolysis degree (approximately 5% and16%) was obtained for both enzymes used, while for rice and wheat starch consider-able hydrolysis degrees were obtained as constant at an approximate value of 30%by using Bacillus licheniformis at a temperature of 40�C, at any sonifier duty cyclesand acoustic power rates. At a temperature of 50�C, the hydrolysis degrees for wheatstarch obtained were quite high for both enzymes. On the other hand, the enzymesdid not lose their stabilities significantly depending on duty cycle and acoustic power

Table I. Estimated parameters and statistical values for wheat starch hydrolysis at atemperature of 50�C

Acousticpower (W)

a-Amylasesource Amax

kD

(min�1)Standard error

(r)R2

statistic

20 B. subtilis 94.1029 0.1959 0.9058 0.9903100 B. subtilis 91.3171 0.2009 0.3016 0.998920 B. licheniformis 90.6076 0.1062 1.0898 0.9550100 B. licheniformis 91.2758 0.1260 1.1861 0.9618

Table II. Starch hydrolysis without sonication

Starch typeCorn starch

(40�C)

Ricestarch(40�C)

Wheatstarch(40�C)

Wheat starch(50�C)

Enzymesource

B. subs. B. lich. B. subs. B. lich. B. subs. B. lich. B. subs. B. lich.

Hydrolysisdegree (%)

13 21 21 32 23 32 75 90

Residualenz. act. (%)

94 83 87 87 96 85 89 78

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rates, except at a temperature of 50�C. At this temperature for wheat starch hydroly-sis, the relation between duty cycle and enzyme stability for each enzyme wasrepresented by the zero-order inactivation model.

The experiments of starch hydrolysis without sonication with alpha-amylasefrom two bacterial sources at temperatures of 40� and 50�C were also performedto show the effect of sonication. The higher starch hydrolysis degrees and lowerresidual enzyme activity values were obtained without sonication. These resultsshow that sonication did not contribute to increase hydrolysis degrees at the givenexperimental conditions.

Acknowledgements

This work was supported by the Yildiz Technical University Research Fund (ProjectNo: 21-07-01-03).

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