CORN GLUTEN HYDROLYSIS BY ALCALASE: KINETICS OF HYDROLYSIS

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CORN GLUTEN HYDROLYSIS BYALCALASE: KINETICS OF HYDROLYSISDilek Kiliç Apar a & Belma Özbek aa Department of Chemical Engineering , Yıldız Technical University,Davutpaşa Campus , Esenler/Istanbul, TurkeyPublished online: 03 Feb 2010.

To cite this article: Dilek Kiliç Apar & Belma Özbek (2010) CORN GLUTEN HYDROLYSIS BYALCALASE: KINETICS OF HYDROLYSIS, Chemical Engineering Communications, 197:7, 963-973, DOI:10.1080/00986440903359368

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Corn Gluten Hydrolysis by Alcalase:Kinetics of Hydrolysis

DILEK KILIC APAR AND BELMA OZBEK

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

In the present study, the reaction kinetics of corn gluten hydrolysis by Alcalase,a bacterial protease produced by Bacillus licheniformis, was investigated. The reac-tions were carried out for 10min in 0.1 L of aqueous solutions containing 10, 20, 30,40, and 50 g protein L�1 corn gluten at various temperature and pH values. Theamount of enzyme added to the reaction solution was 0.25% (v=v). Also, to deter-mine decay and product inhibition effects for Alcalase, a series of inhibition experi-ments were conducted with the addition of various amounts of hydrolysate. For eachexperimental run, both the amount of hydrolysis (meqv L�1) and the soluble proteinamount (g L�1) were investigated with respect to time, and the initial reaction rateswere determined from the slopes of the linear models that fitted to these experi-mental data. The kinetic parameters, Km and Vmax were estimated as 53.77 g L�1

and 5.94meqv L�1min�1. The type of inhibition for Alcalase was determined asuncompetitive, and the inhibition constant, Ki, was estimated as 44.68% (hydrolysate=substrate mixture).

Keywords Alcalase; Corn gluten; Hydrolysis kinetics; Product inhibition

Introduction

Protein hydrolysis is carried out for many reasons, including improving nutritionalvalue, retarding deterioration, imparting texture, increasing solubility, adding foam-ing or coagulation properties, adding emulsifying capacity, preventing undesiredinteractions, removing off-flavors or odors, removing toxic or inhibitory ingredients,and isolation of bioactive peptides (Lahl and Braun, 1994; Kim et al., 2004; Ferreiraet al., 2007). Traditionally, proteins are hydrolyzed by chemical means. However,hydrolysis by chemical reagents produces no selective by-products. The use ofenzymes provides milder process conditions and allows for a selective hydrolysisof protein (Kim et al., 2004). New sources of food proteins are needed because ofincreasing global demand. Hence, over the past decade, plant proteins as alternativesto animal proteins are increasingly being used since they are an economic and versa-tile substitute for animal proteins (Friedman, 1996; Wang et al., 2006).

Corn gluten is a by-product from the production of starch from maize. On drybasis, it contains 67–71% proteins (Kim et al., 2004). Low water solubility of corn

Address correspondence to Belma Ozbek, Department of Chemical Engineering, YıldızTechnical University, Davutpasa Campus, 34210, Esenler=Istanbul, Turkey. E-mail: [email protected]

Chem. Eng. Comm., 197:963–973, 2010Copyright # Taylor & Francis Group, LLCISSN: 0098-6445 print=1563-5201 onlineDOI: 10.1080/00986440903359368

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gluten limits its use in food products; the utilization of corn gluten in food productswill be increased by improving its solubility with enzymatic modification. For thisreason, knowledge of the kinetics of the reaction will be essential to optimize thehydrolysis process. In the literature, the hydrolysis of corn gluten was performedby Kim et al. (2004) and Suh et al. (2003) with various enzymes by consideringthe solubility and angiotensin I converting enzyme inhibitory activity at constantprocess conditions and by Apar and Ozbek (2007, 2008) with enzymes Neutraseand Alcalase at various process conditions concerning the hydrolysis degree andsolubilization. However, limited studies have been performed concerning thehydrolysis kinetics of corn gluten (Mannheim and Cheryan, 1992; Hardwick andGlatz, 1989). On the other hand, enzyme inhibition is one of the constraints of thereactions catalyzed by the enzymes; hence, to optimize the process, information isrequired that describes the inhibition mechanisms that affect the process yield.Therefore, aims of the present study are to investigate the reaction kinetics of corngluten hydrolysis and to determine decay and product inhibition effects for alcalase.

Materials and Methods

Materials

Corn gluten used in this research, which contains 62.22% protein, was obtainedfrom Cargill (Istanbul). The enzyme used in this work was Alcalase 2.4 L (2.4 Ansonunit=g), a bacterial protease produced by Bacillus licheniformis, obtained fromNovozymes (Istanbul).

Methods

The reactions were monitored by using pH-stat method. The adjustment of pHduring hydrolysis was made with 0.2M KOH. The amount of hydrolysis wascalculated by Equation (1):

AH ¼ B NB1

V

1

að1Þ

where AH is the amount of hydrolysis (meqv L�1), B is the volume of baseconsumed during the hydrolysis (L), NB is the normality of the base (meqv L�1), Vis the processing volume (L), and a is the degree of dissociation of the a-NH2 groups.

The degree of dissociation of a-NH2 groups was computed from the followingequation:

a ¼ 10pH�pK

1þ 10pH�pKð2Þ

By comparing the pairs of hydrolysis at different pH values (pH1 and pH2), forwhich free amino groups, Leu-NH2 eqv, determined by the TNBS (trinitrobenzenesulfonic acid) reaction, and base consumptions, B eqv, are linearly correlated withthe slope b, pK was calculated from the following equation (Alder-Nissen, 1986):

pK ¼ pH2 þ logðb1 � b2Þ � logð10pH2�pH1 � b2 � b1Þ ð3Þ

964 D. K. Apar and B. Ozbek

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Soluble protein concentration was determined by the Lowry method (Lowry et al.,1951) using bovine serum albumin as standard. Prior to hydrolysis, thebackground protein of corn gluten and the protein concentration of enzyme werecalculated and subtracted from the overall protein. For each sample, the assaywas carried out in triplicate and their averages were taken.

Enzymatic Hydrolysis

Hydrolysis experiments were carried out in a 200mL jacketed reactor with magneticstirring and automatic pH and temperature control.

A measured amount of corn gluten was added to the reactor containing 100mLof distilled water and allowed to disperse, and then the pH and temperature of thereaction solution were set. The experiment was initiated by the addition of enzymesolution. In this study, all experiments were carried out at least in duplicate, andthe reproducibility between trials was within �5%.

Preparation of Hydrolysate

A 30 g amount of protein L�1 of corn gluten was hydrolyzed by 0.25% (v=v) Alcalaseat 55�C and pH 8 for 120min (Apar and Ozbek, 2008). Then, the reaction wasstopped by inactivating the Alcalase via decreasing the pH. After that, the particu-late material was removed by filtration, and hydrolysate solutions (hydrolysisdegree %¼ 28.4%, including 25.86 g L�1 soluble protein) were stored in a freezer.The enzyme-inactivated hydrolysates were then used as a source of inhibitor in thefurther study.

Computational Work

The software package MATLAB 5.0 was used in the numerical calculations. Theparameters were evaluated by the nonlinear least squares method of Marquardt-Levenberg until minimal error was achieved between experimental and calculatedvalues. The residual (SSR) is defined as the sum of the squares of the differencesbetween experimental and calculated data and is given by

SSR ¼XNd

m¼1

ðCobsm � Ccal

m Þ2 ð4Þ

where m is the observation number and Nd is the total number of observations. Theestimated variance of the error (population variance) is calculated by the SSR at itsminimum divided by its degrees of freedom:

r2 � s2 ¼ ðSSRÞmin

ðm� pÞ ð5Þ

where p is the number of parameters and s2 is the variance. The standard error, r(the estimated standard deviation), is calculated by taking the square root of theestimated variance of the error.

Corn Gluten Hydrolysis Kinetics by Alcalase 965

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Results and Discussion

To predict the effect of gluten concentration on hydrolysis, a series of experimentswas conducted at various protein concentrations (10–50 g L�1) under the standardconditions of 55�C and pH 8 with addition of 0.25% (v=v) enzyme. During theexperiments, to monitor the progress of the reaction, both the amount of hydrolysis(meqv L�1) and soluble protein amount (g L�1) were investigated with respect totime; the results are given in Figures 1(a) and 1(b). As it can be seen from thesefigures, during the 10min of processing time, the amounts of hydrolysis and

Figure 1. At various substrate concentrations with respect to time: (a) amount of hydrolysis,(b) soluble protein concentration. T¼ 55�C, pH¼ 8, E¼ 0.25% (v=v), ^ S¼ 10 g protein L�1,& S¼ 20 g protein L�1, ~ S¼ 30 g protein L�1, � S¼ 40 g protein L�1, S¼ 50 g protein L�1,— models.


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solubilization linearly increased with time and reaction rates increased by increasingthe substrate amount. The initial reaction rates were determined from the slopes ofthe linear models that fitted the experimental data. The models show highconvenience with the experimental data. For the amount of hydrolysis and solubleprotein concentration data, the models gave values for the coefficient of determi-nation greater than 0.9869 and 0.9788 and standard errors lower than 0.7570 and0.5786, respectively. By increasing the amount of substrate concentration from 10to 50 g protein L�1, the determined initial rates were increased from 0.93 to 3.10meqv L�1 min�1 and from 0.39 to 1.14 g L�1 min�1 for the amount of hydrolysisand soluble protein concentration data.

It is generally accepted that serine proteases like Alcalase, when acting onpeptide bonds, follow the Michaelis-Menten kinetic (Equation (6)) (Svendsen,1976; Alder-Nissen, 1986; Postolache and Oncescu, 1989; Sousa et al., 2004; Tardioliet al., 2005). Hence, to examine the conformity of the corn gluten hydrolysis to theMichaelis-Menten kinetic and to determine the kinetic parameters, the Lineweaver-Burk equation (Equation (7)) was used; the plots drawn (by using the initial ratesobtained from the modeling study) are presented in Figures 2(a) and 2(b). Theestimated constants, (Km and Vmax) and standard error (r) and R2 statistic values

Figure 2. Lineweaver-Burk plots: (a) plot drawn by using the initial rates obtained from theamount of hydrolysis data, (b) plot drawn with the initial rates obtained from the solubleprotein concentration data; — models.


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for the Lineweaver-Burk plots are given in Table I. As can be seen from this table,the Km value obtained from the hydrolysis data was almost the same as the valueobtained from the data of solubilization, which indicates that either the amount ofhydrolysis or solubilization data could be used to examine the hydrolysis kineticof the corn gluten and to determine the kinetic constants.

V ¼ Vmax½S�Km þ ½S� ð6Þ

1

V0¼ Km

Vmax

1

½S�0þ 1

Vmaxð7Þ

In order to investigate the effects of temperature and pH on the hydrolysis kinetics,the initial reaction rate assays with different substrate concentrations were per-formed at various temperatures and various pH values. For each experimentalrun, the amount of hydrolysis (meqv L�1) with respect to time was investigated,and the initial reaction rates were obtained by linear regression (models gave valuesof the coefficient of determination greater than 0.9716 and standard errors lowerthan 1.4403). The initial reaction rates increased with the increase of temperatureand pH. By using the initial rates obtained from the modeling study, theLineweaver-Burk plots (Figures 3(a) and 3(b)) were drawn, and the estimated con-stants, standard error (r), and R2 statistic values for the plots are given inTable II. As can be seen from Table II, Vmax values increased with temperature whilethe Km values decreased. Hence, the catalytic yield of the reaction (as the enzymeconcentration is constant the catalytic yield is taken as Vmax=Km) increased withthe temperature. On the other hand, both of Vmax and Km values increased withthe pH. The catalytic yield also increased with the pH, as the increase rate of Vmax

is higher than the increase rate of Km for all the values of pH examined.In the previous studies given in the literature on the hydrolysis of proteins cat-

alyzed with Alcalase, it is reported that the reaction products competitively inhibitthe hydrolytic reaction (Alder-Nissen, 1986; Sousa et al., 2004; Tardioli et al.,2005; Moreno and Cuadrado, 1993; Gonzalez-Tello et al., 1994). For this reason,in order to determine the type of inhibition of Alcalase by hydrolysis products ofcorn gluten, the initial reaction rate assays with different substrate concentrationswere conducted at 55�C and pH 8 with addition of various amounts of hydrolysate

Table I. Constants and statistical data calculated by using LWB plots

Constants andstatistical data

From amount ofhydrolysis data

From soluble proteinconcentration data

Km ( gL�1) 53.77 49.45Vmax

a 5.94 2.32r 0.0352 0.0468R2 0.9946 0.9985

aUnits for Vmax: meqv L�1min�1 for that obtained from amount of hydrolysis data,g L�1min�1for that obtained from soluble protein concentration data.


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Figure 3. Lineweaver-Burk plots: (a) for initial reaction rate assays performed at various tem-peratures, pH¼ 8, E¼ 0.25% (v=v) (� 40�C, ~ 45�C, � 50�C, & 55�C), (b) for initial reactionrate assays performed at various pH values, T¼ 55�C, E¼ 0.25% (v=v) (� pH 6.5, ~ pH 7,

� pH 7.5, & pH 8, — models).

Table II. Constants and statistical data calculated by using LWB plots at differenttemperatures and pH values

Constantsandstatisticaldata

Temperature (�C)(at pH 8)

pH (at temperature55�C)

40 45 50 55 6.5 7.0 7.5 8.0

Km (g L�1) 80.63 61.27 56.24 53.77 32.85 35.86 47.00 53.77Vmax

a 4.01 4.44 5.32 5.94 3.00 3.28 4.69 5.94Vmax=Km 0.0497 0.0725 0.0946 0.1105 0.0913 0.0915 0.0997 0.1105r 0.0558 0.0262 0.0218 0.0352 0.0264 0.0601 0.0427 0.0352R2 0.9973 0.9987 0.9985 0.9946 0.9979 0.9894 0.9936 0.9946

aUnit for Vmax: meqv L�1min�1.


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solutions (15, 30, and 45% (volume of hydrolysate=volume of substrate mixture) intothe reaction medium. The amount of enzyme added to these solutions was 0.25%(v=v). For each experimental run, both the amount of hydrolysis (meqv L�1) andsoluble protein amount (g L�1) with respect to time were investigated; the initialreaction rates were obtained by linear regression. For the amount of hydrolysisand soluble protein concentration data, the linear models gave values for the coef-ficient of determination of greater than 0.9859 and 0.9357 and standard errors lowerthan 1.3133 and 0.8669, respectively. By increasing the amount of hydrolysate, thedetermined initial rates decreased. By using the initial rates obtained, theLineweaver-Burk plots (Figures 4(a) and 4(b)) were drawn; the type of inhibitionfor Alcalase was determined as uncompetitive. The kinetic equation for the uncom-petitive inhibition is given in Equation (8) (Segel, 1993; Kuchel and Ralston, 1988).The values of Kapp

m and Vappmax were determined from the Lineweaver-Burk plots, and

inhibition constant Ki was estimated from Equation (8). The estimated constantsand standard error (r) and R2 statistic values for the Lineweaver-Burk plots aregiven in Table III.

Figure 4. Lineweaver-Burk plots for initial reaction rate assays performed with the addition ofvarious hydrolysate amounts: (a) plot drawn by using the initial rates obtained from theamount of hydrolysis data, (b) plot drawn with the initial rates obtained from the solubleprotein concentration data, T¼ 55�C, pH¼ 8, E¼ 0.25% (v=v) (� control, ~ 15% (v=v),

� 30% (v=v), & 45% (v=v), — models).


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V ¼ Vmax½S�Km þ ð1þ ½I�

KiÞ½S�

¼Vmax

ð1þ½I�=KiÞ ½S�Km

ð1þ½I�=Kiþ ½S�

¼ Vappmax½S�

Kappm þ ½S� ð8Þ

As can be seen from Table III, the Ki value obtained from the amount of hydrolysisdata was almost the same as the value obtained from the data of solubilization,which indicates that either the amount of hydrolysis or solubilization data couldbe used to examine the hydrolysis kinetic of the corn gluten and to determine thekinetic constants. As can be seen from Figures 1(a) and 1(b), both the amount ofhydrolysis and soluble protein concentration linearly increased with time. Also, inour previous study (Apar and Ozbek, 2008), the relationship between the hydrolysisdegree and solublization were investigated at different process conditions; it wasfound that the solubility increased in a linear relationship with degree of hydrolysis.In addition, the linear relationship between the degree of hydrolysis and solubilitywas also stated by Soral-Smietana et al. (1998) for hydrolysis of pea protein by tryp-sin and by Bombara et al. (1997) for the modification of wheat flour with protease.Hence, these results also supported the statement given above.

Conclusions

In this study, the kinetic parameters of the enzymatic hydrolysis of corn gluten wereestimated by using the pH-stat technique and also by analyzing the soluble proteinconcentration during the reaction. It was shown that either the amount of hydrolysisor solubilization data could be used to examine the hydrolysis kinetic of the corn glu-ten. The Michaelis-Menten constants, Km and Vmax, were obtained as 53.77 g L�1

and 5.94 meqv L�1 min�1 and 49.45 g L�1 and 2.32 g L�1 min�1 in the case of usingthe amount of hydrolysis and solubilisation data, respectively. The influence of tem-perature and pH on the kinetic parameters in the range of 40� to 55�C and 6.5 to 8was also examined, and it was found that the catalytic yield of the reaction increased

Table III. Constants and statistical data calculated by using LWB plots withaddition of hydrolysate

Hydrolysateaddition % (v=v)

From amount ofhydrolysis data

From soluble proteinconcentration data

15 30 45 15 30 45

Kappm (g=L) 40.34 32.06 26.02 37.06 30.80 24.99

V appa

max 4.43 3.45 2.74 1.74 1.42 1.17Ki % 45.06 44.32 42.19 44.49 46.26 45.79Ki % (average) 43.85 45.51r 0.0143 0.0204 0.0223 0.1061 0.0965 0.0237R2 0.9991 0.9983 0.9980 0.9913 0.9930 0.9996

aUnits for Vappmax: meqv L�1min�1 for that obtained from amount of hydrolysis data,

g L�1min�1for that obtained from soluble protein concentration data.


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as the temperature and pH values increased. The experiments conducted withaddition of various amounts of hydrolysate show that the hydrolysis products areinvolved in the inhibition of Alcalase. The type of inhibition for Alcalase wasdetermined as uncompetitive; the value of the inhibiton constant, Ki, was estimatedas 44.68% (volume of hydrolysate=volume of substrate mixture).

Acknowledgments

The authors gratefully acknowledge Novoyzmes and Cargill for their support.

Nomenclature

E enzyme concentration, (v=v) %I inhibitor amount, volume of hydrolysate=volume of substrate mixture %Kapp

m apparent value of the Michealis-Menten constant, g L�1

Ki inhibition constant, volume of hydrolysate=volume of substrate mixture %Km Michealis-Menten constant, g L�1

S0 initial substrate concentration, g L�1

V0 initial reaction rate, meqv L�1min�1 or g L�1min�1

V appmax apparent value of maximum reaction rate, meqv L�1min�1 or g L�1min�1

Vmax maximum reaction rate, meqv L�1min�1 or g L�1min�1

r standard error

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CORN GLUTEN HYDROLYSIS BY ALCALASE: KINETICS OF HYDROLYSIS