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Brazilian Journal of Chemical Engineering

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

Braz. J. Chem. Eng. vol. 14 no. 4 São Paulo Dec. 1997 



H. C. Trevisan1, E. P. Bergamo1, J. Contiero1, O. Hojo1 and R. Monti2

1Depto. de Química Tecnológica e de Aplicação, Unesp, Rua Prof. Francisco Degni, s.n.,
Araraquara, SP, Brazil, CEP 14.800-900, Phone (016)2322022/133, Fax (016)2227932
2Depto. de Alimentos e Nutrição, Unesp, Rod. Araraquara-Jaú, km 1, Brazil, CEP 14801-902


(Received: June 11, 1997; Accepted: October 30, 1997)


Abstract - The immobilization of b -galactosidase from Kluyveromyces fragilis on controlled pore silica was investigated. Immobilization was performed on amino silica activated with glutaraldehyde and the product was applied to the hydrolysis of lactose of whey. The behaviors of the soluble and immobilized enzyme were compared by using whey and a lactose solution as the substrate. With the aim of optimizing the method, parameters such as the amount of glutaraldehyde and the size of the particles were evaluated by comparing activities and stabilities on batch and continuously fluidized bed reactors.
b -galactosidase, immobilization, whey, lactose hydrolysis.




Whey is the major by-product of the dairy industry. In spite of its nutritive value (4.5% lactose, proteins and mineral salts), its direct consumption or addition to foods is relatively small. Whey may be processed onto hydrolyzed whey, permeate or syrup (Baret, 1987). These derivatives, with equimolar amounts of glucose and galactose, are sweeter and more soluble than whey and, thus, have advantages and properties that permit their use in new economically attractive products (Holsinger, 1978; Woychik et al., 1974). The hydrolysis of milk and whey lactose also results in alternative products for people with low levels of intestinal lactase, who are estimated to constitute half of the world’s adult population. (Paige et al., 1971).

The production of cheese by the Brazilian dairy industry was 310 thousand tons in 1993, with an estimated increase of 13% per year. From the 440 thousand tons of cheese produced in 1996, approximately 3 million tons of whey resulted (Rodrigues, 1995).

The technology for processing whey has already been developed in some countries, where plants are operating on either a pilot or an industrial scale (Gekas and Lopes-Leiva, 1985), which is indicative of the economical viability of the process, as well as the fact that the production of whey is increasing. Another advantage of this process is the conversion of an effluent, that must be treated, into a commercial product.

Controlled pore silica is, in most cases, a suitable support for enzyme immobilization, after silylation with g -aminopropyltrietoxysilane and activation with glutaraldehyde. Considering the importance of the process for lactose hydrolysis and the fact that the technology needed for preparation of this support has already been developed (Trevisan, 1993), we decided to work on a method for b -galactosidase immobilization and the use of the resulting biocatalyst in whey processing.



The support for enzyme immobilization was a silica with a pore diameter of 450Å , as measured by mercury porosimeter, prepared and treated according to a method described previously (Trevisan, 1993). The amino derivative of the support was activated with glutaraldehyde in a 0.1M phosphate buffer, pH=7.0. The enzyme was a b -galactosidase from Kluyveromyces fragilis, obtained from NOVO Nordisk (Lactozym 3000 HP-G), diluted in a lactic buffer, pH=6.5. Immobilization was performed by adding 60 mg of protein per gram of silica and letting it stand during 24 hours at 4° C, occasionally stirring. The amount of protein immobilized was calculated by the mass balance of the supernatant before and after the reaction, using the Bradford method for dosage (Bradford, 1976). The activity assays were conducted in a bath or a continuously fluidized bed reactor. The amount of glucose produced was analysed using an enzymatic method (Harry et al., 1974) and plotted as a function of time. The activity was calculated from the slope of the fitted straight line. One unit (U) of enzyme is the amount that produces 1 m mol of glucose per minute.



Behavior of the Soluble and Immobilized b -Galactosidase in Lactose Whey and Lactose Solution

For the purpose of obtaining figures on the influence of the substrate composition on enzyme activity and final conversion of the reaction, assays were performed using soluble b -galactosidase in both substrate solutions (Figures 1 and 2) and immobilized b -galactosidase in whey (Figure 3).

The assays with the soluble b -galactosidase, using whey and lactose in a lactic buffer as the substrate, resulted in specific activities of 59 U/mg and 70 U/mg, respectively. In this way, enzyme activity could be evaluated under both conditions, with a decrease of about 20% in the case of the whey. The main reason for such assays was to establish the method for determining enzyme activity using whey, the natural substrate in process applications. The behavior of the hydrolysis was similar for soluble and immobilized enzymes, but final conversion was achieved quickly due to the larger amount of enzyme added.

Figure 1: Lactose whey hydrolysis with soluble b -galactosidase.


Figure 2: Lactose solution hydrolysis with soluble b -galactosidase.


Figure 3: Lactose whey hydrolysis with immobilized b -galactosidase.


It’s worth pointing out that the behavior observed applies only in short-term reactions. For operations in continuous reactors with immobilized enzymes, a relatively rapid drop in activity was observed, due to protein denaturation and the deposit of whey impurities on the surface of the catalyst particles.

Effect of Particle Size on Activity of the Immobilized Enzyme

It was observed that in the assays the smaller the particle size of the support, the greater the activity of the immobilized enzyme (Figure 4). These results, typical of heterogeneous catalysts, are due to an increase in diffusional resistance as particle size increases. Considering the tendency of the curve in Figure 4, when particle size approaches zero, specific activity is at least 30 U/mg, which corresponds to 65% of the soluble enzyme activity.

The total activity (U/g of silica) for immobilization on particles with a 45 m m diameter was 1550 U/g. The evaluation of these data is very important for purposes of reactor dimensioning, as there is a compromise amongst activity, particle size, flow rate and pressure drop.

Effects of Some Immobilization Parameters on b -Galactosidase

In an attempt to improve the activity and stability of the immobilized enzyme (IME), it was adsorbed on silica and afterwards cross-linked by glutaraldehyde. This procedure was already described for b -galactosidase from Escherichia coli (Gekas and Lopes-Leiva, 1985). The result with the lactase from K. fragilis was total inactivation. In order to better evaluate the mechanism of inactivation, 3.75 m mol of glutaraldehyde per gram of protein were added to a enzyme solution. Under this condition, there was a rapid decay in enzyme activity, followed by a stabilization step after about 30 min. As was observed, the decrease in activity depends on the amount of glutaraldehyde; this was attributed to the reaction with the activity site or groups that directly affect it. This behavior is not exactly the same during immobilization, where the glutaraldehyde is linked to the support, but it may occur to some extent.

Another parameter considered for the enzyme immobilization was the buffer adopted. The lactic buffer, recommended by the b -galactosidase producer, is complex and relatively expensive, compared to the phosphate buffer. Also, the addition of substrate during immobilization may have the effect of protecting the enzyme and improving the IME. Immobilization with both buffers resulted in equivalent activities (2200 U/g). Independent of the immobilization method used, the better buffer for IME storage was the lactic one; there was a significant decay in activity after one month in the phosphate buffer, contrary to what occurred in the other. IME activity was lower when immobilized in the presence of lactose than it was when immobilized without it (1790 U/g).

Effect of the Amount of Glutaraldehyde on the Activity and Stability of the IME

In previous assays it was noticed that the excess of glutaraldehyde decreased the activity of the immobilized enzyme, but it was worth evaluating its effect on the stability of the enzyme. For this purpose, amino silica, with a particle size of 150-250 m m, was activated using different amounts of glutaraldehyde (expressed as m mol/gram of silica) and the stabilities of the IMEs were assayed by measuring the activities during continuous operation in a fluidized bed reactor. The effect observed was a decrease in activity and an increase in stability as the amount of glutaraldehyde was increased, which is interpreted as a compromise between its degenerative effect on enzyme activity and its strengthening effect on the structure of the protein (Figure 5).

The stability of the IME in a fluidized bed reactor with whey as the substrate was lower than expected, resulting in a half-life time of 40 min at 40° C. After the initially rapid decay, residual activity was 700 U/g and 400 U/g for the IMEs with more and with less glutaraldehyde, respectively. The greater activity at 25° C (Figure 5) may be explained as a consequence of the higher rate of inactivation at 40° C, overlapping the increase in activity at higher temperatures.


Figure 4: Effect of particle size on the specific activity of the immobilized b -galactosidase.


Figure 5: Effect of the amount of glutaraldehyde on the activity and stability of the IME, in a fluidized bed reactor with whey as substrate.



For the optimization of immobilization, ativties of 1900 U/g were obtained using particles with an average diameter of 200 m m and activities of 3100 U/g were obtained for 50 m m particles.

Glutaraldehyde deactivated the b -galactosidase, whether in solution or immobilized, and its direct addition is not recommended as a stabilizing agent.

The lactic buffer is the best environment for b -galactosidase storage, but is not necessarily the best for the immobilization step.

Despite the high level of activity of the IMEs obtained, the stability assays in a continuous fluidized bed reactor, using whey as the substrate, resulted in a product not yet suitable for industrial purposes. Studies are being conducted in order to discover and overcome the causes of inactivation. The residual activity of 700 U/g, despite being much lower than the initial activity, is comparable to that of IME preparations in other work (Gekas and Lopes-Leiva, 1985) and is still under consideration for the process of lactose hydrolysis. Future work will be directed towards stabilization of the IME.



We would like to thank Novo Nordisk for supplying the enzyme and CNPq, FAPESP and FUNDUNESP for providing the financial support.



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Woychik, J.H.; Honolowisk, M.V. and Dahl, K.J., Preparation and Application of Immobilized b -galactosidase of Saccharomyces lactis, In:, "Immobilized Enzymes in Food and Microbial Process." Olson, A.C. and Cooney, C.L., ed., Plenum Press, New York, p.41 (1974)

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