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Brazilian Journal of Chemical Engineering

Print version ISSN 0104-6632On-line version ISSN 1678-4383

Braz. J. Chem. Eng. vol.17 n.4-7 São Paulo Dec. 2000 



F.J.Bassetti1, R.Bergamasco2, F.F.Moraes2 and G.M.Zanin2*
1CEFET, PR - UNED, Campo Mourão, P.O. Box 271, 87301 - 005 - Campo Mourão - PR, Brazil
2 Maringá State University, Chemical Engineering Department, Av. Colombo 5790,
Bloco E46-09, 87020-900, Maringá - PR, Brazil


(Received: October 19, 1999 ; Accepted: April 18, 2000)



Abstract - The thermal stability and the energy of deactivation of free invertase and the immobilized enzyme (IE) was measured at temperatures in the range of 35 to 65°C for the hydrolysis of a 5% w/v sucrose solution. The free enzyme at pH 5.0 is stable up to 50°C for a period of 4 h. Invertase immobilized in controlled pore silica by the silane-glutaraldehyde covalent method is stable up to 55ºC, in pH 4.5 for the same period. For higher temperatures the enzyme deactivation follows the exponential decay model and half-lives are 0.53, 1.80, and 13.9 h for free invertase, at 65, 60, and 55ºC, respectively. For the IE half-lives are 0.48, 1.83, and 20.9 h, at 65, 60, and 55ºC, respectively. The IE is more stable than the free invertase; the energy of deactivation being 83.1 kcal/mol for the IE and 72.0 kcal/mol for the free enzyme.
Keywords: invertase, thermal stability, immobilized invertase, sucrose, energy of deactivation




Enzymatic hydrolysis of sucrose produces an equimolar mixture of glucose and fructose known as inverted sugar that is sweeter and easier to incorporate to industrial preparations than granulated sugar. Because of that it has many applications in the food industry, particularly in the production of soft drinks.

The enzymatic hydrolysis of sucrose has good potential for the application of the technology of immobilized enzyme, since it may offer technical and economical advantages, such as the reduction of enzyme usage, because in the immobilized form the enzyme can be used for a longer period than in the soluble form, i.e. it has a longer half-life. In addition, the immobilized enzyme can lead to preferred continuous processes that may use either fixed or fluidized bed reactors, in which it is possible to use higher enzyme dosage per volume of reactor than in the soluble enzyme process, and this contributes to high reaction rates and consequently, small reactor sizes (Zanin and Moraes, 1994; Bassetti et al., 1997)). These technical advantages allow a reduction in the operational and capital process costs, if the immobilized enzyme half-life is sufficiently long (Messing, 1975; Daniels, 1985; Zanin and Moraes, 1994).

This article presents results on the thermal stability and energy of deactivation for the enzyme invertase that was studied in two forms: free in solution and immobilized in controlled pore silica. These data are useful for the modeling and design of fixed and fluidized-bed reactors to be used in the enzymatic hydrolysis of sucrose.

Deactivation Hypothesis and Modeling

As temperature is raised in a reaction catalyzed by free or immobilized enzyme two opposing effects are observed: (i) the reaction rate increases, because the enzyme activity is a function of temperature, and, (ii) the stability of the protein enzyme decreases by thermal denaturation (Chibata, 1978; Dixon and Webb, 1979; Messing, 1975). The energy of deactivation can be determined from residual activity data obtained by incubating the enzyme for a certain period of time, within specified conditions. Normally, it is assumed that enzyme thermal denaturation is a reaction in which the rate of enzyme deactivation (rd) is first order in relation to the concentration of the active enzyme (E):

rd = - kd E

and the deactivation constant (kd) is a function of temperature as given by Arrhenius equation:

kd = kdo exp( - Ed/RT)

where Ed is the energy of deactivation, R the universal gas constant (1.987 cal/mol K) and T the absolute temperature. For a batch reactor of constant liquid density the rate of reaction from Eq. (1) is:

dE/dt = -kd E

which integrated with the initial condition E = E0 for t = 0, gives:

E = E0 exp ( - kd t )

where E0 is the initial active enzyme concentration, and t is time elapsed during reaction.

The residual enzyme activity (Ar) is directly proportional to the concentration of the active enzyme (E):


where Ar is the initial enzyme activity observed with the initial enzyme concentration (E0). From Eq. (4) and (5) the residual enzyme activity follows as:

Ar =A0 exp (-kd t )

This result is known as the exponential decay model. Plotting residual activity data (log of Ar / A0) as a function of time, the deactivation constant (kd) is obtained. From Eq. (2), and, as experimentally observed, it can be seen that the deactivation constant increases with temperature. Plotting log of kd as a function of the inverse of the absolute temperature the energy of deactivation (Ed) is obtained as the product of the angular coefficient of the adjusted straight-line times R, the universal gas constant

Another important parameter related to enzyme stability is the enzyme half-life (t1/2), which corresponds to the time period necessary for the residual enzyme activity to decrease to 50% of its initial value. From Eq. (6) it results that the half-life (t1/2) can be calculated by:

t1/2= -ln (0.5)/kd = 0.693 / kd

The energy of deactivation (Ed) of most enzymes is normally within the range from 47 to 96 kcal/mol, hence higher than the energy of activation (Ea) that is normally lower than 25 kcal/mol.




Yeast Invertase (b-D-frutofuranosidase, E.C. from Novo Nordisk (Brazil) was used as free enzyme in solution and immobilized.


The immobilization support particles were controlled pore silica (CPS) from Corning Glass Works (USA), with mean particle diameter equal to 0.351 mm, average pore size of 37.5 nm, and internal porosity of 56%.


The substrate solution contained 5% w/v sucrose at pH 5.0 for the free enzyme and pH 4.5 for the immobilized enzyme (IE). This solution was buffered with disodium phosphate - citric acid buffer (Morita and Assumpção, 1972) at 50 mM.

Assay Methods

The sum of the concentrations of glucose and fructose was measured by a modified DNS method (Zanin and Moraes, 1987) and protein by the method of Lowry, et al. (1951).

Enzyme immobilization

Invertase was immobilized in CPS with the silane-glutaraldehyde covalent method of Weetall (1976, 1993) and Zanin and Moraes (1998) that consists of the following steps: (a) silanization of the support with 0.5% (v/v) g-aminopropyl-trietoxisilane during 3 h at 75°C; (b) wash the silanized support with distilled water and dry it for 15 h at 105°C; (c) activate the dried silanized support with a solution of 2.5% (v/v) glutaraldehyde, pH 7.0, during 45 min at 20°C; (d) wash with distilled water; (e) contact the activated support with the enzyme solution for 15 h at 20°C; (f) wash the IE with distilled water and stock it in buffer solution in a refrigerator for later use. The IE was kept in 0.02 M acetate buffer, pH 4.5, at 4°C.

Enzyme Activity

One unity (U) of enzyme activity corresponds to the quantity of enzyme that produces one 1 micromol of both glucose and fructose/min with the given substrate at the specified temperature.

Thermal Deactivation Test

Free or IE was incubated in the substrate solution at temperatures in the range from 35 to 65°C, for a period of 4 h. At intervals of 40 min a sample was taken, and the residual activity (Ar) was determined. The following conditions were used for the thermal deactivation test: (a) Free enzyme: 10 mL of the aging solution contained 3.14×10-4 mg of protein/mL of solution (Bergamasco, 1989). (b) Immobilized enzyme: seven stainless steel baskets containing 1.000 g wet weight IE were used, six being aged in 200 mL of substrate at the test temperature, and one used to determine A0 (Bassetti, 1995).

Enzymatic Activity Determination

The enzyme residual activity (Ar) was determined by the method of initial velocities (Dixon and Webb, 1979). Free and IE samples were put in contact with 50 mL of the substrate solution in a batch reactor at a specified temperature, and aliquots were taken at regular intervals for determining the concentration of glucose/fructose produced. Total reaction time was 30 min in this test to give maximum substrate conversion below 20%. Samples of 0.5 mL were collected at 5 min intervals.



Immobilized invertase was produced by offering 15.75 mg of protein/g of support, and 5.55 mg were retained, giving a protein yield of 35.2%, with 98.2 U/mg of protein at 55°C, pH 5.0. The activity yield, i.e. the percentage of activity recovered in the IE, was 22.4%.

The thermal deactivation test has shown that free invertase looses little of its activity in a period of 4 h at the temperatures of 35 to 50ºC, whereas for the IE deactivation remains small up to 55°C. Free enzyme thermal denaturation at 55°C is already noticeable, retaining 83% of its activity after 4 h in contact with the substrate. At the higher temperatures of 60 and 75°C the denaturation of the free invertase is much more pronounced than that of the IE. Equation (6) adjusted to data obtained at the higher temperatures gave the results shown in Table 1. Immobilized enzyme deactivation at 50 and 55°C is too slow, and experimental errors can be greater than the observed reduction in activity.



Figures 1 and 2 compare the exponential decay model with the deactivation data obtained at the higher temperatures for free and IE, respectively. There is a general good fit. Exception was seen at 65°C for the free enzyme. At this temperature free invertase has a fast deactivation. The general good fit lends support to the application of the exponential decay model for the thermal deactivation of invertase aged in sucrose solutions.




Figure 3 compares the Arrhenius plot of the deactivation constant (kd) for free and IE invertase. Equation (2) adjusted to this data gives:



free invertase:

kd = 5.720 x 1046 exp ( - 72,039/RT )

immobilized enzyme:

kd = 7.972 x 1053 exp ( - 83,085 / RT )

Therefore, comparing Eqs. (8) and (9) with Eq. (2) it follows that the adjusted values for the deactivation energy (Ed) is 72.0 kcal/mol for free invertase and 83.1 kcal/mol for the IE. These values found for the deactivation energy show that there is a 15.3% increase in Ed as invertase is immobilized in CPS, confirming that immobilization confers more stability to the enzyme, as observed with other enzyme studies (Araujo and Schimdell, 1987; Brillouet, et al. 1977; Cabral, 1982; Cabral, et al. 1982; Johnson, 1979; Johnson and Costelloe, 1972; Maeda and Suzuki, 1972).

Equations (8) and (9) also allow to calculate the value of the deactivation constant (kd) for free and immobilized invertase for all temperature, and this value of kd can be substituted in Eq. (7) to calculate the corresponding enzyme half-life (tl/2). Experimental and predicted values for invertase tl/2 are compared in Table 2. Agreement is satisfactory, and they deviate less than 30%. Table 2 also shows that the stabilization by immobilization is more effective at the lower temperatures, the ratio of experimental half-lives decreases from 1.5 to approximately 1 as the temperature is raised from 55 to 65º. This trend was also observed for the enzymatic hydrolysis of cellobiose with cellobiase (Calsavara, et al., submitted for publication).




1. Invertase free in a solution of 5% (w/v) sucrose, pH 5.0, is stable up to 50°C for a period of 4 h.

2. When invertase is immobilized in controlled pore silica by the silane-glutaraldehyde covalent method it is stable up to 55°C in the same substrate and pH condition.

3. This immobilized procedure gave protein and activity yields of 35.2 and 22.4%, respectively.

4. For higher temperatures the enzyme deactivation follows the exponential decay model and half-lives are satisfactorily predicted for free or IE using Eqs. (8) or (9) and then Eq. (7).

5. The immobilized invertase is more stable than the free enzyme, and their energy of deactivation are 83.1 and 72.0 kcal/mol, respectively.

6. Enzyme stabilization by immobilization was shown to be more effective at the lower temperatures.



The authors thank the financial support received from CNPq, CONCITEC, PADCT/CAPES and the State University of Maringá. The companies that supplied materials (Novo Nordisk and Corning Glass Works) are also acknowledged.



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