Kinetic study of α-amylase in the process of starch hydrolysis by microcalorimetry

Thermochimica Acta 579 (2014) 70–73

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Kinetic study of ␣-amylase in the process of starch hydrolysis by microcalorimetry Xiang-yang Li a , Jun Wang b,∗ , Hai-zhou Dong a,∗ , Hong-lin Zhang c a b c

College of Food Science & Engineering, Shandong Agricultural University, Tai’an, Shandong 271018, PR China College of Environmental Science and Engineering of Donghua University, Shanghai 201620, PR China Department of Chemistry, Qufu Normal University, Qufu, Shandong 273165, PR China

a r t i c l e

i n f o

Article history: Received 3 June 2013 Received in revised form 20 January 2014 Accepted 23 January 2014 Available online 1 February 2014 Keywords: ␣-Amylase Microcalorimetry Optimum temperature Optimum acidity Metal ions (Ag+ , Cu2+ , Zn2+ )

a b s t r a c t Thermokinetic behaviors of ␣-amylase in the process of starch hydrolysis were studied under the conditions of different temperatures, different pH and metal ions by using thermokinetics theory and reduced extent method; the Michaelis constant (Km ), apparent Michaelis constant (K  m ), maximum velocity (Vmax ) and apparent maximum velocity (V  max ) of the ␣-amylase catalyzed reaction were obtained, Km –T and Km –pH equation were deduced. According to Km –T and Km –pH equation, the optimum temperature is 315.04 K and the optimum acidity is pH = 5.08. At the conditions of T = 313.15 K and pH = 5.02, the influence of metal ions (Ag+ , Cu2+ and Zn2+ ) in ␣-amylase catalyzed reaction were studied; according to the data, it was discovered that the inhibitory type were versa-competitive inhibition and inhibitory consecution were Ag+ > Cu2+ > Zn2+ at the same concentration. © 2014 Published by Elsevier B.V.

1. Introduction The most complicated reactions have been found in living creatures. Among these reactions, enzyme-catalyzed reactions are very important. ␣-Amylase is a type of catalytic enzyme that hydrolytically breaks, the glucosidal bonds of the starch molecule and its derivatives [1–3]. It was extracted from a stain of fungus screened from deep Huanghai Sea mud. Due to highly production and lower optimum temperature, it has been extensively researched and applied in the industries of medicine, food and beverage, washing, leather, and so on [4–6]. The activity of the ␣-amylase is influenced directly by temperature, pH and concentration of metal ions. Therefore, the thermodynamic and kinetic properties of ␣-amylase need to be further studied to improve its many applications. Many analytical methods have been used to study the activity of ␣-amylase, but sometimes the results obtained by different methods cause confusion. The various methods differ from one another with regard to the detecting reaction, the instrumental techniques, the analytical criteria or the choice of substrate [7,8]. Among the reported methods [9–13], the final results are not reliable because of the accumulation of errors: too many reaction

∗ Corresponding authors. Tel.: +86 21 67792539; fax: +86 21 67792522. E-mail addresses: [email protected] (J. Wang), [email protected] (H.-z. Dong). 0040-6031/$ – see front matter © 2014 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.tca.2014.01.015

steps combined, many and tedious pretreatment of the sample, interferences due to several reagents needed [14]. Sometimes, these methods cannot be applied to high levels of activity and not give very accurate results. It is still necessary and useful to develop a new method for determining the properties of ␣-amylase. Microcalorimetry is a reliable and direct method, due to minimize errors and to obtain accurate and unambiguous results. Microcalorimetry has already attracted wide attention, because it is an integral, universal, non-invasive and non-destructive method for many investigations in recent years [15–27]. For example, Kabanova et al. [18] studied the growth of bacterial colonies of Lactococcus lactis IL1403 in agar gels by microcalorimetric method. Haluk Ertan et al. [23] developed isothermal titration calorimetry (ITC) for measuring lignin peroxidase (LiP) and manganese peroxidase (MnP) activities of versatile peroxidase (VP) from Bjerkandera adusta. ITC approach provided an alternative to colorimetric methods that enabled reaction kinetics to be accurately determined. Guigón-López et al. [25] had measured the growth of six Trichoderma spp. warm-weather strains (WWS) and temperateweather strains (TWS) was determined by microcalorimetry at different temperatures from 10 to 35 ◦ C. The metabolic heat production (p) and the apparent activation energy (Ea ) were calculated. Microcalorimetry also is an effective approach to provide some thermodynamic information of the enzyme-catalyzed reactions [28,29]. Thermokinetics theory of enzyme-catalyzed reaction has been developed greatly these years [30–37].

X.-y. Li et al. / Thermochimica Acta 579 (2014) 70–73

71

2

The purpose of this work is to study the influence of the optimum temperature, optimum acidity, inhibitory rule of metal ions of ␣-amylase catalyzed reaction by using titration microcalorimetric method. Studies of this aspect have not been previously reported.

3 1 4

2. Thermokinetics theory Michaelis–Menten equation of enzyme-catalyzed reaction of single substrate is: 1

v

=

Km 1 1 · + Vmax [S] Vmax

5 7

Coexistence of single substrate and inhibitor, enzyme-catalyzed reaction has three inhibitory types: competitive inhibition (Eq. (2)), non-competitive inhibition (Eq. (3)) and versa- competitive inhibition (Eq. (4)) [38]. 1

v

=

Km Vmax



1+

 1 [I] Ki

·

[S]

+

1 Vmax



or

1

v

=

K m 1 1 · + Vmax [S] Vmax (2)



where K  m = Km 1 + ([I]/Ki ) , so K  m > Km , and Vmax is constant. 1

Km = v Vmax or

1

v



[I] 1+ Ki

=

v

=

·

[S]

Vmax , 1+([I]/Ki )

Km 1 1 · + Vmax [S] Vmax

where K  m =

+



1 Vmax

[I] 1+ Ki



Km 1 1 · +  V  max [S] V max

where V  max = 1

 1

Km , 1+([I]/Ki )

(3)

so V  max < Vmax , and Km is constant.



1+

[I] Ki

V  max =

 or

Vmax , 1+([I]/Ki )

1

v

=

K m 1 1 · +  V  max [S] V max (4)

so K  m < Km and V  max <

Vmax . Compare Eqs. (1)–(4), data (Vmax , V  max , Km , K  m ) of enzymecatalyzed reaction are different. When the temperature and concentration of inhibitors are constant, V  max and K  m also are constant. Eqs. (1)–(4) are similar. Eq. (1) can be changed into: 1 1 Km 1 + = · Vmax [S] Vmax d[S]/dt

(5)

Integration of Eqs. (5) and (6) can be derived: Km k2 [E0 ] [S] [S] − [S0 ] ln + =− t [S0 ] [S0 ] [S0 ] [S0 ]

(6)

If ([S0 ] − [S])/[S0 ] = ϕ, then ([S]/[S0 ]) = 1 − ϕ, Vmax = k2 [E0 ], where ϕ is the reduced extent. Hence, the following reduced extent equation can be derived: Km Vmax ϕ− ln (1 − ϕ) = t [S0 ] [S0 ]

(7)

According to the area under power–time curve, Q and Q∞ could be gotten. Q represents the thermal effect at the time of t, and Q∞ is the total thermal effect of enzyme catalyzed reaction without inhibitor, and ϕ = Q/Q∞ . Choosing three points (ϕ1 , ϕ2 and ϕ3 ) from the ϕ–t curve with identical time intervals (t = t3 − t2 = t2 − t1 ), the following equations can be derived from Eq. (7). Km =

2ϕ2 − ϕ1 − ϕ3 [S0 ] 2 ln (1 − ϕ2 ) − ln (1 − ϕ1 ) − ln (1 − ϕ3 )

Vmax =

6 8

(1)

9 10 Fig. 1. Schematic diagram of titration operation: (1) titration solution, (2) micro perpex pump, (3) stirrer motor, (4) titration tube for cannula, (5) main tube, (6) heat sink, (7) stirrer shaft, (8) ampoule lid coned, (9) 4 mL ampoule and (10) turbine stirrer.

Using similar method treated Eqs (2)–(4), results can be derived into the same type, just replacing Vmax with V  max , and Km with K  m . S is substrate, I is inhibitors, E is enzyme, ES is complex, P is product, Km is Michaelis–Menten constant, v is reaction velocity, Vmax is reaction velocity of maximum value, Vmax = k2 [E0 ], k2 is velocity constant, [E0 ] is total enzyme concentration, ϕ is reduced extent, V  max is apparent maximum velocity, K  m is apparent Michaelis constant. 3. Experimental 3.1. Instrument The 2277 thermal activity monitor is used an isothermal instrument with 23 L water bath, equipped with four independent calorimetric units and operated at working temperatures between 20 ◦ C and 80 ◦ C with an external water circulator. The working temperature is maintained constant to within ±2 × 10−4 ◦ C of the given temperature. The detection limit is 0.15 ␮W and the baseline stability is 0.2 ␮W over a period of 24 h. 2251–3104 mL stainless steel ampoule titration calorimetric unit is independent calorimetric unit, titration unit have stirrers, it is equipped with a motor that rotates the stirrer shaft at the desired speed (usually between 0 and 120 rpm). A Kelf turbine is used for a 4 mL ampoule filled with 2.0–3.0 mL of solution. Schematic diagram of titration operation is shown in Fig. 1. 3.2. Materials All chemicals for the preparation of Britton–Robinson buffer and soluble starch were commercial products of analytical grade. ␣-Amylase was obtained from State Key Lab of Microbial Technology, Shandong University. It was stored at 5 ◦ C until used. The concentration of ␣-amylase solution is 4 × 10−2 g L−1 , and the contrast activity is 2.94 × 103 U mg−1 . The soluble starch solution was prepared daily and stored at 0 ◦ C, whose starch concentration is 5 × 10−3 g L−1 .

(8)

Km (ϕ3 − ϕ1 ) ln (1 − ϕ2 ) − (ϕ3 − ϕ2 ) ln (1 − ϕ1 ) − (ϕ2 − ϕ1 ) ln (1 − ϕ3 ) · 2ϕ2 − ϕ1 − ϕ3 t

(9)

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X.-y. Li et al. / Thermochimica Acta 579 (2014) 70–73

Table 1 Values of Km and Vmax of ␣-amylase catalyzed reaction at 313.15 K and pH 5.02. ϕ1

ϕ2

ϕ3

t (s)

Km (×103 g L−1 )

Vmax (×105 g L−1 s−1 )

0.2580 0.3960 0.5210 0.2580 0.3960

0.3960 0.5210 0.6312 0.5210 0.6312

0.5210 0.6312 0.7251 0.7251 0.8020

60 60 60 120 120

2.4948 2.5084 2.5117 2.5054 2.5022

2.0077 2.0110 2.0239 2.0090 2.0129

2.5045

2.0129

Average

Table 3 Values of Km and Vmax of ␣-amylase catalyzed reaction at 313.15 K and different acidity. pH

4.10

4.56

5.02

5.72

6.37

Km (×103 g L−1 ) Vmax (×105 g L−1 s−1 )

2.1873 1.9948

2.4105 2.0043

2.5045 2.0129

2.3896 2.0257

2.1078 1.9974

Table 4 Values of K  m and V  max of ␣-amylase catalyzed reaction at 313.15 K and pH 5.02 with 2.5 m mol L−1 Ag+ .

3.3. Method

ϕ2

ϕ3

t (s)

Km (×103 g L−1 )

Vmax (×105 g L−1 s−1 )

0.3620 0.5210 0.6620 0.7797 0.3620 0.5210

0.5210 0.6620 0.7797 0.8695 0.6620 0.7797

0.6620 0.7797 0.8695 0.9301 0.8695 0.9301

120 120 120 120 240 240

1.4537 1.4661 1.4610 1.4500 1.4618 1.4588

1.0097 1.0137 1.0117 1.0069 1.0112 1.0111

1.4586

1.0109

Average

4. Results and discussion

a

200

b c d

150

Pt/uW

In the experiment 4 mL ampoule units were used. Put 2 mL starch solution contained metal ion (2.5 mmol. L−1 ) in the sample ampoule and 2 mL starch solution without metal ion in the reference ampoule. Put 0.10 mL ␣-amylase solution into sample ampoule with peristaltic pump at the constant temperature and the rotating speed of 120 rpm of the stirrer system. The monitor began to record the power–time curve. When the recording pen returned to the baseline and stabilized, the ␣-amylase-catalyzed reaction was considered complete. All measurements were made three times with the average values of power–time curves obtained, the experimental relative error was less than ± 2%.

ϕ1

100

4.1. Experiments of different temperatures The power–time curves of ␣-amylase catalyzed reaction were determined at a temperature range of 303.15–321.15 K, pH 5.02 with 5 × 10−3 g L−1 starch and 4 × 10−2 g L−1 ␣-amylase solution. From data of curves by using thermokinetics theory and reduced extent method, the Michaelis constant (Km ) and maximum velocity (Vmax ) of the ␣-amylase catalyzed reaction were obtained, the partial data are shown in Table 1. With the same method, Km and Vmax at different temperatures can be gotten, and the data are shown in Table 2. From the result in Table 2, the non-linear equation Km = −8.245 × 10−8 T3 + 7.545 × 10−5 T2 − 2.299 × 10−2 T + 2.335 can be established, when T = 315.04 K, Km is the maximum, therefore the optimum temperature of the ␣-amylase catalyzed reaction is 315.04 K. 4.2. Experiments of different acidity (pH) The power–time curves of ␣-amylase catalyzed reaction were measured at 313.15 K and different acidity (pH 4.10–6.37) with 5 × 10−3 g L−1 starch and 4 × 10−2 g L−1 ␣-amylase solution. With the same method, Km and Vmax of different acidity (pH) can be gotten, and data are shown in Table 3. From the data in Table 3, equation of Km –pH was derived: Km = 3.9732 × 10−5 pH3 − 8.9285 × 10−4 pH2 + 5.9957 × 10−3 pH − 1.0126 × 10−2 , when pH = 5.08, Km is the maximum, therefore the optimum acidity (pH) of the ␣-amylase catalyzed reaction is pH = 5.08.

Table 2 Values of Km and Vmax of ␣-amylase catalyzed reaction at different temperatures and pH 5.02. T (K)

303.15

310.15

313.15

317.15

321.15

Km (×103 g L−1 ) Vmax (×105 g L−1 s−1 )

2.3105 1.9942

2.4493 2.0058

2.5045 2.0129

2.4720 2.0070

2.3738 1.9918

50

0

0

300

600

900

1200

t/s Fig. 2. Power–time curves of ␣-amylase catalyzed reaction at 313.15 K and pH5.02: (a) without metal ion, (b) 2.5 mmol L−1 Zn2+ , (c) 2.5 mmol L−1 Cu2+ and (d) 2.5 mmol L−1 Ag+ .

4.3. Inhibitory results of metal ions When substance (metal ion) is fed into ␣-amylase catalyzed reaction system, the enzyme reaction activity reduced and even lost, this substance called enzyme inhibitor. The power–time curves of ␣-amylase catalyzed reaction were measured at 313.15 K, pH5.02, and existence inhibitor (2.5 m mol L−1 Zn2+ , 2.5 m mol L−1 Cu2+ , 2.5 m mol L−1 Ag+ ). From data of curves the apparent Michaelis constant (K  m ) and apparent maximum velocity (V  max ) of the ␣-amylase catalyzed reaction system were obtained, using thermokinetics theory and reduced extent method, The data are shown in Table 4, Curves are shown in Fig. 2. Using Eqs. (8) and (9), values of K  m and V  max also were gained. The data are shown in Table 5. According to Eqs. (2)–(4), compare the results of Tables 1 and 5, we defined inhibitory type and inhibitory consecution. From data in Table 5 Values of K  m and V  max of ␣-amylase catalyzed reaction at 313.15 K and pH5.02 with 2.5 m mol L−1 Cu2+ , Ag+ , or Zn2+ .

Km (×103 g L−1 ) Vmax (×105 g L−1 s−1 )

Cu2+

Ag+

Zn2+

1.2052 1.0154

1.4586 1.0109

1.1256 1.0180

X.-y. Li et al. / Thermochimica Acta 579 (2014) 70–73

Table 5 these metal ions have inhibitory action. Because K  m < Km and V  max < Vmax , therefore the inhibitory type of Cu2+ , Ag+ and Zn2+ are versa-competitive inhibition. From the data in Table 5, K  m of Ag+ is the maximum value, the second one is Cu2+ , and the minimal one is Zn2+ . The relative order of different metal ion were Ag+ > Cu2+ > Zn2+ at the same concentration. 5. Conclusion 1. The microcalorimetric method is simple, reliable and novel method for the study of the enzyme catalysis reaction system. The reaction system was not destroyed after measurement, so it could be used for further researched. 2. From the power–time curves, we defined that the optimum temperature (T = 315.04 K) and optimum acidity (pH = 5.08). These results are useful for further industrial application of enzyme catalysis. 3. The influence of metal ions (Ag+ , Cu2+ and Zn2+ ) were studied in the ␣-amylase catalyzed reaction, we defined that the inhibitory type were versa-competitive inhibition and inhibitory consecution were Ag+ > Cu2+ > Zn2+ at the same concentration. In general, it is a new method to define the optimum temperature, optimum acidity, inhibitory rule and inhibitory type. It has important theory value and broad application for further research of enzyme catalyzed reaction. Acknowledgement The authors are deeply grateful to the financial support of Independent Inovation Special Project of Shan Dong Province 2013CXA05029Fund, the Natural Science Foundation of China and Development projects of Shandong Province Science and Technology Fund. The support of the College of Food Science & Engineering, Shandong Agricultural University as well as the support of the College of Environmental Science and Engineering of Donghua University and the Department of Chemistry, Qufu Normal University are also acknowledged. References [1] R. Gupta, P. Gigras, H. Mohapatra, V.K. Goswami, B. Chauhan, Microbial ␣-amylases: a biotechnological perspective, Process Biochem. 38 (2003) 1599–1616. [2] A. Pandey, P. Nigam, C.R. Soccol, V.T. Soccol, D. Singh, R. Mohan, Advances in microbial amylases, Biotechnol. Appl. Biochem. 31 (Pt 2) (2000) 135–152. [3] G. Salieri, G. Vinci, M.L. Antonelli, Microcalorimetric study of the enzymatic hydrolysis of starch: an ␣-amylase catalyzed reaction, Anal. Chim. Acta 300 (1995) 287–292. [4] G. Bayramo˘glu, M. Yilmaz, M. Yakup Arica, Immobilization of a thermostable ␣-amylase onto reactive membranes: kinetics characterization and application to continuous starch hydrolysis, Food Chem. 84 (2004) 591–599. [5] M.J. van der Maarel, B. van der Veen, C.M. Joost, J.C. Uitdehaag, H. Leemhuis, L. Dijkhuizen, Properties and applications of starch-converting enzymes of the ␣-amylase family, J. Biotechnol. 94 (2002) 137–155. [6] J.L. Uma Maheswar Rao, T. Satyanarayana, Improving production of hyperthermostable and high maltose-forming ␣-amylase by an extreme thermophile Geobacillus thermoleovorans using response surface methodology and its applications, Bioresour. Technol. 98 (2007) 345–352. [7] K. Lorentz, ␣-Amylase determination using maltopentaose as substrate, J. Clin. Chem. Lab. Med. 21 (1983) 45–48. [8] R. McCroskey, T. Chang, H. David, E. Winn, p-Nitrophenylglycosides as substrates for measurement of amylase in serum and urine, Clin. Chem. 28 (1982) 1787.

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