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ESTIMATION OF KINETIC PARAMETERSFOR RICE STARCH HYDROLYSIS INHIBITEDBY ADDED MATERIALSDilek Kiliç Apar a & Belma Özbek aa Department of Chemical Engineering , Davutpaşa Campus, YιldιzTechnical University , Esenler/Istanbul, TurkeyPublished online: 05 Dec 2006.

To cite this article: Dilek Kiliç Apar & Belma Özbek (2007) ESTIMATION OF KINETIC PARAMETERS FORRICE STARCH HYDROLYSIS INHIBITED BY ADDED MATERIALS, Chemical Engineering Communications,194:3, 334-344, DOI: 10.1080/15397730600830039

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Estimation of Kinetic Parameters for Rice StarchHydrolysis Inhibited by Added Materials

DILEK KILIC APAR AND BELMA OZBEK

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

In the present study, glucose, maltose, and glycerol inhibition effects for alpha-amylase hydrolyzing rice starch were investigated. For the enzymatic hydrolyzsisof rice starch, a-amylase enzyme produced from Bacillus licheniformis was used.During the starch hydrolysis experiments, the temperature and pH were kept con-stant at 60�C and 6.5 respectively. The reactions were carried out in 0.5 L of aqueoussolutions containing 1, 2, 4, and 8% (w=v) rice starch. The amount of a-amylaseenzyme added to this solution was 5 mL per liter (approximately 130500 units=L).The amounts of glucose, maltose, and glycerol added to the reaction solution were4 and 8% (w=v), 4 and 8% (w=v), and 5 and 10% (v=v), respectively. An empricalmodel effectively simulated the data of residual starch concentration as a functionof processing time for each condition. The Lineweaver-Burk plots showed that theinhibition effects of these added materials were uncompetitive.

Keywords Alpha-amylase; Hydrolysis; Inhibition kinetic; Rice starch; Stirredbatch reactor

Introduction

Starch-containing crops form an important constituent of the human diet, and a largeproportion of the food consumed by the world’s population originates from them.Besides the use of the starch-containing plant parts directly as a food source, starchis harvested and chemically or enzymatically processed into a variety of different pro-ducts such as starch hydrolysates, glucose syrups, fructose, starch or maltodextrinderivatives, and cyclodextrins. In spite of the large number of plants able to producestarch, only a few plants are important for industrial starch processing. The majorindustrial sources are maize, tapioca, potato, and wheat (van der Maarel et al., 2002).

Amylases are widely used enzymes that can hydrolyze the glucosidic bonds in starchand related glucose-containing compounds. There are two major types of amylases:alpha-amylase and beta-amylase. The action of alpha-amylase on starch moleculesreduces the solution viscosity by acting randomly along the glucose chain at a-1,4glucosidic bonds; alpha-amylase is often called the starch-liquefying enzyme for thisreason. Beta-amylase can attack starch a-1,4 bonds only on the non-reducing ends ofthe polymer and always produce maltose when a linear chain is hydrolyzed. Becauseof the characteristic production of the sugar maltose, beta-amylase is also called thesaccharifying enzyme (Bailey and Ollis, 1987; Salieri et al., 1995; Schuster et al., 2000).

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

Chem. Eng. Comm., 194:334–344, 2007Copyright # Taylor & Francis Group, LLCISSN: 0098-6445 print/1563-5201 onlineDOI: 10.1080/15397730600830039

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Several industrial processes use alpha-amylaze to hydrolyze starch prior tofermentation or production of added-value biochemicals. Therefore, the enzymatichydrolysis of starch has been studied by many investigators (van der Maarel et al.,2002; Bailey and Ollis, 1987; Salieri et al., 1995; Schuster et al., 2000; Hill et al., 1997;Colonna et al., 1988; Komolprasert and Ofoli, 1991; Apar and Ozbek, 2004, 2005;Graber and Combes, 1989; Somers et al., 1991). As both proteins and hydrolysisproducts can inhibit the action of alpha-amylase on starch, information is requireddescribing phenomena that affect the kinetics of starch hydrolysis bya-amylase enzyme.

In the present study, the decay and product inhibition effects for a-amylasehydrolyzing rice starch are presented. The degree of rice starch hydrolysis (%) wasinvestigated at constant process conditions such as temperature 60�C, pH 6.5, impellerspeed 300 rpm, processing time 10 minutes, and enzyme concentration 5 mL=L(approximately 130500 units enzyme per liter). Glucose, maltoses and glycerol wereused as the inhibiting chemicals. An emprical model, which represents the residualstarch concentration as a function of the processing time, was used to simulate thedata obtained from the hydrolysis experiments.

Materials and Methods

Bioreactor

A Gallenkamp Modular Bioreactor system (Model No. FER-195-010, manufacturedby Sanyo Gallenkamp PLC, Loughborough, UK) was used for the starch hydrolysisexperiments. Control of various parameters including impeller speed, pH, and tem-perature were performed by its modules. The 1.0 L vessel (round-bottom design)was constructed of glass and stainless steel with an aspect ratio (height=diameter)of 1.545. The important design details were as follows: operating volume, 0.5 L; inter-nal diameter, 11 cm; height, 17 cm; number of baffles, 4; baffle height, 13.5 cm; bafflewidth, 1.5 cm, number of impellers, 1; location of impeller from top plate, 14 cm;location of impeller from bottom plate, 3 cm; type of impeller, Rushton disc turbine;impeller diameter of disc, 4.8 cm; impeller blade width, 1.4 cm; impeller blade length,1.9 cm; number of blades, 6. The ratio of diameter of impeller to diameter of tank(D=T) is 0.436.

Materials Used

The a-amylase used was commercially available from Sigma Company, productcode: A3403, with unit definition: one unit of enzyme will liberate 1 mg of maltosefrom starch in three minutes at pH 6.9 at 20�C. Starch used was insoluble and com-mercially available from Calbiochem Company, product code: 569380. Maltose(product code: 1.05912), glucose (product code: 1.08337), and glycerol (productcode: 1.04093) were obtained from Merck Company.

Determination of Residual Starch Concentration

For determination of the residual starch concentration (Astolfi-Filfo et al., 1986;Mian et al., 2002), samples were taken at timed intervals. A 5 mL of iodine solution(0.5% KI and 0.15% I2) and known volumes of the samples were mixed. The finalvolume was completed to 15 mL by addition of distilled water. The absorbencieswere measured at 550 nm against a blank containing 5 mL of iodine solution and

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10 mL of distilled water. Then, absorbencies were converted to starch concentrationusing the calibration chart prepared. At least five measurements were made for eachcondition and the data were averaged. The reproducibility of the experiments waswithin the range of �5%.

Results and Discussion

Effect of Starch Concentration on Hydrolysis

To predict the effect of starch concentration on hydrolysis, a series of experimentswere conducted at various starch concentrations (10–80 g=L) under standard con-ditions (Figure 1). By increasing the amount of starch from 10 to 80 g=L, the hydroly-sis degree was decreased from 83.5 to 75.9% (reduced 9.1%) at the end of 10 minutesprocessing time due to production inhibition as higher concentration of starchresulted in higher concentrations of inhibitors. Yankov et al. (1986) also reported thatthe enzyme action was inhibited by substrate at high concentrations of starch.

After evaluation of the experimental data, the following residual starch concen-tration-processing time expression (an empirical model) given by Komolprasert andOfoli (1991) was used for each experimental set:

S1 ¼ a� expð�b�½t�Þ þ c ð1Þ

Figure 1. Starch concentration vs. processing time (without any addition) at various initialstarch concentrations.

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where S1 is the residual starch concentration (g=L) and t is processing time (min). Theconstants in Equation (1), a (g starch=L), b (min�1), and c (g starch=L); standard error(r), and R2 statistica values were estimated and are given in Table I.

Inhibition Effect of Glucose on Starch Hydrolysis

Inhibition experiments were conducted in order to determine the possible type ofproduct inhibition for a-amylase at various starch concentrations (10–80 g=L) inthe presence of glucose (see Figures 2 and 3). At 10 g=L initial starch concentration

Table I. Estimated parameters and statistical data for starch concentration withoutany addition at various initial starch concentrations

Parameters and statistical data

Starch concentration (without additive)

1% (w=v) 2% (w=v) 4% (w=v) 8% (w=v)

a 7.6551 15.1093 29.7814 57.8055b 1.9914 1.9490 1.9342 1.4010c 2.2357 4.6842 9.8858 21.6139d 0.6146 0.9797 1.6661 2.3093R2 0.9818 0.9880 0.9910 0.9956

Figure 2. Starch concentration vs. processing time with addition of 4% glucose (w=v) atvarious initial starch concentrations.

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in the presence of 4% w=v of glucose, the degree of hydrolysis was decreased2.3%; in the presence of 8% w=v of glucose, the degree of hydrolysis was decreased11.4%, with respect to the experiment performed without any addition, at the endof 10 minutes processing time. At 80 g=L initial starch concentration in the presenceof 4 and 8% w=v of glucose, the degrees of hydrolysis were decreased 35.3% and39.5%, respectively, compared to the experiment performed without any addition.As a result, hydrolysis degree of rice starch decreased as glucose inhibited thecatalytic action of the enzyme. The glucose inhibition was also reported by Hillet al. (1997) and Yankov et al. (1986) in their work. And also, the decrease in therate of mass transfer due to the addition of glucose may cause lower hydrolysisdegree, as reported by Hill et al. (1997).

After evaluation of the experimental data, again Equation (1), the residualstarch concentration-processing time expression, accurately represented the data inthe presence of glucose for each experimental set. The constants a, b, and c, standarderror (r), and R2 statistica values were estimated and are given in Table II.

Inhibition Effect of Maltose on Starch Hydrolysis

The effect of maltose addition (4% and 8% w=v) on hydrolysis was also investigated.By addition of maltose into the reaction solution, similar inhibition trends wereobserved as in the case of the presence of glucose. This conclusion was also reported

Figure 3. Starch concentration vs. processing time with addition of 8% glucose (w=v) atvarious initial starch concentrations.

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by Hill et al. (1997). At the end of 10 minutes processing time, in the presence of 4%w=v of maltose, the degree of hydrolysis was decreased 19.6%; in the presence of 8%w=v of maltose, the degree of hydrolysis was decreased 31.5%, at 10 g=L initialstarch concentration, with respect to the experiment performed without anyaddition. At 80 g=L initial starch concentration in the presence of 4 and 8% w=vof maltose, the degrees of hydrolysis were decreased 44.1% and 54.4%, respectively,compared to the experiment performed without any addition, as inhibition occurred.And also, the decrease in the rate of mass transfer due to the high sugar concentra-tions may cause lower hydrolysis degree, as claimed by Hill et al. (1997).

In the presence of maltose, to predict the effect of processing time on starchhydrolysis, again Equation (1), the residual starch concentration-processing timeexpressions was used for each experimental set. The constants a, b, and c, standarderror (r), and R2statistica values were estimated and are given in Table III.

Inhibition Effect of Glycerol on Starch Hydrolysis

Glycerol was claimed to be a competitive inhibitor for a-amylase by Graber andCombes (1989). And also, Somers et al. (1991) in their study added glycerol to theprocess medium to protect the enzyme from inactivation and cross-linked potatostarch powder (used as an adsorbent) from enzymatic degradation. Therefore, a seriesof experiments were performed in the presence of glycerol (5% and 10% v=v)under standard conditions to investigate the effect of glycerol on hydrolysis andagain similar inhibition trends with glucose and maltose were observed. At theend of 10 minutes processing time, in the presence of 5 and 10% v=v of glycerol,the degrees of hydrolysis were decreased 13.9% and 24.7%, at 10 g=L initial starchconcentration, with respect to the experiment performed without any addition. At80 g=L initial starch concentration in the presence of 5 and 10% v=v of glycerol,the degrees of hydrolysis were decreased 27% and 53.2%, respectively, comparedto the experiment performed without any addition.

Table II. Estimated parameters and statistical data for starch concentration withaddition of 4 and 8% glucose (w=v) at various initial starch concentrations

Parameters andstatistical data

Starch concentration (with 4% (w=v) glucose addition)

1% (w=v) 2% (w=v) 4% (w=v) 8% (w=v)

a 7.4897 13.3447 23.8498 36.2787b 1.7615 1.8079 1.6115 0.9993c 2.3677 6.4155 15.5994 42.2186d 0.5738 0.9845 2.0232 2.3896R2 0.9836 0.9847 0.9802 0.9888

Starch concentration (with 8% (w=v) glucose addition)

a 6.8586 11.9792 22.6476 34.3598b 2.0002 1.9771 1.7748 1.1946c 3.0390 7.8775 16.9998 45.3100d 0.5901 0.8384 1.5580 1.7857R2 0.9792 0.9861 0.9867 0.9927

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Starch hydrolysis was decreased because of inhibition of the enzyme by theaddition of glycerol into the reaction solution. Also, the decrease in hydrolysis couldbe related to mass transfer limitation as the viscosity of the solution increased byaddition of glycerol. At low viscosity, starch and enzyme molecules move freely tocontact each other; therefore, reaction takes place. On the other hand, at high vis-cosity, the molecules cannot move freely because of viscous effects and=or reducedwater activity; therefore, the rate of reaction decreases.

Equation (1), the residual starch concentration-processing time expression, wasused again for each experimental set in the presence of glycerol; the constants andstatistical values were estimated and are given in Table IV.

Table III. Estimated parameters and statistical data for starch concentration withaddition of 4 and 8% maltose (w=v) at various initial starch concentrations

Parameters andstatistical data

Starch concentration (with 4% (w=v) maltose addition)

1% (w=v) 2% (w=v) 4% (w=v) 8% (w=v)

a 6.2362 10.1637 18.5320 31.4024b 2.4566 1.6725 1.4612 1.0730c 3.7133 9.6927 20.9913 48.0658d 0.4360 0.7260 1.7429 1.6236R2 0.9859 0.9858 0.9762 0.9929

Starch concentration (with 8% (w=v) maltose addition)

a 5.2214 8.7837 16.5522 26.1075b 2.4079 1.7262 1.0929 0.8736c 4.7386 11.0850 22.8224 53.5810d 0.3468 0.6482 1.3565 1.5193R2 0.9873 0.9848 0.9825 0.9914

Table IV. Estimated parameters and statistical data for starch concentration withaddition of 5 and 10% glycerol (v=v) at various initial starch concentrations

Parameters andstatistical data

Starch concentration (5% (v=v) glycerol addition)

1% (w=v) 2% (w=v) 4% (w=v) 8% (w=v)

a 6.6691 13.3319 22.9915 41.4514b 1.8955 1.9242 1.5824 1.0765c 3.2287 6.4697 16.5649 37.6449d 0.4842 0.9907 1.6741 2.5084R2 0.9851 0.9844 0.9853 0.9904

Starch concentration (10% (v=v) glycerol addition)

a 5.6606 9.4372 14.7071 26.5754b 2.0103 1.4343 1.1090 0.9935c 4.2420 10.4061 24.5610 52.7989d 0.5039 0.6426 1.4794 1.4689R2 0.9778 0.9873 0.9742 0.9920

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Estimating of Michealis-Menten Constants

The reaction kinetics of the enzyme was investigated by the initial rate approach.Lineweaver-Burk plots (Figures 4–6) were drawn, and the type of inhibition for

Figure 5. Lineweaver-Burk plot for addition of maltose.

Figure 4. Lineweaver-Burk plot for addition of glucose.

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a-amylase was determined as uncompetitive for all additives. Then, uncompetitiveinhibition kinetic constants were estimated for each additive according to Equation (2)taken from Segel (1993). This uncompetitive product inhibition equation was alsoused by Pasari et al. (1988) and Steverson et al. (1984) in their works for a-amylasesobtained from different sources and for different process conditions.

V ¼ VmaxS

Km þ ð1þ ½I�KiÞSðuncompetitive inhibition kinetic equationÞ ð2aÞ

� 1

Kappm

¼ �1þ ½I�Ki

� �

Kmðwhen 1=V ¼ 0Þ ð2bÞ

� 1

Vappmax

¼ �1þ ½I�Ki

� �

Vmaxðwhen 1=S ¼ 0Þ ð2cÞ

The estimated constants for Equation (2) for each additive standard error (r),and R2 statistica values for Lineweaver-Burk plots are given in Table V(a) and (b).

Figure 6. Lineweaver-Burk plot for addition of glycerol.

Table V(a). Constants and statistical data calculated by usingLWB plot without additive

Constants and statistical data Without additive

Km(g=L) 227.79Vmax(g=L.sec) 4.32d 0.0772R2 0.9995

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[I]=Ki ratio in Equation (2) (given in Table V(b)) represents the inhibition degree(a). It was concluded that maltose is the most effective inhibitor when the inhibitiondegrees were compared at mass basis.

Conclusions

In this study, an evaluation of the experimental and theoretical data showed thatthe used additives, glucose, maltose, and glycerol, inhibited the catalytic action ofa-amylase in the same manner during the hydrolysis of starch.

The modeling study showed that a simple exponential function (an empiricalequation) accurately represented the data of residual starch concentration dependingon processing time for each condition.

The reaction kinetics of the enzyme was investigated by the initial rate approach,and the type of inhibition for a-amylase was determined as uncompetitive for alladditives. The Michaelis-Menten constants, Vmax and Km, were obtained as 4.32(g=L.sec) and 227.79 (g=L), respectively. The inhibition constants (Ki) for glucose,maltose, and glycerol were found as 24.21 g=L, 8.06 g=L, and 14.60 ml=L, respectively.

Acknowledgments

This research has been supported by Yildiz Technical University Scientific ProjectsCoordination Department. Project Number: 20-A-07-01-02.

References

Apar, D. K. and Ozbek, B. (2004). a-Amylase inactivation by temperature during starchhydrolysis process, Process Biochem., 39(9), 1137–1144.

Apar, D. K. and Ozbek, B. (2005). a-Amylase inactivation during rice starch hydrolysis,Process Biochem., 40(3–4), 1367–1379.

Astolfi-Filfo, S., Galembeck, E. V., Faria, J. B., and Frascino, A. C. S. (1986). Stable yeasttransformants that secrete functional a-amylase encoded by cloned mouse pancreaticcDNA, Biotechnology, 4, 311–315.

Table V(b). Constants and statistical data calculated by using LWB plots withadditives

Constants andstatistical data

Glucose addition Maltose addition Glycerol addition

4% (w=v) 8% (w=v) 4% (w=v) 8% (w=v) 5% (v=v) 10% (v=v)

Kappm (g=L) 83.49 52.97 35.70 21.52 52.76 28.75

Vappmax(g=L.sec) 1.61 1.03 0.69 0.41 1.02 0.53

Kai 23.72 24.69 7.70 8.42 15.07 14.13

Ki(average) 24.21 8.06 14.60a¼ [I]=Ki 1.65 3.30 4.96 9.92 3.42 6.84d 0.0289 0.2345 0.3954 0.3748 0.1579 0.2164R2 0.9999 0.9954 0.9870 0.9888 0.9979 0.9964

aUnits for Ki g=L for glucose and maltose; mL=L for glycerol (density of 1.26 g=mL).

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Komolprasert, V. and Ofoli, R. Y. (1991). Starch hydrolysis kinetics of Bacillus licheniformisa-amylase, J. Chem. Biotechnol., 51, 209–223.

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Pasari, A. B., Korus, R. A., and Heimsch, R. C. (1988). Kinetics of the amylase system ofSchwanniomyces castellii, Enzyme Microb. Technol., 10(3), 156–160.

Salieri, G., Vinci, G., and Antonelli, M. L. (1995). Microcalorimetric study of the enzymatichydrolysis of starch: an a-amylase catalyzed reaction, Anal. Cheimi. Acta, 300, 287–292.

Schuster, K. C., Ehmoser, H., Gapes, J. R., and Lendl, B. (2000). On-line FT-Raman spectro-scopic monitoring of starch gelatinisation and enzyme catalysed starch hydrolysis, Vib.Spectrosc., 22(1–2), 181–190.

Segel, I. H. (1993). Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and SteadyState Enzyme Systems, John Wiley, New York.

Somers, W., Rozi, H., Bonte, A., Visser, J., Rombouts, K., and Riet, K. (1991). On the Inter-action of a-amylase with crosslinked starch: evaluation of process conditions, EnzymeMicrob. Technol., 13, 997–1006.

Steverson, E. M., Korus, R. A., Admassu, W., and Heimsch, R. C. (1984). Kinetics of theamylase system of Saccharomycopsis fibuliger, Enzyme Microb. Technol., 12(6), 549–554.

van der Maarel, M. J. E. C., van der Veen, B., Uitdehaag, J. C. M., Leemhuis, H., andDijkhuizen, L. (2002). Properties and applications of starch-converting enzymes of thea-amylase family, J. Biotechnol., 94(2), 137–155.

Yankov, D., Dobreva, E., Beschkov, V., and Emanuilova, E. (1986). Study of optimum con-ditions and kinetics of starch hydrolysis by means of thermostable a-amylase, EnzymeMicrob. Technol., 11(8), 665–667.

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