Biotechnol. Prog. IQQ4, 10, 220-224
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Immobilization of @-Galactosidaseon Chitosan Carlos R. Carrara and Amelia C. Rubiolo* INTEC (U.N.L.-CONICET), Guemes 3450, Santa Fe, Argentina
The j3-galactosidase immobilization on chitosan was studied. Enzymatic activity and stability were determined for the different conditions used. The best results were obtained on chitosan beads of 2.2 mm diameter where the immobilization process was carried out at 37 O C with a 1% glutaraldehyde concentration and the addition of galactose. The higher activity values of the immobilized enzyme compared with those of the free &galactosidase could be obtained over a larger pH range and were a t higher pH’s and temperatures, but the best activity is only 10.7% of the free enzyme values.
Introduction Lactose is the main milk carbohydrate, which is hydrolyzed in isomolecularmixtures of glucoseand galactose by the @galactosidaseenzyme. Persons with an inherent deficiencyof the intestinal enzyme lactase cannot consume milk and dairy products because they cannot tolerate lactose. The low sweetness and the insolubility of lactose limit its use as a food ingredient. Its crystallization in dairy foods can produce defects such as a sandy or gritty texture and deposit formation. Therefore, hydrolyses of lactose by appropriate processes from a nutritional, commercial point of view have received widespread interest. The high lactose content in cheese whey, a byproduct of cheese manufacturing, also makes for the problem of waste water disposal in the dairy industry. Enzyme immobilization has been generally regarded as a solution for the lactose hydrolysis process, but it has remained impracticablebecause of the high cost of support materials that had showed technical success in the immobilization procedure. j3-Galactosidase of different microorganisms has been immobilized through a variety of techniques and support carriers (Gekas, 1985),but few works on Kluyveromyces fragilis enzyme immobilizationcan be found in literature. An enzyme appropriate to the lactose hydrolysis in milk and sweet whey for its optimum pH of catalytic activity had been studied and characterized by Mahoney (1977, 1978). Chitosan is a polysaccharide mainly made up of 2-amino-2-deoxy-~-glucose units, which arejoined by j3-1,4linkages. It is obtained by deacetylation with a drastic alkaline treatment of chitin, which is the principal component in the exoskeletonsof crustaceans and insects and also in the cell walls of some fungi. Chitosan has been used as a carrier in the immobilizationof microorganisms and enzymes, e.g., a-chymotrypsin and acid phosphatase (Muzzarelli,1976),aspergillusj3-glucosidase (Bisset,1978), alkaline phosphatase and pepsin (Hirano, 19791, and penicillin G-acylase (Braun, 1989). The cost of this material with respect to others used as the support is low. Chitin and chitosan have numerous applications, such as the purification of water and beverages (Knorr, 1991),and due to their antimicrobial properties are used to obtain reduction in microbial growth (Popper, 1990) and preservative coating material (Gaouth, 1991). Enzyme immobilization on chitosan can mainly be achieved by means of the glutaraldehyde reaction between the free amino groups of chitosan and the enzyme molecules to form covalent linkages (Bookletof IBF LKB, 1983; Braun, 1989). This biopolymer soluble under pH 8756-7938/94/3010-0220$04.50/0
5.5 can form gels by acting as a polycation with sodium triphosphate as an oppositely charged electrolyte (Knorr, 19851, which permits one to obtain matrices in different shapes and sizes. This work describes &galactosidaseimmobilization on chitosan beads. The results for different glutaraldehyde concentrations, enzyme, dilution, temperature, and the effect of galactose presence during the immobilization process are presented. The protein immobilized, enzyme activity, and half-life in every assay were determined for obtaining the process conditions of the method that produce more enzymatic activity and stability of the immobilized enzyme. Also it is indicated the effect of temperature and pH on immobilized enzyme with better performance, and its activity retention respect to free enzyme.
Materials and Methods Kluyveromyces (saccharomyces)fragilis &galactosidase of commercial name Lactozym 3000 was from Novo (Bagsvaerd, Demark). The enzyme solution in 4.75% lactose solution had a specific activity of 3000 LAU/cm3 (Novo lactase unit). This commercial preparation was used without further purification and has a protein concentration of 35.09 mg/cm3. Crab shellchit” and sodium triphosphate of practical grade were from Sigma Chemical Co. (St. Louis, MO). All other chemicals, lactose, dibasic sodium phosphate, monobasic potassium phosphate, and (glacial)acetic acid, were analytical quality or better from Mallinckrodt (St. Louis, MO) or Merck (Buenos Aires, Argentina). Kits of enzymatic glucosedetermination and biuret reagents were from Wiener Lab (Rosario, Santa Fe, Argentina). Preparation of Support. Chitosan (1.5 g) was dissolved in (50cm3) on aqueous solution of 2.5% (v/v) acetic acid and then filtered to remove insoluble materials. This solution was added dropwise to a gently stirred (1.5% (w/v)) sodium triphosphate solution (150 cm3) using hypodermic needles of two diameters to obtain different bead sizes. Before immobilization,they were washed until neutrality and suspended in 0.025 M KH2P04 and 0.025 M Na2HP04 buffer of pH 6.86. Immobilization Procedure. Activation of the supports was performed with 1,3,and 5% (v/v)glutaraldehyde solutionsin 0.025 M KH2POl and0.025M Na2HP04 buffer of pH 6.86 for 24 h at room temperature. In order to determine the glutaraldehyde concentrationsthat provide greater enzyme activity in the support, three concentrations were used in the range commonly indicated for this
0 1994 American Chemical Society and American Institute of Chemical Engineers
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procedure. After the activation,matrices were thoroughly washed with the same buffer. A total of 1.6 cm3 of @-galactosidasesolution from the commercialpreparation or a dilution of this volume with buffer (pH 6.86)was then added to 1.5 g of the activated support. In assays with galactose, 0.03 g/cm3 initial enzyme solution was added. The preparations were left at 4,24,37,and 43 "C overnight with intermittent mixing. The preparations obtained were washed with the same buffer (pH 6.86) used for the activation until no enzyme remained in the washing solution. Galactose is a competitive inhibitor in the 8-galactosidase kinetic reaction (Mahoney, 1977)and could interact reversibly with an enzyme active site. Its addition to the coupling mixture could protect the active site, preventing it from taking part in the cross-linking reaction (Srere, 1976). The glutaraldehyde reaction with amino groups of chitosan or enzyme is fast in aqueous solution at room temperature. Although this reaction takes place over a fairly wide pH range (5.0-9.0),all of the reactions were carried out at near-neutrality because the @-galactosidase of Kluyueromyces fragilis is stable in only a small pH range (6.5-7.5) (Mahoney, 1977). Determination of Immobilized Protein. Protein concentrations in solution were determined by the biuret method (Rojkin, 1974;Mammarella, 1991). The amount of immobilized protein was estimated by subtracting the amount of protein determined in the supernatant after immobilization from the amount of protein used for the immobilization. Activity Measurement. The activity of soluble and immobilized enzymes was determined with the use of a 5 % solution of lactose as substrate in potassium phosphate buffer, pH 6.86 (0.025M KH2PO4,0.025 M Na2HP04, and 1 mM Mg2+),in a stirred reactor with the temperature controlled at 37 OC. The concentration of glucose was determined enzymatically by using the glucose oxidase method (Trinder, 1969). The enzymatic activity of each preparation was expressed as the glucose concentration (mol/dm3)measured after 30 min of reaction of 1.5 i.O.05 g of immobilized enzyme with 15 cm3 of substrate solution. The activity control of each preparation was made during 30 assays, at most, called cycles, or until the determined activity was 70 f 3% of its initial value. Another value, called the activity retention, was also calculated, which considered the protein content of the soluble or immobilized enzyme used in the determination. In this case, the glucose concentration produced in the hydrolysis was divided by the protein presented in the sample, as follows:
Ar = C / ( E P ) where Ar is the activity in mol of glucose/mg of protein, C is the moles of glucose measured after 30 min with the corresponding enzyme (0.30g of immobilized enzyme or 0.025 g of soluble enzyme) in 20 cm3 of 0.146 M (5%) ladose buffer solution,E is the immobilizeden zyme weight (g) or the solubleenzymevolume (cm3),and Pis the protein content by enzyme weight (immobilized enzyme mg of protein/g of support) or the protein content of the soluble enzyme (35.09mg of protein/cm3 of solution). pH Profile. The 0.146 M (5%)solutions of lactose in buffers of different concentrations of KH2P04 and NaOH for obtaining pH values ranging from 5.8 to 8.0 were prepared to determine the optimal pH of the soluble and immobilized enzyme (Weast, 1982). The activity was
expressed as a glucose concentration after 30 min of enzymatic hydrolysis of lactose. For the assays, 0.025 cm3 of soluble enzyme or 0.52 0.05g of immobilized enzyme (catalyst XI) in 15 cm3 of lactose buffer solution was used at 37 "C. Since the amounts of soluble or immobilized enzyme were not important for this comparative study, they were smaller to ensure no changes in the pH values throughout the reaction. The relative activity was expressed as glucose concentration (MI multiplied by the factor (100/0.146). Temperature Effect on Activity. The effect of temperature on the activity of the immobilized enzyme beads, identified as catalyst XI, was determined with the assay conditions given in the Activity Measurement section, at temperatures between 30 and 57 "C. Also, the 37 and 43°C assays were carried out for determining the activity durinig different storage and working times, up through a 183-dperiod. Half-Life. The half-life was determinedby considering that the activity loss leads to a first-order process: kt
Ao+A where A0 is the activity at time t = 0, A is the activity at time t, and kt is the deactivation rate constant. The mathematical expression used for this process was A = Aoe4tt, where the half-life was t1p = 0.693/kt. A linear regression of the [ln(AdA)] experimental values against time was used to obtain kt and the half-life (t112). This calculationfor each preparation was made with values determinedaccording to the Activity Measurement section and is reported in hours. Results and Discussion The general behavior of &galactosidase immobilized on chitosan beads under different immobilized conditions was studied. The amount of fixed protein was determined and correlated with the activity. The results obtained are listed in Table 1. The effects of immobilizationconditions on the activity of the immobilized enzyme are presented in Figure 1. The rate of activity loss for immobilized enzyme with the cycles for the various concentrations of glutaraldehyde used in the activation of the support was plotted in Figure la. It can be seen that the more commonly used concentrationof 1 % (Praun, 1989)produced higher values of glucose concentration during a larger number of cycles. Table 1 also shows the performanceof these assays. From these data, it can be shown that a higher glutaraldehyde concentration produced an increment of the attached protein, but the activity of the enzyme fixed to the beads rapidly decreases. It appears that the lowest concentration (1%) has less of an affect on the enzyme and produces better covalent binding. The assay results for the different enzyme concentrations are given in Figure lb. Catalyst I, obtained without enzyme dilution, showed less activity loss during the cycles. This behavior was found in direct relation to the protein concentration on matrix given in Table 1 for catalysts I, IV, V, and VI. The different resulta for the four immobilization temperature assays (4,24,37,and 43 "C) are shown in Table 1. The values of half-life and fixed protein show the same behavior: they have similar values at 4 and 24 "C, increese at 37 "C, and then decrease at 43 "C. Generally, this immobilization method is used at 25 "C, but Figure IC shows that the largest yield was for the assay at 37 "C (catalystVIII). The action of the small amountof galactose
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Table 1. ImmobillsetionConditions and Features of Obtained 8-QalactoridareChitosanPreparations. im temp bead diam. protein content % fKd [enzyme] (mgof protein/&) cat. protein half-life (h) [glut] (% 1 (OC) (”) ( m g / g of support) galact 4 I 1 35.09 no 3.8 18.64 i 0.8 42.3 49.8 4 35.09 I1 3 3.8 19.41 i 0.0 no 51.9 23.6 4 I11 35.09 5 3.8 21.7 i 0.8 no 58.0 9.4 4 17.55 14.9 i 1.5 Iv 1 no 3.8 30.4 39.8 8.77 4 1 V no 3.8 5.98 i 0.7 12.3 32.0 4.39 4 1 VI no 3.8 2.87 i 0.6 9.3 30.7 35.09 3.8 VI1 1 24 no 18.64 i 0.8 39.2 49.8 35.09 37 VI11 1 no 3.8 19.03 i 0.4 51.5 60.8 35.09 43 Ix 1 no 3.8 17.08 i 0.8 45.6 46.0 35.09 3.8 37 X 1 50.8 19.03 0.4 50.4 yes 35.09 2.2 37 1 20.86 i 0.1 XI 55.7 92.7 yes O [glut] is the glutaraldehyde concentration; [enzyme] is the enz,yme concentration; im temp ie the immobilization temperature; and galact is the galactose presence in the immobilization.
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added to the enzyme solution before immobilization at the conditions that showed the best results is analyzed in Figure Id. It showsthat catalyst X prepared with galactuse improved the activity values, with respect to the values of catalyst VIII, obtained without galactose. The two matrix sizes assayed were 2.2 and 3.8 mm diameter beads. If the support is assumed to have a smooth surface, since ita “structure” is nonporous (Braun, 19891, and the enzyme molecules are fixed only on the carrier surface, the small beads present 36% more surface area than the larger beads for the same weight of support. However, catalyst XI, which was the small size beads, presented a fixed amount of protein only 8.8 % higher than catalyst X, which had large beads. Nevertheless, a significant difference of 42.3 h was found in the half-life values. Table 1showsthat this value of small bead support is roughly 1.84times that of large beads. Figure Id presents more slowly decreasing rates of enzyme activity during the cycles in catalyst XI than in catalyst X. It appears that these differences could indicate that the greater surface area of small beads allows a better distribution of
enzyme molecules on the support surface and a more effective immobilization process. In all of the assays, after the immobilization process, it was found that the beads in chitosan had lost solubility in acetic acid (2.5% (v/v)) at 100 O C . Experiments conductedwith more dilute chitosan solutions (3% (w/v)) produced beads of quite poor mechanical properties and irregular shape. Also,an increment of matrix mechanical resistance was observed with respect to chitosan/triphosphate gel, when the cross-link between biopolymer chains and glutaraldehyde was made. The best activity and stability results for the enzyme were obtained with catalyst XI. Therefore, the conditions used in that immobilization process were chosen to determine, in the following studies, the behavior of fixed enzyme with respect to soluble enzyme at different pH and temperature values and also the activity retention. Figure 2 showsthe pH influenceon the activity of soluble and immobilized enzymes. The optimal range of pH for soluble enzyme is 6.3-6.9. The optimal pH for the pure enzyme determined by Mahoney (1977) waa 6.81. It can
Bbte~hnoI.Rog., 1994, Vol. 10, No. 2
221)
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be seen that the optimal pH of immobilized enzyme changed with respect to soluble enzyme, since it was displacing a t a more alkaline value. The convenient pH range of immobilized enzyme was within pH 6.9-7.5, and the effect of pH changes on the activity was a smaller variation of these values. This behavior is usually explained by suggesting that the alteration of the microenvironment of the enzyme was modified due to immobilization or support. It was detected in several other studies that the pH profile of the catalytic activity shifts for fixed enzymes on polyelectrolytes. Similar effects were found for the immobilization of other enzymes on chitin and chitosan supports (Stanley, 1975;Muzzarelli,1976;Bisset, 1978). The temperature recommended for the soluble employed enzymewas 37 "C; therefore, catalyst XI was proved at 30,37,43,50,and 57 "C,obtaining the results presented in Figure 3. As was expected, in almost all reactions, higher velocities were obtained when the temperature increased, until this behavior in enzymatic catalysts was limited for the protein denaturalization. Table 2 shows, for 30, 37, and 43 "C,the changes in half-lifewith temperature. These differenceswould be produced by the greater initial activity (% hydrolysis), with assay temperature changes and similar activity loss during the three assays. On the other hand, it exhibited a faster activity loss a t 50 "C and very sharp drop at 57 "C that produced a smaller half-life. Moreover, high retention of catalytic capacity could be seen after 183 days for the 37 and 43 "C assays. In Table 3 are given the activity retention values. The relative activity of immobilized enzyme (catalyst XI) with respect to soluble enzyme activity was only 10.7%. This loss of activity could be explained by the denaturing effect of glutaraldehyde.
Conclusions The data presented for enzyme immobilization dilution indicate very similar values of fixed protein, from 17.08 to 20.86 mg/g of support. But differences in half-life were very significant, ranging from 9.4 to 92.7 h. On the basis of these two parameters, the immobilization conditions of catalyst XI were the more appropriate for the support and enzyme employed. Its best working conditions were a pH value similar to that of the soluble enzyme and 43 "C,which was higher than the temperture used for the soluble enzyme. The percent activity retention was very low, but reasonable for the method applied. This immobilization technique allowed us to fix the enzyme with the possibility of holding more than 75 % of the initial activity during 183 days at a half-life of 108.9 h.
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Figure 3. Activity variation of catalyst XI with temperature. Table 2. Catalyst X I Behavior at Different Temperatures temperature (OC) 30 37 43 50 57 half-life (h) 89.8 92.7 108.9 29.1 1.7 5% initial hydrolysis 82.9 93.8 100.0 100.0 96.6 5% hydrolysis at 183 days 62.7 75.3 ~~
Table 3. Calculated Activity for Soluble and Immobilized Enzyme glucose 10-9 Ar enzyme (mol/L) (mol of glucose/mg of protein) 0.0746 1.70 soluble immobilized 0.0568 0.182
Literature Cited Bisset, F.; Sternberg, D. Immobilization of aspergillus BetaGlucosidaseon chitosan. Appl. Env. Microbiol. 1978,36,760. Booklet of IBF-LKB, Ultrogel, Manogeland Trieacryl: Practical guide for use in affinity chromatography and related techniques; Reactifs IBF-Socibth Chimique: Paris, France, 1983. Braun, J.; Le Chanu, P.; Le Goffic, F. The immobilization of penicillin G-acylase on chitosan. Biotechnol. Bioeng. 1989, 33, 242. Gaouth, A.; Arul, J.; Ponnampalan, R.; Bulet M. Chitosan effect on storability and quality on fresh strawberries. J.Food Sci. 6,1618-1620. Gekas, V.; Lopez-Leiva, M. Hydrolysis of lactose: A literature review. Process Biochem. 1985,20,2. Hirano, S.; Miura, 0. Alkaline phosphatase and pepsin immobilized in gels. Biotechnol. Bioeng. 1979,21,711. Knoor, D.Recovery and utilization of chitin and chitosan in food processing waste management. Food Technol. 1991,45,(1) 114-122. Knoor, D.; Miazga, M. Immobilization and permeabilization of cultured plant cells. Food Technol. 1985,39,(10)139. Mahoney, R.; Whitaker, J. Stability and enzymatic properties of 8-Galactosidase from Kluyveromyces fragilis. J. Food Biochem. 1977,1,327. Mahoney, R.; Whitaker, J. Purification and physiochemical properties of @-Galactosidasefrom Kluyveromyces fragilis. J. Food Sci. 1978,43,584. Mammarella, E.; Carrara, C.; Rubiolo, A. Transferencia al medio de la enzima 8-Galactosidasaentrampada en geles con distintm compuestos. Proceedings I V Congreso Latinoamericano de Transferencia de Calor y Materia, La Serena, Chile, 1991,p 360. Muzzarelli, R.; Barontini, G.; Rocchetti, R. Immobilizedenzymes on chitosan columns a-Chymotrypsin and Acid Phosphatase. Biotechnol. Bioeng. 1976,18,1445. Popper, L.; Knorr, D. Combined application of high pressure homogenization and lytic enzymes or chitosan for food stekilization. Food Technol. 1990,44(7),84-89.
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Rojkin, M.; Drappo, G. Fraccionamiento proteico por determinaci6n directa de albbina. Bioquim. Atlbtico 1974,W 6 3 , 1931.
Srere, P.; Uyeda, K. 1: Functional groups on enzymes suitable for the binding to matrices. In Methods in Enzymology: Immobilized Enzymes;AcademicPress: New York, 1976;Vol. 44,pp 11-18. Stanley,W.; Watters, G. Lactase and other enzymes bound to chitinwith glutaraldehyde. Biotechnol. Bioeng. 1975,17,315.
Trinder,P. Enzymaticmethodfordeterminationof glucose.Ann. Clin. Biochem. 1969,6, 24.
Weaat, R.;Melvin,A. CRC Handbook of Chemistry andPhysics, 69th ed.; CRC Press: Boca Raton, FL, 1989;pp D125-127. Accepted November 4,1993.. Abstract published in Adoance ACS Abstracts, December 16, 1993.
