A comparison of the kinetic properties of free and immobilized Aspergillus oryzae β-galactosidase

Biochemical Engineering Journal 58–59 (2011) 33–38

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A comparison of the kinetic properties of free and immobilized Aspergillus oryzae ␤-galactosidase Fernanda F. Freitas ∗ , Líbia D.S. Marquez, Gustavo P. Ribeiro, Gabriela C. Brandão, Vicelma L. Cardoso, Eloízio J. Ribeiro Faculty of Chemical Engineering, Uberlândia Federal University, P.O. Box 593, Av. João Naves de Ávila 2121, Campus Santa Mônica, Bloco 1K, 38400-902 Uberlândia, MG, Brazil

a r t i c l e

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Article history: Received 17 November 2010 Received in revised form 8 August 2011 Accepted 12 August 2011 Available online 8 September 2011 Keywords: Immobilized enzymes ␤-galactosidase Alginate Glucose Kinetic parameters Lactose

a b s t r a c t The objective of this work was to compare the properties of free and immobilized ␤-galactosidase (Aspergillus oryzae), entrapped in alginate–gelatin beads and cross-linked with glutaraldehyde. The free and immobilized forms of the enzyme showed no decrease in enzyme activity when incubated in buffer solutions in pH ranges of 4.5–7.0. The kinetics of lactose hydrolysis by the free and immobilized enzymes were studied at maximum substrate concentrations of 90 g/L and 140 g/L, respectively, a temperature of 35 ◦ C and a pH of 4.5. The Michaelis–Menten model with competitive inhibition by galactose fit the experimental results for both forms. The Km and Vm values of the free enzyme were 52.13 ± 2.8 mM and 2.56 ± 0.3 gglucose /L min mgenzyme , respectively, and were 60.30 ± 3.3 mM and 1032.07 ± 51.6 glactose /min m3 catalyst , respectively, for the immobilized form. The maximum enzymatic activity of the soluble form of ␤-galactosidase was obtained at pH 4.5 and 55 ◦ C. Alternatively, the immobilized form was most active at pH 5.0 at 60 ◦ C. The free and immobilized enzymes presented activation energies of 6.90 ± 0.5 kcal/mol and 7.7 ± 0.7 kcal/mol, respectively, which suggested that the immobilized enzyme possessed a lower resistance to substrate transfer. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The dairy industry has heavily invested in the development and modification of milk-based products. A significant portion of the world’s population is lactose intolerant and cannot enjoy the benefits of milk and lacteal products [1–3]. Lactose has limited use in lacteal products due to its low solubility, low sweetening power and laxative effect at high concentrations [4,5]. Every year, several tons of milk whey are produced worldwide, and the recovery of its lactose content by bioconversion is a promissory strategy [6]. Lactose hydrolyze can be catalyzed by acids, cationic resins or enzymatic processes [7]. In its basic components, lactose hydrolysis by ␤-galactosidase is a suitable alternative to acid catalysis, as enzymes do not alter the nutritional and technological characteristics of food [6–9]. Several microbial sources of ␤-galactosidase have been studied for food applications. Currently, the main producers of ␤-galactosidase, Aspergillus niger, Aspergillus oryzae, Kluyveromyces lactis and Kluyveromyces fragilis, are considered to be GRAS and are used in the food industry [10]. Fungal ␤-d-galactosidases are more suited for acidic whey hydrolysis than yeast enzymes, due to their

∗ Corresponding author. E-mail address: [email protected] (F.F. Freitas). 1369-703X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2011.08.011

thermostability; however, these galactosidases are more sensitive to product inhibition, mainly by galactose [2,7,9,13]. The milk has a pH near 6.7. With neutral pH conditions, the yeast lactases are well suited for hydrolysis of lactose in milk and are widely accepted as safe for use in food products [12,14]. Industrially, both soluble and immobilized ␤-galactosidases are used for lactose hydrolysis; however, some properties of soluble enzymes, such as their non-reusability, may hamper their usefulness [6,15,16]. The use of a relatively expensive catalyst, such as enzymes, requires, in many instances, its recovery and reuse to make the process economically feasible. The idea of enzyme reuse implicitly means that the stability of the final enzyme preparation should be sufficiently high to permit its reuse [17]. However, recent works have shown that enzyme immobilization is a very powerful tool for improving almost all enzyme properties, including stability, activity, specificity, selectivity and inhibition reduction [18,19]. Another alternative that has been studied to reduce the inhibition of ␤-galactosidase by galactose is the use of engineered microorganisms [20].Of all the various applications, the industrial applications of immobilized enzymes are the most important, and they are much discussed in the literature. In industrial processes, immobilized enzymes are used in chemical reactors similar to those normally used in chemical catalysis [21–23]. Another important application of immobilized enzymes is their use in biosensors, which are analytical devices that incorporate biological

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F.F. Freitas et al. / Biochemical Engineering Journal 58–59 (2011) 33–38

materials such as enzymes, tissues and organisms. The immobilization of enzymes in various matrices has been used for the fabrication of biosensors to estimate levels of urea and glucose–cholesterol [24]. The immobilization of ␤-galactosidase for lactose hydrolysis in milk and cheese whey has been described in different works, and different methods have been employed [15,25]. Entrapment in calcium alginate gel is one of the simplest methods of enzyme immobilization. Natural polymers, like alginate, are particularly suitable as matrix materials, due to their non-toxicity, and the methods used in their gelation are very mild [12]. The major advantage of the entrapment technique is the simplicity in which particles can be obtained by dripping a polymer–enzyme suspension into a medium containing positively charged ions, such as Ca+2 . However, due to the high porosity of alginate beads, the entrapped enzymes may be leached out of the polymer matrix [9,11,12,26,27]. Several studies have been performed attempting to avoid enzyme leaching from the calcium alginate beads [9,25]. A. oryzae ␤-galactosidase is a monomeric extracellular enzyme, presents a molecular weight of 90,000 and can leak out of alginate beads due to their high porosity [16,20,28]. An approach to circumvent this problem consists of cross-linking the enzyme and gelatin with glutaraldehyde, which forms a structure that can stop enzyme leakage. Glutaraldehyde treatment also stabilizes the alginate gel [11,27]. Gelatin is used for enzyme immobilization cross-linked with glutaraldehyde under mild conditions, and the reaction between gelatin and glutaraldehyde involves only the lysine residues of the protein [27,29]. The glutaraldehyde molecule that is bound to the ␧-amino groups of the enzyme lysines can covalently react with the glutaraldehyde molecule bond of the primary amino groups of the support, which establishes a multi-point covalent enzyme-support attachment [30,31]. The objective of this study was to compare the effects of temperature and pH on the enzymatic activity, as well as the kinetic parameters of free and immobilized A. oryzae ␤-galactosidase. The study employed a combined immobilization process consisting of entrapment in sodium alginate and cross-linking the enzyme and the gelatin with glutaraldehyde. 2. Experimental 2.1. Enzyme A. oryzae ␤-galactosidase (3.2.1.23) was obtained from the Sigma Chemical Co. The enzyme is available in the form of a white powder and has an activity for lactose hydrolysis of 9 units per mg of commercial product. The unit of activity (U) was defined as 1 ␮mol of lactose per minute at 30 ◦ C, using a 50 g/L lactose solution as a substrate in an acetate buffer with pH 4.5. The enzyme was diluted to 1% (w/v) in a 4.5 pH acetate buffer, and contained 0.14 mg/mL of protein, determined by Lowry’s method [32]. 2.2. Immobilization of ˇ-galactosidase in alginate, gelatin and glutaraldehyde Sodium alginate (1.65 g) and gelatin (1.01 g) were dissolved in distilled water, in order to generate a suspension with a final weight of 20 g. To allow the alginate to completely dissolve, the mixture was heated to 80 ◦ C. Subsequently, the suspension was cooled to 40 ◦ C, and 5 mL of an aqueous solution of 1% (w/v) ␤-galactosidase (450 U) was added. In this immobilization protocol, the final alginate concentration was 6.6% (w/w). With a peristaltic pump, the resulting suspension was added to a stirred solution of 0.05 mol/L of CaCl2 and 3.64% of glutaraldehyde (v/v). The resulting biocatalyst beads were hardened for 12 h in the immobilization medium,

and their activity was determined. The spherically formed particles presented an average diameter of 4.4 mm and were resistant to the reaction medium conditions. 2.3. Determination of the activity of free and immobilized enzymes The enzymatic activity on lactose hydrolysis was determined by the initial rates procedure. The glucose-oxidase method was used to measure the glucose produced by the enzymatic reaction [33]. Moreover, protein determination measurements were conducted according to Lowry’s method [32]. The hydrolysis reactions were performed in a reactor containing 50 mL of buffered lactose solution at the appropriate conditions of lactose concentration, pH and temperature. For the immobilized form of ␤-galactosidase, 15 cm3 of the immobilized enzyme beads, determined by volume displacement on a measuring cylinder, was added to the solution. In each experiment, the buffered solution had different conditions of pH, temperature and lactose concentration. All of the experiments were performed in triplicate. The alginate gel particles without ␤-galactosidase did not present any lactose hydrolysis activity. The unit of specific activity of the free enzyme (UF ) was defined as the grams of glucose produced per liter per minute per milligram of protein (gglucose /L min mgenzyme ). The unit of activity (UI ) of the immobilized form was defined as the grams of lactose consumed per minute and cubic meter of biocatalyst (glactose /min m3 biocatalyst). 2.4. The stability of the free and immobilized enzymes as a function of pH Samples of the enzyme with known activity were incubated in an appropriate buffer at 35 ◦ C for 18 h. Upon completion, the beads residual enzymatic activity was determined by the method of initial rates. All experiments were carried out in triplicate. The activity experiments were conducted in an acetate buffer with a pH of 4.5 and an initial lactose concentration of 50 g/L at 35 ◦ C. The relative (Ai /A0 ) activity associated with each pH was determined by the ratio of the residual activity (Ai ) with the activity without incubation (A0 ). The activity of the incubation medium after the removal of the immobilized enzyme particles was also determined. The conditions used to determine the activity without incubation were identical to the aforementioned conditions. To assess the soluble form of ␤-galactosidase, the enzyme was diluted to 1% (w/v) and was incubated in citrate-phosphate buffer at pH values ranging from 3.0 to 7.0. Alternatively, the immobilized enzyme was incubated in buffers at pH values between 2.6 and 8, and a 10−1 mol/L. A citrate-phosphate buffer was used to reach pH values from 2.6 to 3.6 as well as 5.6 to 7.0, and, a 10−1 mol/L acetate buffer was used to reach pH values ranging from 3.6 to 5.6. 2.5. The effect of temperature and pH on the activity of the free and immobilized enzymes All of the experiments were performed in triplicate with a lactose concentration of 50 g/L and a reaction volume of 50 mL. To study the effect of pH on the free enzyme, the temperature of the reaction medium was maintained at 35 ◦ C. The pH of the reaction medium was varied by employing the buffer solutions described in Section 2.4. To study the effect of temperature, the pH of the reaction medium was maintained at 4.5 in an acetate buffer, and the temperature was varied from 5 to 70 ◦ C. The objective of this study was to evaluate the simultaneous effect of temperature (X1 ) and pH (X2 ) on the activity of the immobilized enzyme. To determine the optimal conditions of lactose hydrolysis with immobilized lactase, a central composite design (CCD) with

F.F. Freitas et al. / Biochemical Engineering Journal 58–59 (2011) 33–38 Table 1 The central composite design used to study the effect of temperature and pH on the enzymatic activity of immobilized lactase.

1 2 3 4 5 6 7 8 9 10 11

1.2 1.0

Real value (codified value) Temperature (◦ C)

pH

30 (−1) 58 (1) 30 (−1) 58 (1) 44 (0) 44 (0) 44 (0) 28 (−˛) 60 (+˛) 44 (0) 44 (0)

3.2 (−1) 3.2 (−1) 5.7 (1) 5.7 (1) 4.4 (0) 4.4 (0) 4.4 (0) 4.4 (0) 4.4 (0) 3 (−˛) 5.9 (+˛)

Relative Activity

Experiments

35

0.8

Immobilized Free

0.6 0.4 0.2 0.0 0

1

2

3

4

5

6

7

8

pH

2.6. Kinetic study of lactose hydrolysis by free and immobilized ˇ-galactosidase The effect of the lactose concentration on the activity of ␤galactosidase in the free and immobilized forms was determined experimentally by the method of initial rates. For the free enzyme, the concentration of the substrate in acetate buffer was varied from 10 to 90 g/L at 35 ◦ C and a pH of 4.5. For the immobilized form of the enzyme, the concentration of the substrate in acetate buffer was varied from 10 to 140 g/L at 35 ◦ C and a pH of 4.5. The velocity of the reaction as a function of the substrate concentration fit the Michaelis–Menten model, and values of Km and Vm were obtained through non-linear regression using Statistica® 7.0 software. The influence of glucose and galactose on the kinetics of the reaction of free enzyme was studied in the range of 15–55 g/L for lactose and 1.25–11.26 g/L for glucose and galactose. These values were selected based on preliminary assays and in the literature [35,36]. 3. Results and discussion 3.1. Immobilization yield A. oryzae ␤-galactosidase was immobilized in alginate and gelatin and hardened with glutaraldehyde. The immobilization yield, which is defined as the ratio of the immobilized enzyme activity to the total activity of soluble enzyme used [16,37,38], was 30%. The immobilization yield depends on the load of enzyme offered for immobilization and showed a low value, possibly due

Fig. 1. Enzymatic stability of the free and immobilized forms of ␤-galactosidase as a function of the pH. Bars indicate the standard deviation from triplicate determinations.

to interactions between enzyme–alginate–gelatin–glutaraldehyde, involving the active site. The enzyme activities determined after the gel formation and after the hardening of the gel particles presented practically the same results. The enzyme activity of the supernatant after the immobilization procedure was insignificant. 3.2. Stability of the enzyme as a function of the pH Compared to the initial activity of ␤-galactosidase (A0 ), which was determined at a pH of 4.5, the enzyme retained its full activity after 18 h of incubation, at a pH range of 4.5–7.0, in its free and immobilized forms. No enzyme activity was found in the incubation medium. The relative activity (Ai /A0 ) of the free and immobilized enzymes, which is defined as the ratio of activity to initial activity, is presented in Fig. 1. A. oryzae ␤-galactosidase was stable in pH ranges of 4.0–8.0. 3.3. The effect of temperature and pH on the activity of the free and immobilized enzymes It is well-known that pH has a significant effect on the stability of an enzyme, and the activity of most enzymes is highly dependent on the pH of the solution. For the free enzyme, a pH of 4.8 provided the highest enzymatic activity, as shown in Fig. 2. Also, the enzyme was completely stable at this pH value, as shown in Fig. 1.

2 1.8

Activity (U F)

two variables (temperature and pH) was implemented, and 11 experiments were conducted. To investigate the linear model, 22 experiments were performed, and three experiments corresponding to the central point were conducted. In addition, four experiments consisting of rotationally distributed (axial points) at a distance of ˛ from the central point were also performed. An orthogonal CCD was employed in this study, which is an experimental design with a diagonal matrix of variance and covariance in which the estimated parameters are not correlated. To achieve the orthogonal CCD, an ˛ value of 1.1474 was employed. In addition, the Statistica® 7.0 software was used to conduct a multiple regression analysis on the experimental results. As shown in Table 1, this was achieved by using a central composite design. A simultaneous analysis of the effect of pH and temperature on the activity of ␤-galactosidase from A. oryzae has not been previously reported. Thus, the initial pH and temperature range was obtained from the results of preliminary tests and previous studies on the isolated effect of pH and temperature on the activity of the enzyme [13,26,27,34,35].

1.6 1.4 1.2 1 2.5

3

3.5

4

4.5

5

5.5

6

6.5

pH Fig. 2. The effect of the pH on the activity of free ␤-galactosidase. Bars indicate the standard deviation from triplicate determinations.

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Fig. 3. Response surfaces and contour curves of the effect of the temperature and pH on the enzymatic activity of immobilized lactase.

Y = 1499.18 + 171.2X1 + 79.95X2 + 354X1 X2 + 99.46X12 − 249.16X22

(1)

All of the terms in Eq. (1) were evaluated with Student’s t-test, and significance levels lower than 10% were observed. Therefore, all of the parameters were considered to be significant. The determination coefficient (R2 ) was 97.4%, which indicated that the experimental data were appropriately adjusted. Moreover, this result revealed that 97.4% of the variability of the data was explained by Eq. (1). It should be emphasized that this equation is valid for the conditions employed in these experiments, such as the particle size of the biocatalyst, the immobilized enzyme activity, the buffer solutions used and the lactose concentration in the ranges of temperature and pH employed in the statistical design. The response surface diagrams generated by the experiments shown in Table 1 are presented in Fig. 3. As shown in Fig. 3, the greatest activities were observed at the highest temperatures and at intermediate and high pH values. In the optimal pH range, the greatest enzymatic activity was observed at the highest temperature, starting at 58 ◦ C (level +1). An intermediate pH value provided optimal enzymatic activities (from level 0 to +˛). Therefore, the temperature of the solution had the strongest effect on the activity of ␤-galactosidase. To identify the variables that maximized the response and to calculate the maximum point of the enzymatic activity, an algorithm of the complete equation model was implemented. The objective of this work was to increase enzymatic activity (Y), and the results indicated that a temperature of 60 ◦ C and a pH of 5.0 provided optimal results. The conditions

corresponding to the optimal point on the surface plot displayed an enzymatic activity of 1932 UI . The activity calculated by the model presented in Eq. (1) was 2034 UI ; thus, a 5% deviation was observed. In addition, both forms of the enzyme were stable at their pH of maximum activity, 4.8 for the free and 5.0 for the immobilized enzyme, respectively. In most published studies, the optimal temperature and pH were similar to those of this investigation. In the work of Ates and Mehmetoglu [26], Tanriseven and Dogan [27], Prashanth and Mulimani [39], Gaur et al. [40] and Haider and Hussain [6], the effect of temperature and pH were analyzed separately; however, these authors obtained results similar to those of the present study. The temperature range resulting in the greatest enzymatic activity was between 45 and 60 ◦ C, and the range of optimal pH values was between 4.0 and 5.0. The results of previous studies have suggested that enzyme immobilization may alter the temperature of maximum activity. For enzymes immobilized by gel entrapment and covalent or ionic bonds, the optimal temperature can be greater than that of the native form.

1.0 Free Immobilized

0.8

Relativity Activity

The results of different studies have indicated that the optimal pH values for A. oryzae ␤-galactosidase activity ranges from 4.5 to 5.0; this observation was confirmed in the present study. The simultaneous effect of pH and temperature on enzymatic activity was analyzed by a CCD, according to Table 1. Based on the experimental results, a multiple regression was performed, which generated Eq. (1) with coded significant variables (X1 = temperature and X2 = pH). The activity of immobilized enzyme Y in (glactose /min m3 biocatalyst) is presented in Eq. (1).

0.6 0.4 0.2 0.0 0

10

20

30

40

50

60

70

80

90

Temperature (Celsius) Fig. 4. The effects of temperature on the enzymatic activity of the free and immobilized enzymes. Bars indicate the standard deviation from triplicate determinations.

F.F. Freitas et al. / Biochemical Engineering Journal 58–59 (2011) 33–38

As shown in Fig. 4, the free and immobilized enzymes presented similar profiles; however, the optimal temperature for the immobilized enzyme was greater than that of the free enzyme. These results suggest that the immobilized enzyme was more tolerant to higher temperatures than the soluble form in the studied temperature range. These results are in accordance with those of Zhou and Chen [41] and Kennedy [42]. Based on these results, immobilization improved the thermal enzyme stability, which was possibly due to the multi-point covalent attachment of the enzyme to the gelatin [17,18,31]. Additionally, the results in Fig. 4 indicate that the maximum activity for free ␤-galactosidase was 55 ◦ C and 60 ◦ C for the immobilized enzyme. These findings show that the immobilized ␤-galactosidase possessed better heat tolerance than the soluble enzyme, indicating that enzyme immobilization can protect the active conformation of the enzyme from damage by heat exchange [25,41]. However, at temperatures greater than 60 ◦ C, enzymatic activity rapidly decreased due to denaturation. The results of the aforementioned temperature studies were used to determine the activation range of the free and immobilized enzymes, and the activation energy of lactose hydrolysis was determined using the Arrhenius equation. In this work, the activation energies were 6.9 ± 0.5 kcal/mol and 7.7 ± 0.7 kcal/mol for the free and immobilized enzymes, respectively. The similarity of these values suggested a low resistance of the immobilized enzyme to substrate transfer with similar enzyme catalytic efficiency. According to David et al. [43], in non-porous matrixes, the enzyme is immobilized on the outer surface of the matrix, and the activation energies of free and immobilized forms are similar. Therefore, enzymes entrapped in a gel or bound to a porous matrix or support should possess lower activation energies. In many cases, the activation energy of the immobilized form was equal to or greater than that of the native enzyme [42].

3.4. Kinetic study of lactose hydrolysis by free and immobilized ˇ-galactosidase For both forms of the enzyme, the effect of substrate concentration on the hydrolysis of lactose fit the Michaelis–Menten model, and values of Km and Vm were obtained through nonlinear regression using Statistica® 7.0 software. The coefficients of determination (R2 ) of 96% and 90% were observed for the free and immobilized forms, respectively. According to the Student t test, only the parameters with reliability over 95% were considered statistically significant (p-value < 5%). The values of Km for the free and immobilized forms were statistically significant, and values of 52.13 ± 2.8 and 60.30 ± 3.3 mM, respectively, were obtained, suggesting they have basically the same substrate affinity. According to Prashanth and Mulimani [39] and Reshmi et al. [44], an increase in the value of Km when the enzyme is immobilized can be partially attributed to the occurrence of mass transfer resistance from the substrate to the immobilized enzyme and to a decreased affinity of the enzyme for the substrate, which may be due to the effects of intraparticle mass transfer. The aforementioned authors reported that low mass transfer rates are characteristic of systems that use alginate as the immobilization medium due to the presence of strong crosslinks, which limit the velocity of substrate and product permeation. The results of the present work demonstrate the existence of competitive inhibition by galactose for both free and immobilized ␤-galactosidase. The values of the inhibition parameter Ki for the free and immobilized enzyme were 1.02 ± 0.08 and 9.6 ± 0.72 mM respectively. This fact suggests that the inhibition by galactose was smaller in the immobilized form, thus making it more suitable for industrial application. The inhibitory effect was lower for the immobilized enzyme, as seen

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in other works [17,18,45,46]. Glucose did not present any inhibitory effect on enzymatic activity [36,47,48]. 4. Conclusions The results of this study revealed that the pH corresponding to the greatest free ␤-galactosidase activity was 4.5, whereas the activity of the immobilized form of the enzyme was optimal at a pH of 5.0. The temperatures of maximum activity for the free and immobilized enzymes were 55 and 60 ◦ C, respectively. The activation energies of lactose hydrolysis by the free and immobilized forms of the enzyme were 6.9 and 7.7 kcal/mol, respectively, which suggests that the immobilized enzyme possessed a low resistance to substrate transfer. Within the studied range of lactose concentrations, the Michaelis–Menten model fit the experimental data. The Km of the free and immobilized enzymes was significant, and values of 52.13 and 60.30 mM, respectively, were obtained. Galactose behaved as a competitive inhibitor in the hydrolysis of lactose by the free and immobilized enzyme, with Ki values of 1.02 and 9.6 mM, respectively. Acknowledgements The authors acknowledge the Federal University of Uberlândia and FAPEMIG for financial support. References [1] M. Di Serio, C. Maturo, E. De Alteriis, P. Parascandola, R. Tesser, E. Santacesaria, Lactose hydrolysis by immobilized ␤-galactosidase: the effect of the supports and kinetics, Catalysis Today 79–80 (2003) 333–339. [2] I. Roy, M.N. Gupta, Lactose hydrolysis by Lactosym TM immobilized on cellulose beads in batch and fluidized bed modes, Process Biochemistry 39 (2003) 325–332. [3] Scientific opinion on lactose thresholds in lactose intolerance and galactosaemia, EFSA Journal. 8 (9) (2010) 1777. [4] J. Rogalski, A. Dawidowicz, A. Leonowicz, Lactose hydrolysis in milk by immobilized ␤-galactosidase, Journal of Molecular Catalysis B: Enzymatic 3 (1994) 223–245. [5] J. Szczodrak, Hydrolysis of lactose in whey permeate by immobilized ␤galactosidase from Kluyveromyces fragilis, Journal of Molecular Catalysis B: Enzymatic 10 (2000) 631–637. [6] T. Haider, Q. Hussain, Calcium alginate entrapped preparation of A. oryzae ␤-galactosidase: its stability and applications in the hydrolysis of lactose, International Journal of Biological Macromolecules 41 (2007) 72–80. [7] D.G. Hatzinikolaou, E. Katsifas, D. Mamma, A.D. Karagouni, P. Christakopoulos, D. Kekos, Modeling of the simultaneous hydrolysis-ultrafiltration whey permeate by a thermostable ␤-galactosidase from A. oryzae, Biochemical Engineering Journal 24 (2005) 161–172. [8] A.E. Al-Muftah, I.M. Abu-Reesh, Effects of internal mass transfer and product inhibition on a simulated immobilized enzyme-catalyzed reactor for lactose hydrolysis, Biochemical Engineering Journal 23 (2005) 139–153. [9] Z. Grosová, M. Rosenberg, M. Rebros, Perspectives and applications of immobilised ␤-galactosidase in food industry – a review, Czech Journal of Food Sciences 26 (1) (2008) 1–14. [10] N.A. Greenberg, R.R. Mahoney, Immobilisation of lactase (␤-galactosidase) for use in dairy processing: a review, Process Biochemistry (1981) (Fevereiro/Marc¸o). [11] M.R. Kosseva, P.S. Panesar, G. Kaur, J.F. Kennedy, Use of immobilised biocatalysts in the processing of cheese whey, International Journal of Biological Macromolecules 45 (2009) 437–447. [12] P.S. Panesar, R. Panesar, R.S. Singh, J.F. Kennedy, H. Kumar, Microbial production, immobilization and applications of ␤-d-galactosidase – review, Journal of Chemical Technology and Biotechnology 81 (4) (2006) 530–543. [13] M. Portaccio, S. Stellato, S. Rossi, U. Bencivenga, M.S. Mohy Eldin, F.S. Gaeta, D.G. Mita, Galactose competitive inhbition of ␤-galactosidase (A. oryzae) immobilized on chitosan and nylon supports, Enzyme and Microbial Technology 23 (1998) 102–106. [14] M. Ladero, A. Santos, F. García-Ocha, Kinectic modeling of lactose hydrolysis with an immobilized ␤-galactosidase from Kluyveromyces fragilis, Enzyme and Microbial Technology 27 (2000) 583–592. [15] S.A. Ansari, Q. Hussain, Immobilization of A. oryzae ␤-galactosidase ion exchange resins by combined ionic-binding method and cross-linking, Journal of Molecular Catalysis B: Enzymatic 63 (2010) 93–101. [16] C.Z. Guidini, J. Fisher, L.N.S. Santana, V.L. Cardoso, E.J. Ribeiro, Immobilization of A. oryzae ␤-galactosidase in ion exchange resins by combined ionic-binding method and cross-linking, Biochemical Engineering Journal 52 (2010) 137–143.

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