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Characterization of free and immobilized invertase regarding activity and energy of activation


Invertase from NOVO Nordisk has been immobilized in controlled pore silica particles (diameter: 0.351 mm and mean pore size: 37.5 nm) by covalent binding with the silane-glutaraldehyde method. The activity of the free and immobilized enzyme (IE) was determined with 5% (w/v) sucrose, at 35 to 65ºC and pH from 3 to 7. Maximum activities were found in the pH range from 5 to 6 for free invertase, and pH 4.5 for the IE. Activity yield for the IE was 24%. The Energy of Activation (Ea) was found to be a function of pH, giving for free invertase, Ea = 7.0 and 6.86 kcal/mol at pH 5.0 and 5.5, respectively, whereas for the immobilized enzyme, Ea = 6.55 and 5.93 kcal/mol at pH 4.5 and 5.0, respectively.

invertase; immobilized invertase; energy of activation; sucrose; controlled pore silica


R.Bergamasco1, F.J.Bassetti2, F,F. de Moraes1 and G.M.Zanin1* * To whom correspondence should be addressed

1Chemical Engineering Department, State University of Maringá,

Av. Colombo 5790, Bloco E46-09, 87020-900, Maringá - PR, Brazil


2CEFET – PR/UNED, Campo Mourão P.O. Box 271,

87301 - 005, Campo Mourão - PR, Brazil

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

Abstract - Invertase from NOVO Nordisk has been immobilized in controlled pore silica particles (diameter: 0.351 mm and mean pore size: 37.5 nm) by covalent binding with the silane-glutaraldehyde method. The activity of the free and immobilized enzyme (IE) was determined with 5% (w/v) sucrose, at 35 to 65ºC and pH from 3 to 7. Maximum activities were found in the pH range from 5 to 6 for free invertase, and pH 4.5 for the IE. Activity yield for the IE was 24%. The Energy of Activation (Ea) was found to be a function of pH, giving for free invertase, Ea = 7.0 and 6.86 kcal/mol at pH 5.0 and 5.5, respectively, whereas for the immobilized enzyme, Ea = 6.55 and 5.93 kcal/mol at pH 4.5 and 5.0, respectively.

Keywords: invertase, immobilized invertase, energy of activation, sucrose, controlled pore silica.


Inverted sugar is a mixture of glucose and fructose produced by the hydrolysis of sucrose, and it is used to a great extent in the food industry. Processes for hydrolyzing sucrose that use the enzyme invertase can lead to a product of higher quality, without colored by-products and less salts as normally produced by acid hydrolysis. However, the enzymatic process is more expensive than acid hydrolysis, owing to the relatively high cost of invertase.

This may constitute a good industrial opportunity for the application of the technology of immobilized enzyme, because it may offer technical and economical advantages, such as:

(1) Reduction of enzyme usage, because once immobilized, the enzyme can be used for a much longer period than in the soluble form;

(2) Immobilized enzyme can lead to preferred continuous processes that may use either fixed or fluidized bed reactors;

(3) 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;

(4) These technical advantages allow a reduction in the operational and capital process costs, if the immobilized enzyme half-life is sufficiently long (Lartigue, 1975; Daniels, 1985).

This article presents results concerning the activity and activation energy of a commercial invertase. The data presented contain results on both the free enzyme in solution, and the immobilized enzyme (IE), that was produced with controlled pore silica (CPS) as support.


A brief discussion of two main variables that affect the activity of free and immobilized enzyme, and the model for correlating enzyme activity as a function of temperature are presented below.

Effect of pH

Enzymes normally contain various amino acid residues in their active site, and the interaction among them, and with the substrate, influences the catalytic process. Consequently, enzymes are only active in a restricted range of pH, and for most cases, show a definite optimum pH where activity is maximal (Dixon and Webb, 1979; Segel, 1975). The graph of enzymatic activity as a function of pH may produce curves of various shapes, in particular, some are bell-shaped. These curves vary according to three fundamental effects that depend on the concentration of the ion H+ in the enzyme molecule microenvironment (Ballesteros, et al. 1994).

(1) The protein structure of an enzyme molecule is influenced by the alkalinity or acidity of the solution, because its various amino acid residues are in different states of ionization. This dependence on pH is highly complex and there is not a general equation to describe it satisfactorily;

(2) In some enzymes the active sub-sites, involved in binding with the substrate, contain acid or basic groups, and the particular balance of charges affected by pH play an essential role in the formation of the enzyme-substrate complex; and

(3) The pH may influence the values of the maximum velocity of reaction (Vmax) and the Michaelis-Menten constant (Km), because the dissociation of some groups may be necessary to confer activity to the active site, whereas the dissociation of other groups may hinder enzymatic activity.

The bulk pH of the solution in contact with the IE may differ from the pH at the IE microenvironment, owing to partition effects and diffusional limitations that may alter the hydrogen ion concentration in the IE vicinity. The main consequence of this difference is a shift in the IE pH activity profile with respect to the free enzyme. This shift occurs in the direction of more alkaline or acidic pH, in the case of immobilization supports that have negative or positive charges, respectively (Hartmeier, 1978).

Effect of Temperature

The increase in activity as temperature is raised in an enzymatic reaction is modeled by the Arrhenius equation:

A=A0 exp(-Ea/RT)



A = enzymatic activity measured by the initial rate of reaction;

A0 = pre-exponential factor; Ea = energy of activation;

R = universal gas constant (1.987 cal/mol K);

T = absolute temperature in Kelvin.

The energy of activation normally observed for enzymes are in the range of 4 to 20 kcal/mol, therefore a 10ºC increase in temperature from 25ºC raises the enzymatic activity by a factor of 1.2 to 3.0, respectively (Dixon and Webb, 1979; Hartmeier, 1988; Cheetham, 1985).


Enzymatic Activity

For the free enzyme, one unity of enzymatic activity (U) corresponds to the quantity of enzyme that produces one micromol of glucose and fructose in the hydrolysis of a 5% (w/v) sucrose solution, at 55ºC and pH 5.0. The conditions for activity measurements with the IE were the same, except for the pH that was 4.5.

Enzyme and Carrier

The enzyme used was invertase from yeast, β-D-frutofuranosidase (E.C., a kind gift from NOVO Nordisk (Copenhagen, Denmark). The carrier used for immobilization was controlled pore silica (CPS), also a kind gift but from Corning Glass Works (USA), having mean pore size of 37.5 nm and average particle diameter of 0.351 mm.


The substrate was commercial household refined sugar-cane sucrose, acquired from Açucar União. This was used in a 5% (w/v) solution, containing McIlvaine (Morita and Assumpção, 1972) dissodium phosphate-citric acid buffer, 0.1 M, in the pH range of 3 to 7.

Enzyme Immobilization

Invertase was immobilized in CPS by the covalent method of Weetall (1993) and Zanin and Moraes (1998) with the following steps: (a) silanization of the carrier, with a 0.5% (v/v) solution of γ-aminopropyl trietoxisilane, for 3h at 75ºC; (b) washing with distilled water and drying for 15h at 105ºC; (c) activation with a 2.5% (v/v) solution of glutaraldehyde, pH 7.0 for 45 min at 20ºC; (d) washing with water, (e) contacting the activated carrier with a solution of the enzyme for 15h at 20ºC, and, (f) washing the immobilized enzyme with distilled water, and stocking it under sodium acetate buffer (0.2 M) pH 4.5 at 4ºC.

Activity Measurements

A batch reactor with temperature control and containing 50 mL of substrate solution was used for measuring enzymatic activity as a function of pH and temperature for the free and immobilized enzymes. Data processing followed the method of initial velocities (Dixon and Webb, 1979, Hawcroft, 1987). For the test with free enzyme, 0.5 mL of invertase solution with a concentration of 0.1 g/L was used, whereas for the immobilized enzyme, 0.5g dry weight IE was used inside a stainless steel screen basket. Reaction in both cases proceeded under intense agitation during half an hour, while 0.5 mL samples of the reactor solution were taken at regular intervals. These samples were collected in 2.5 mL of distilled water, boiled for 10 min, cooled to room temperature and stocked in the refrigerator for later assay of the glucose and fructose produced during hydrolysis (Bergamasco, 1989; Bassetti, 1995).

Activity tests were carried out at the following temperatures: 35, 40, 45, 50, 55, 60, 65ºC, and pHs: 3.0, 4.0, 5.0, 5.5, 6.0, and 7.0, both for the free and immobilized enzymes.

Assay Methods

Glucose and fructose concentrations in the samples collected during sucrose hydrolysis were measured by the reducing sugar method of DNS (Miller, 1959), as modified by Zanin and Moraes (1987). Protein was determined according to Lowry et al (1951).


Figure 1 shows the results for the specific activity of the free invertase, as a function of pH and temperature. Figure 2 has the same kind of results, but for the immobilized enzyme.

For all temperatures studied, the maximum specific activity observed for the free invertase with 5% sucrose occurs around pH 5, and the optimum temperature was 55ºC (Fig. 1).

For invertase from Saccharomyces cerevisae other workers have found the optimum pH in the range of 3.5 to 6.0, depending on the concentration of substrate, buffers and the temperatures used (Simionescu et al., 1987; Wiseman and Woodward, 1975). For Aspergillus athecius at pH 4.5, the optimum temperature found was 55ºC (Monsan and Combres, 1984), and also for the external enzyme of Aspergillus niger at pH 4.7 (Woodward and Wiseman, 1975). These results demonstrate that the optimum pH for invertase depends on the enzyme source, and to some extent, also on experimental conditions, because the same enzyme from different microorganisms normally have different amino acid composition which affects its state of ionization in solution. The temperature optimum is also a function of enzyme source, and experimental conditions, values having been reported in the range of 45 to 73ºC (Bergamasco, 1989; Woodward and Wiseman, 1975).

Increased substrate concentration has a very clear protection effect against invertase thermal denaturation, as shown by Woodward and Wiseman (1975). They have obtained optimum temperatures for invertase from S. cerevisae at 55 and 65ºC, respectively for sucrose concentrations of 2 and 60%.

For immobilized invertase, Fig.2 shows that the optimum pH is found at pH 4.5, for all temperatures considered by this work. Other published works on immobilized invertase report optimum pHs in the range of 3.0 to 6.1. Literature shows that the support and method used for immobilization are the factors that most influence the optimum pH, the highest value being obtained with covalent binding with diazonium salts on corn grits (Monsan et al., 1984) and the lowest with ionic binding on ion exchange resin (Ooshima and Harano, 1980).

The comparison of Figs. 1 and 2 show that the immobilization of invertase in controlled pore silica by the covalent method shifts the optimum pH of the enzyme about 0.5 point, from 5.0 to 4.5. The maximum specific activity observed for free invertase, occurred at 55ºC, pH 5.0, reaching 409.6 U/mg of protein (Fig. 1), whereas the immobilized invertase has reached 98.2 U/mg of protein at the same temperature and pH 4.5 (Fig. 2). Disregarding the pH shift, the activity yield upon immobilization is low: 24.0%. For the same pH, namely pH 5.0, the activity yield is lower: 22.4%. Comparing the IE with free enzyme it was observed that there was no alteration in the optimum temperature, which for the IE was also 55ºC.

Figures 3 and 4 present the Arrhenius plot for the free and immobilized invertase. It can be observed that enzyme activity first increases with temperature as predicted by Eq. (1) but for higher temperatures there is a point where activity deviates from Eq. (1) and begins to fall as temperature is increased.

The explanation for this behavior comes from the fact that enzyme catalytic activity is highly dependent on temperature, similarly to the case of conventional catalysts, but as temperature is raised in an enzymatic reaction, two effects occur simultaneously: (1) the reaction rate increases, and (2) the enzyme stability decreases because of its denaturation resulting from stretching of the three dimensional protein structure, caused by the availability of excess energy which favors the rupture of covalent bonds that hold the enzymatic lateral chains together (Lartigue, 1975; Dixon and Webb, 1979; Ballesteros et al., 1994; Chibata, 1978). Consequently, Eq. (1) applies only for the lower temperatures below the point in which thermal denaturation of the enzyme becomes too severe. For higher temperatures, above this point, thermal deactivation becomes too fast, and the experimental activity values deviate from Eq. (1), giving lower results and shaping the Arrhenius plot with a maximum that corresponds to the optimum temperature for maximal activity. Beyond the maximum, the gains in activity given by higher temperatures are offset by decreased concentration in active enzyme, caused by thermal denaturation, and this explains the observed decreased activity in spite of the higher temperatures.

From the straight-line portion of the plots in Figs. 3 and 4 the energy of activation (Ea) was calculated, and the results are shown in Fig. 5. It can be observed that the lowest energy of activation occurred at pH 6.0 for both free and immobilized enzyme, giving 5.23 and 4.94 kcal/mol, respectively.

The plot of Ea as function of pH in Fig. 5 reveals a complex dependence of the energy of activation on pH and shows that Ea for the immobilized enzyme is generally lower. The difference in Ea for free invertase for pHs 5.0 and 5.5 is only 2%, which is, in this range Ea is almost constant, but for pH values above 5.5 or below 5.0, Ea variation is more pronounced. For pH values below 4.0 or above 6.0, two factors seem to contribute to increase Ea: (1) an increasingly higher contribution of the acid or alkaline hydrolysis, and (2) a partial denaturation of the enzyme at these extreme pHs. Other works have found invertase Ea values in the range of 7.32 to 7.85 kcal/mol (Dickensheets et al., 1977).

In Fig. 6 the activity data, for pH 5.0 in the case of the free invertase, and pH 4.5 in the case of the immobilized enzyme, as a function of temperature, are plotted in the form of the Arrhenius plot giving the following adjusted equations for invertase specific activity ( Ae = U/mg of protein) as a function of temperature (T) in the straight-line portion of the curve:

free enzyme, pH 5.0:

immobilized enzyme, pH 4.5:

Ae = 2.31 × 105 exp( -6,554 / RT ), r = 0.9993


Therefore, the free and immobilized energy of activation, Ea, observed in both cases, are very close, deviating only by 6.4%. The proximity of these values indicates that the hydrolysis reaction with the immobilized invertase is not limited by mass transfer restrictions (Messing, 1978). This result should be a consequence of the wide and regular pore structure of the controlled pore silica.


(1) The invertase studied in this work has properties that are similar to published data on various invertases.

(2) Immobilization of the enzyme with covalent bonds, as given by the silane-glutaraldehyde method, gives relatively low activity yield around 24%.

(3) At 55ºC, maximum free and immobilized enzyme activity are 409.6 and 98.2 U/mg of protein, at pH 5.0 and 4.5, respectively.

(4) The immobilized enzyme produced according to the procedure of this work, does not seem to be restricted by diffusional limitations, and the energies of activation for free and immobilized enzyme, are very close, namely 7.00 and 6.55 kcal/mol, at pH 5.0 and 4.5, respectively.

(5) The energy of activation observed experimentally is a complex function of pH.


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

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  • *
    To whom correspondence should be addressed
  • Publication Dates

    • Publication in this collection
      16 Mar 2001
    • Date of issue
      Dec 2000


    • Received
      19 Oct 1999
    • Accepted
      18 Apr 2000
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