Process Biochemistry 39 (2004) 11371144

-Amylase inactivation by temperature during starch hydrolysis

Dilek Kl Apar, Belma zbek
Department of Chemical Engineering, Yldz Technical University, Davutpa a Campus, 34210 Esenler, Istanbul, Turkey s Received 29 January 2003; received in revised form 20 May 2003; accepted 12 June 2003

Abstract The work reported here investigates the effects of temperature on the enzymic hydrolysis of corn, rice and wheat starch. Three commercial -amylases, from Bacillus sp., Aspergillus oryzae and Bacillus licheniformis were used for starch hydrolysis. For each starch hydrolysis process, the residual starch concentration and the residual -amylase activity (%) were investigated at temperature of 50 and 60 C depending on the processing time in a stirred batch reactor. Mathematical models were derived using experimental data of residual concentration of each starch used. Some inactivation models were tested to gain an understanding of the relation between temperature and enzyme stability during hydrolysis for each enzyme used. 2003 Elsevier Ltd. All rights reserved.
Keywords: Corn, rice and wheat starch hydrolysis; -Amylase; Inactivation; Temperature; Processing time; Mathematical modelling; Stirred batch bioreactor

1. Introduction The rapid enhancement of many biotechnology processes has been severely constrained by enzyme deactivation. An improved knowledge of enzyme deactivation would significantly enhance the feasibility of a number of few biotechnological processes [13]. Starch [2] has become a very important biopolymer and is used in many industries as a feedstock material. In several industrial processes, enzymes are used to transform starch molecules to useful, value-added biochemicals. Examples of two industries that have taken advantage of this development are the sweetener and ethanol industries. -Amylase is also widely used in industries such as food, textile, etc. For example, the chemical materials used in the textile industry, which are able to remove starch sizes from the textiles, are harmful for fabrics and environment. Therefore, alternative desizing materials are used such as -amylases which have been produced from different sources and have different properties. -Amylase is known to attack both insoluble starch and starch granules held in aqueous suspension. Before designing a successful hydrolysis system, information is required

describing phenomena which affect the kinetics of starch hydrolysis such as temperature, pH, enzyme concentration, etc. [19]. In the present study, the effects of temperature on maximal efciency of active -amylase was investigated using a stirred batch reactor system. The -amylases used were obtained from Sigma (Product Code: A6814, A6211 and A3403) and produced from Bacillus sp., Aspergillus oryzae and Bacillus licheniformis, respectively. The effects for -amylase hydrolysing wheat, rice and corn starch at temperature values 50 and 60 C are presented by investigating the residual starch concentration and residual -amylase activity depending on the processing time. Mathematical models, which represent the residual starch concentration and residual -amylase activity as a function of the processing time, were developed according to the data obtained from the experiments.

2. Materials and methods 2.1. Bioreactor A Gallenkamp Modular Bioreactor system (Model No: FER-195-010, manufactured by Sanyo Gallenkamp PLC, Loughborough) was used for starch hydrolysis. The controls

Corresponding author. Fax: +90-212-449-1895. E-mail address: bozbck@yildiz.edu.tr (B. zbek).

0032-9592/$ see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0032-9592(03)00224-3

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of various parameters including impeller speed, pH and temperature 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; internal diameter, 11 cm; height, 17 cm; number of bafes, 4; bafe height, 13.5 cm; bafe width, 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 proportionality of diameter of impeller to diameter of tank (D/T) was 0.436. 2.2. Determination of residual starch concentration For determination of the residual starch concentration in the reaction solution [10], samples were taken at timed intervals. Five milliliter of iodine solution (0.5% KI and 0.15% I2 ) and known volumes of the samples were mixed. The nal volume was adjusted to 15 ml by addition of distilled water. The absorbance was measured at 550 nm against a blank containing 5 ml of iodine solution and 10 ml of distilled water. Absorbances were converted to starch concentration using a calibration chart. At least 5 measurements were made for each condition and the data given are an average of these results. 2.3. Determination of -amylase activity The method used to determine the -amylase activity of the samples was described by De Moraes et al. [11]. 0.2 g of soluble starch was dissolved in 100 ml boiling 50 mM sodium acetate buffer (pH 5.9). The solution was cooled to 40 C. Two hundred microliter of the appropriately diluted enzyme solution was added to 1 ml of starch solution and the mixture was incubated at 40 C in a water bath for 10 min. Iodine reagent was prepared by addition of 1 ml stock solution (0.5% I2 in 5% KI) and 5 ml 5 M HCl to 0.5 l distilled water. Two hundred microliter incubated reaction mixture was added to 5 ml of iodine solution to stop the reaction. The degradation of starch by the enzyme was measured at 620 nm against 200 l water in 5 ml of iodine solution as blank. At least 5 measurements were made for each condition and the data given are an average of these results. An original sample was kept as a control activity measurement.

of the squares of the differences between experimental and calculated data and is given by
Nd

SSR =
m=1

obs cal Cm Cm

where m is observation number and Nd is total number of observations. The estimated variance of the error (population variance) was calculated by the SSR at its minimum divided by its degrees of freedom: 2 s2 = (SSR)min (m p) where p is the number of parameters and s2 is the variance. The standard error, , (estimated standard deviation) was calculated by taking the square root of the estimated variance of the error.

4. Results and discussion Experiments were performed at temperatures of 50 and 60 C for a processing time of 90 min at an impeller rate 300 rpm and pH 6.5 0.05. Reactions were carried out in 0.5 l aqueous solutions containing 10 g l1 starch. These reaction solutions contained approximately 90 000 units enzyme per litre for each experiment. The degree of corn, rice and wheat starch hydrolysis (%) and residual -amylase activity (%) were investigated. Enzyme activities prior to hydrolysis were also determined and used as the control. In calculations, Amax was denoted as the -amylase activity prior to the hydrolysis process. This value was then considered as 100% activity. Activity at any operational condition (A) was then given in terms of percentage values of the control. 4.1. Effect of temperature on the activity of -amylase produced by Bacillus sp. and corn, rice and wheat starch hydrolysis In order to investigate the effect of the temperature on the hydrolysis of corn, rice and wheat starch, -amylase produced by Bacillus sp. was used. Figs. 13 show the effect of temperature on the residual -amylase activity and residual starch concentration. The residual enzyme activity (from Bacillus sp.) decreased as the temperature increased from 50 to 60 C due to the inhibitory effects of the temperature. Decreases in the residual starch concentration were found by increasing the temperature for each starch used. To predict the effect of the temperature on enzyme stability, the data of residual -amylase activity versus processing time were evaluated for each starch hydrolysis process. Some inactivation models were tested to understand the relationship between the residual enzyme activity and the temperature depending on the processing time during the hydrolysis process [12,13]. For this purpose, residual -amylase activity-processing time expressions were used as follows.

3. Computational work The software package matlab 5.0 was used in the numerical calculations. The parameters were evaluated by the nonlinear least-squares method of MarquardtLevenberg until minimal error was achieved between experimental and calculated values. The residual (SSR) is dened as the sum

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Fig. 1. Effect of temperature on the activity of -amylase produced by Bacillus sp. during corn starch hydrolysis (expm.; enzyme act. at 50 C, enzyme act. at 60 C, residual starch conc. at 50 C, residual C; solid lines: model for enzyme act., dotted lines: starch conc. at 60 model for starch conc.).

Fig. 3. Effect of temperature on the activity of -amylase produced by Bacillus sp. during wheat starch hydrolysis (expm.; enzyme act. at 50 C, enzyme act. at 60 C, residual starch conc. at 50 C, residual starch conc. at 60 C; solid lines: model for enzyme act., dotted lines: model for starch conc.).

4.1.1. Single step non-rst-order inactivation model For some enzymes, Sadana and Henley [14] reported that the single step inactivation leads to a nal state that does exhibit some residual activity. E E1 1 In above reactions, E and E1 are enzyme states of different specic activities and are homogeneous. 1 is the ratio of the specic activity of the nal state to the initial state. kD is the degradation coefcient (min1 ). The activity-processing time relationship is then as follows: A = (1001 )exp(kD [t]) + 1 (1)
kD

where A is residual enzyme activity (percentage values after hydrolysis). 4.1.2. Quadratic t for inactivation model A = Amax + kD1 t + kD2 t 2 (2)

where Amax is enzyme activity (percentage value prior hydrolysis); kD1 , kD2 are the coefcients dependent on the processing time ((min)1 and (min)2 ), respectively. 4.1.3. Zero-order inactivation model A = Amax kD t (3)

where Amax is enzyme activity (percentage value prior hydrolysis); kD is the zero-order inactivation coefcient dependent on the processing time (min1 ). 4.1.4. First-order inactivation model A = Amax exp (kD t) (4)

Fig. 2. Effect of temperature on the activity of -amylase produced by enzyme act. at 50 C, Bacillus sp. during rice starch hydrolysis (expm.; enzyme act. at 60 C, residual starch conc. at 50 C, residual starch conc. at 60 C; solid lines: model for enzyme act., dotted lines: model for starch conc.).

where Amax is enzyme activity (percentage value prior hydrolysis); kD is the rst-order inactivation coefcient dependent on the processing time (min1 ). After evaluation of the data of -amylase from Bacillus sp., a single-step non-rst-order inactivation model (Eq. (1)), zero-order inactivation model (Eq. (3)) and rst-order inactivation model (Eq. (4)) did not simulate the inactivation data of -amylase during hydrolysis of corn, rice and wheat starch. However, for Eqs. (1), (3) and (4), the program either estimated negative values or resulted in inconsistent values. The data for -amylase for each starch were then subjected to quadratic t (Eq. (2)) for each temperature value depending on the processing time (from 0 to 90 min). The parameters, kD1 and kD2 were estimated using the MarquardtLevenberg optimisation routine of the matlab

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Table 1a Estimated parameters of residual -amylase activity (A) produced by Bacillus sp. Starch Corn Temperature ( C) 50, Eq. (2) 60, Eq. (2) 50, Eq. (2) 60, Eq. (2) 50, Eq. (2) 60, Eq. (2) Amax 99.08 100.59 101.45 101.65 100.87 101.58 kD1 (min1 ) 0.8136 1.9147 0.7325 0.8742 0.4000 2.1869 kD2 (min2 ) 0.0007 0.0107 0.0028 0.0007 0.0002 0.0125 Standard error () 2.5405 2.3844 3.1902 3.4459 1.0970 2.6413 R2 statistic 0.9961 0.9981 0.9855 0.9954 0.9972 0.9981

Rice

Wheat

5.0 software and are presented in Table 1a for each starch used. The values of standard error () and R2 statistical are also given in Table 1a for each experimental set. 4.1.5. Starch hydrolysis To predict the effect of the processing time on the starch hydrolysis, the data of residual starch concentration versus the processing time at different temperature values were evaluated. The following residual starch concentration-processing time expression (an empirical rst-order model) given by Komolprasert and Ofoli [3] was used: S1 = aexp(bt) + c (5)

where S1 is the residual starch concentration (g l1 ) and t is processing time (min). The parameters in Eq. (5), a (g starch l1 ), b (min1 ) and c (g starch l1 ) were estimated using the MarquardtLevenberg optimisation routine of matlab 5.0 software. The estimated parameters for corn, rice and wheat starch for each temperature values depending on the processing time are given in Table 1b. In this table, the residual (SSR), standard error () and R2 statistic values for each experimental set are also given. 4.2. Effect of temperature on the activity of -amylase produced by Bacillus licheniformis and corn, rice and wheat starch hydrolysis Using -amylase produced by Bacillus licheniformis, the effect of temperature on the hydrolysis process of corn, rice

Fig. 4. Effect of temperature on the activity of -amylase produced by Bacillus licheniformis during corn starch hydrolysis (expm.; enzyme act. at 50 C, enzyme act. at 60 C, residual starch conc. at 50 C, residual starch conc. at 60 C; solid lines: model for enzyme act., dotted lines: model for starch conc.).

and wheat starch was investigated and results are shown in Figs. 46. Again, the residual enzyme activity (from Bacillus licheniformis) decreased as the temperature increased from 50 to 60 C due to inhibitory effects of temperature. For each starch used, the decreases in the residual starch concentration were found by increasing the temperature. In this study, after evaluation of the data of -amylase from Bacillus licheniformis, the quadratic t for the

Table 1b Estimated parameters of residual starch concentrations (S1 ) by using -amylase produced by Bacillus sp. Starch Corn Temperature ( C) 50, Eq. (5) 60, Eq. (5) 50, Eq. (5) 60, Eq. (5) 50, Eq. (5) 60, Eq. (5) a (g starch l1 ) 2.3343 4.4231 4.3979 8.2089 7.8108 9.5182 b (min1 ) 0.0854 0.1839 0.0752 0.2280 0.1693 0.3606 c (g starch l1 ) 7.5793 5.5672 5.4422 1.7882 2.1708 0.4817 SSR 0.4004 0.4358 0.8060 0.1560 0.7109 0.0257 Standard error () 0.3164 0.3301 0.4489 0.1975 0.4216 0.0802 R2 statistic 0.9571 0.9868 0.9749 0.9986 0.9929 0.9998

Rice

Wheat

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Fig. 5. Effect of temperature on the activity of -amylase produced by Bacillus licheniformis during rice starch hydrolysis (expm.; enzyme act. at 50 C, enzyme act. at 60 C, residual starch conc. at 50 C, residual starch conc. at 60 C; solid lines: model for enzyme act., dotted lines: model for starch conc.).

Fig. 6. Effect of temperature on the activity of -amylase produced by Bacillus licheniformis during wheat starch hydrolysis (expm.; enzyme enzyme act. at 60 C, residual starch conc. at 50 C, act. at 50 C, residual starch conc. at 60 C; solid lines: model for enzyme act., dotted lines: model for starch conc.).

inactivation model (Eq. (2)), zero-order inactivation model (Eq. (3)) and rst-order inactivation model (Eq. (4)) did not simulate the inactivation data of -amylase. A single-step non-rst-order model (Eq. (1)) simulated very effectively the inactivation data of -amylase. The parameters 1 and kD estimated for each temperature and each starch are given in Table 2a. The values of standard error () and R2 statistical for each experimental set are also given in this table.

The data of residual starch concentration versus processing time at each temperature were evaluated to predict the effect of the processing time on starch hydrolysis. For this study, an empirical rst-order model (Eq. (5)) given by Komolprasert and Ofoli [3] was used for the residual starch concentration-processing time expression. The estimated parameters for each starch at temperatures of 50 and 60 C are given in Table 2b. The residual (SSR), standard error () and R2 statistic values for each experimental set are also given.

Table 2a Estimated parameters of residual -amylase activity (A) produced by Bacillus licheniformis Starch Corn Temperature ( C) 50, Eq. (1) 60, Eq. (1) 50, Eq. (1) 60, Eq. (1) 50, Eq. (1) 60, Eq. (1) 1 77.47 74.33 75.01 69.66 72.56 72.33 kD (min1 ) 0.0919 0.0822 0.0711 0.0559 0.0578 0.0708 SSR 0.0008 0.0244 0.0008 0.0244 0.0008 0.0244 Standard error () 1.6224 2.2137 1.7709 2.5767 1.0071 1.9749 R2 statistic 0.9836 0.9763 0.9839 0.9757 0.9956 0.9834

Rice

Wheat

Table 2b Estimated parameters of residual starch concentrations (S1 ) by using -amylase produced by Bacillus licheniformis Starch Corn Temperature ( C) 50, Eq. (5) 60, Eq. (5) 50, Eq. (5) 60, Eq. (5) 50, Eq. (5) 60, Eq. (5) a (g starch l1 ) 8.0464 9.2856 7.1065 9.4931 9.3753 9.8363 b (min1 ) 0.0267 0.1079 0.0909 0.2639 0.2049 0.3693 c (g starch l1 ) 1.8451 0.6315 2.7532 0.5057 0.6173 0.1637 SSR 0.0726 0.7454 1.1012 0.1326 0.4055 0.0102 Standard error () 0.1347 0.4317 0.5247 0.1821 0.3184 0.0506 R2 statistic 0.9991 0.9947 0.9867 0.9991 0.9972 0.9999

Rice

Wheat

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D. Kl Apar, B. zbek / Process Biochemistry 39 (2004) 11371144

Fig. 7. Effect of temperature on the activity of -amylase produced by Aspergillus oryzae during corn starch hydrolysis (expm.; enzyme act. at 50 C, enzyme act. at 60 C, residual starch conc. at 50 C, residual starch conc. at 60 C; solid lines: model for enzyme act., dotted lines: model for starch conc.).

Fig. 8. Effect of temperature on the activity of -amylase produced by Aspergillus oryzae during rice starch hydrolysis (expm.; enzyme act. at 50 C, enzyme act. at 60 C, residual starch conc. at 50 C, residual starch conc. at 60 C; solid lines: model for enzyme act., dotted lines: model for starch conc.).

4.3. Effect of temperature on the activity of -amylase produced by Aspergillus oryzae and corn, rice and wheat starch hydrolysis The effect of temperature on the hydrolysis of corn, rice and wheat starch using -amylase produced by Aspergillus oryzae was investigated. Experimental results are shown in Figs. 79. As expected, due to inhibitory effects of temperature, the residual enzyme activity (from Aspergillus oryzae) decreased. However, the residual starch concentrations decreased on increasing the temperature. The evaluation of the data of -amylase (from Aspergillus oryzae) for corn and rice starch hydrolysis process showed that the rst-order model (Eq. (4)) simulated the inactivation data of -amylase very effectively. The parameter kD estimated for each temperature value is given in Table 3a. The values of standard error () and R2 statistical are also given in this table for each experimental set. After evaluation of the data of -amylase (from Aspergillus oryzae) for wheat starch hydrolysis process,

Fig. 9. Effect of temperature on the activity of -amylase produced by Aspergillus oryzae during wheat starch hydrolysis (expm.; enzyme act. enzyme act. at 60 C, residual starch conc. at 50 C, at 50 C, residual starch conc. at 60 C; solid lines: model for enzyme act., dotted lines: model for starch conc.).

Table 3a Estimated parameters of residual -amylase activity (A) produced by Aspergillus oryzae Starch Corn Temperature ( C) 50, Eq. (4) 60, Eq. (4) 50, Eq. (4) 60, Eq. (4) Temperature ( C) Wheat 50, Eq. (2) 60, Eq. (2) Amax 98.11 100.80 99.39 103.61 Amax 98.37 105.23 kD (min1 ) 0.0047 0.0997 0.0013 0.0694 kD1 (min1 ) 0.1882 1.3088 Standard error () 1.2044 2.7259 0.5729 6.8954 kD2 (min2 ) 0.0009 0.0027 R2 statistic 0.9957 0.9978 0.9911 0.9872 Standard error () 1.3682 6.7249 R2 statistic 0.9521 0.9866

Rice

D. Kl Apar, B. zbek / Process Biochemistry 39 (2004) 11371144 Table 3b Estimated parameters of residual starch concentrations (S1 ) by using -amylase produced by Aspergillus oryzae Starch Corn Temperature 50, Eq. (5) 60, Eq. (5) 50, Eq. (5) 60, Eq. (5) 50, Eq. (5) 60, Eq. (5) a (g starch l1 ) 1.5695 1.8089 1.5575 6.1727 4.8727 9.0748 b (min1 ) 0.0539 0.1674 0.2226 0.3600 0.0809 0.4139 c (g starch l1 ) 8.4315 8.1867 8.4425 3.8273 5.0588 0.9251 SSR 0.0008 0.0244 0.0253 0.0030 0.3012 0.0056 Standard error () 0.0144 0.0781 0.0796 0.0275 0.2744 0.0374

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R2 statistic 0.9998 0.9955 0.9952 0.9999 0.9923 0.9999

Rice

Wheat

quadratic t inactivation model (Eq. (2)) simulated the inactivation data of -amylase very effectively. The parameters 1 and kD1 estimated for each temperature are given in Table 3a. The values of standard error () and R2 statistical are given in the same table for each experimental set. To predict the effect of processing time on starch hydrolysis, the data of residual starch concentration versus processing time at different temperature values were evaluated. For the residual starch concentration-processing time expression, the Eq. (5) given by Komolprasert and Ofoli [3] was used again. The estimated parameters for each starch at temperature values 50 and 60 C depending on the processing time are given in Table 3b. In this table, the residual (SSR), standard error () and R2 statistic values for each experimental set are also given.

5. Conclusions Experimental data show that temperature and processing time are involved in the inactivation of -amylases (produced from different sources) after the starch hydrolysis process. The residual enzyme activity values obtained for each starch (corn, rice and wheat) decreased on increasing the temperature (from 50 to 60 C) for each enzyme used. The hydrolysis degree values obtained for each starch (corn, rice and wheat) increased on increasing the temperature for each enzyme used. The highest hydrolysis degree values for each starch using each -amylase were obtained at a temperature of 60 C. These values for corn, rice and wheat starch were 49.49, 84.51 and 96.2% using -amylase from Bacillus sp., 97.81, 97.21 and 99.02% using -amylase from Bacillus licheniformis and 18.14, 61.70 and 91.14% using -amylase from Aspergillus oryzae; respectively, after 90 min of processing time. At the same temperature and processing time, the residual enzyme activity values for corn, rice and wheat starch were 14.7, 18.89 and 6.2% for -amylase produced by Bacillus sp.; 71.82, 67.14 and 70.24% for -amylase produced by Bacillus licheniformis; 0, 0 and 12.32% for -amylase produced by Aspergillus oryzae, respectively.

The lowest hydrolysis degree values were obtained for -amylase produced by Aspergillus oryzae after hydrolysis of each starch used at temperatures of 50 and 60 C. The highest hydrolysis degree values were obtained for -amylase produced by Bacillus licheniformis after hydrolysis of each starch at temperatures of 50 and 60 C. This enzyme had the highest stability at these temperatures. The inactivation mechanisms of each -amylase studied at various temperatures depending on the processing time were different and was specic to the enzyme. Various kinetic parameters dependent on the processing time and enzyme stability at temperature values of 50 and 60 C were estimated. Modelling studies showed that the quadratic t inactivation model accurately represented the inactivation data for -amylase from Bacillus sp. during hydrolysis of corn, rice and wheat starch. On the other hand, the data for -amylase produced from Bacillus licheniformis were tted to a single-step unimolecular non-rst-order enzyme inactivation model during hydrolysis of corn, rice and wheat starch. The data for -amylase produced from Aspergillus oryzae resulted in rst-order inactivation model for corn and rice starch hydrolysis and quadratic t inactivation model for wheat starch hydrolysis. For modelling studies of the residual starch concentration, the empirical rst-order model given by Komolprasert and Ofoli [3] accurately represented the data for each starch hydrolysed. Finally, the stability behaviour observed was different for each -amylase. These could be related to molecular weights or sources of the -amylases and starches used. Thus, the operational parameters of the starch hydrolysis process should be optimised specically for each enzyme and each starch used, as these parameters could cause degradation of the -amylase during the starch hydrolysis.

Acknowledgements This work was supported by the Yldz Technical University Research Fund. Project Number: 20-A-07-01-02.

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D. Kl Apar, B. zbek / Process Biochemistry 39 (2004) 11371144 enzymes of the alpha-amylase family. J Biotechnol 2002;94(2): 13755. Godfrey T, Reichelt J. Industrial Enzymology. Stockton: Macmillan Publishers, 1983. Hill GA, Macdonald DG, Lang X. -Amylase inhibition and inactivation in barley malt during cold starch hydrolysis. Biotechnol Lett 1997;19(11):113941. Astol-Filfo S, Galembeck EV, Faria JB, Frascino ACS. Stable yeast transformants that secrete functional -amylase encoded by cloned mouse pancreatic cDNA. Biotechnology 1986;4:3115. De Moraes LMP, Astol-Filho S, Oliver SG. Development of yeast strains for the efcient utilisation of starch: evaluation of constructs that express -amylase and glucoamylase separately or as bifunctional fusion proteins. Appl Microbiol Biotechnol 1995;43:106776. zbek B, lgen K. The stability of enzymes after sonication. Process Biochem 2000;35(9):103743. zbek B, Yceer S. -Amylase inactivation during wheat starch hydrolysis process. Process Biochem 2001;37(1):8795. Sadana A, Henley JM. Single step unimolecular non-rst-order enzyme deactivation kinetics. Biotechnol Bioeng 1987;30:71723.

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