Enzyme immobilization on a low-cost magnetic support: Kinetic studies on immobilized and coimmobilized glucose oxidase and glucoamylase

Enzyme immobilization on a low-cost magnetic support: Kinetic studies on immobilized and coimmobilized glucose oxidase and glucoamylase Bernard R. Pieters and Gilbert Bardeletti Laboratoire de Gdnie Enzymatique, Universitd Claude Bernard-Lyon, Villeurbanne, France

Glucose oxidase (GOx) and glucoamylase (GA) were immobilized and coimmobilized through their carbohydrate moieties onto polyethyleneimine-coated magnetite crosslinked with glutaraldehyde and derivatized with adipic dihydrazide. The carbohydrates were oxidized with sodium periodate, and at optimal concentration, their Vm increased up to 18%for GOx and up to 16%for GA. After immobilization, a remaining activity as high as 88% and 70%for GA with maltose and maltodextrin respectively as substrates was obtained, independently of the particle loading. On the contrary, the remaining activity of GOx strongly decreased at high particle loading. Nevertheless, half of its initial activity was recovered at low loading and was not significantly affected when GA was coimmobilized by saturating the reactive groups left on the particle. The V,, of both immobil&ed enzymes was improved by crosslinking their carbohydrates with adipic dihydrazide, a treatment which allows further coimmobilization of the other enzyme on a second layer.

Keywords:Magnetite;glucoseoxidase;glucoamylase;glycoenzymesoxidation,crosslinking,immobilization,coimmobilization Introduction Immobilization of glycoenzymes onto particles such as glucoamylase and glucose oxidase is an important tool in the sugar industry. Recovery and stabilization of the enzyme and reduction of the reactor size are the principal advantages. Among the reactors suitable for processing immobilized enzymes, the fluidized bed reactor has been proven optimal. ~However, the economic viability of this technique is often compromised by the low remaining activity of the enzyme after immobilization and by the cost of the carrier and of the immobilization procedure. To compensate for the loss in activity, microparticles are suitable to give a high enzyme activity per particle volume, but their density is often too low to compensate for the high drag force of highly viscous liquors such as those encountered in the sugar industry, leading to their washout from the reactor.

Address reprint requests to Dr. Bardeletti at Laboratoire de G6nie Enzymatique, UMR 106-CNRS, Universit6ClaudeBernard-Lyon 1 (B~t. 308-ESCIL),43 Boulevarddu 11 Novembre 1918, 69622, Villeurbanne Cedex, France Received 8 July 1991; revised 19 November 1991

©

1992Butterworth-Heinemann

This problem can be circumvented by the use of magnetic supports, which exhibit high settling rates due to the high density of the magnetite entrapped in their core (5.1 g cm 3). Magnetic particles have the additional advantage of being easily and gently separated by simple application of a magnetic field, which facilitates their manufacture, the immobilization procedure, and their handling in the reaction phase. Magnetic separation prevents the loss in enzyme activity encountered with classical separation techniques such as centrifugation or filtration, damaging their weak tridimensional structure by particle compaction. They are particularly attractive in highly viscous sugar liquors, because they still can be easily separated without entrapping undesirable colloidal matter that is generally present. These advantages may considerably reduce the capital and operational costs of the whole process. In the perspective of designing a fluidized bed containing enzymes, covalently immobilized on a nonrecyclable magnetic particle, and producing low-value products such as sugar derivatives, the challenge is to succeed in tailoring a low-cost magnetic microparticle having good mechanical and surface properties and high re-

Enzyme Microb. Technol., 1992, vol. 14, May

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Papers sidual activity. Recent researches have significantly improved the conversion in such a reactor by surrounding the column with electromagnets which stabilize and compact the particle bed. 2 Back-mixing and channeling effects are substantially reduced in the reactor, which consequently tends to behave in a plugflow fashion. Higher particle loading is allowed, which increases the enzyme activity per reactor volume. Magnetic particles have been successfully used in many important biotechnological fields, such as wastewater treatment, enzyme immobilization, affinity separation, cell sorting, immunoassays, and drug delivery. 3,4 Recently, comprehensive and critical reviews have been written on Magnetic Carrier Technology, which give to the end user some fundamental and practical information concerning magnetic separation techniques, magnetic particles, and their appropriate applications. 3-5 Since they are cheap, small, and easy to manufacture, particles made of polymer-coated magnetite are the most appropriate for enzyme immobilization, although they all are of irregular shape and size. Three attractive manufacturing methods have been developed and recently improved.4 Silanization of crushed magnetite, 6 synthetic magnetite, 7 or biomagnetite 7 with aminopropyltriethoxysilane further derivatized with glutaraldehyde has been widely used but does not yield a highly stable silane coating. Particles made by coprecipitation of dicarboxypolyethyleneglycol with Fe3+/Fe 2+ ions at basic pH have been reported successful for lipase immobilization. ~ However, these particles of around 100 nm in diameter require a very high field for separation, expensive derivatization reagents, 8 and may be subjected to stability problems. 9 Polyamine coating of magnetite further crosslinked with glutaraldehyde was first attempted with albumin, ]°,jj a polymer far too expensive for enzyme immobilization, but this technique has recently been promoted by Dekker for the immobilization of lactase using polyethyleneimine, which yields extremely cheap particles. 12.~3 However, direct coupling on this particle gave a remaining enzyme activity of only 20%, j2 possibly due to coupling of the active site and multisite attachment on this highly reactive carrier. Moreover, lactase being a glycoenzyme, direct coupling is difficult to achieve since the amino acid side chains are shielded by the carbohydrate residues. Because this particle seemed to be economically the most attractive, the purpose of this work was to improve the system for two other glycoenzymes (glucose oxidase and glucoamylase) by introducing a diamine as spacer. This reduces multisite attachment and steric hindrance of the enzyme and diffusional limitation of the substrate. Immobilization through their carbohydrates previously oxidized with sodium periodate ~4-19 prevents coupling through their respective active sites. Both enzymes were coimmobilized onto the particle, together in a monolayer and separately in a bilayer. The second layer was immobilized after crosslinking of the first layer with adipic dihydrazide, a treatment

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which has recently been found to stabilize glycoenzymes.2° The kinetic studies have been carried out with biosensors, a fast and reliable analytical technique particularly appropriate for immobilized enzymes. The sensors directly and continuously measure the evolution of the product concentration in the reaction phase.2J,22

Materials and methods

Chemicals Glucoamylase (GA) E C 3.2.1.3, 6 U mg -L, (25°C; glycogen as substrate) and glucose oxidase (GOx) E C 1.1.3.4, 250 U mg -~ (25°C; and glucose as substrate (grade I) from Aspergillus niger) were purchased from Boehringer. o(+)-Giucose was provided by Prolabo; maltose monohydrate for biochemistry, soluble starch MW 5,000, glutaraldehyde (GAD), and sodium periodate by Merck; magnetite by Alfa Products; polyethyleneimine (PEI) MW 30,000-40,000 by Serva; ethylenediamine (EDA) and hexamethylenediamine (HMDA) by Fluka; and adipic dihydrazide (ADH) by Aldrich. Maltodextrin MDO2 was a gift from Roquette Fr6res S.A. (France).

Particle manufacture The procedure for particle manufacture is based on the work of Sato ~j and Dekker. j2 One gram of magnetite was sonicated 10 rain in 10 ml of a 5% PEI solution pH 8.5 at room temperature. After removal of the supernatant, the PEI-coated magnetite was resuspended in 10 ml of 0.2 M borate buffer pH 8.5 containing 5% GAD. The suspension was sonicated 5 rain and kept under fast stirring for 1 h. Particles were washed twice with water and stored at 4°C in a 50 mM acetate buffer pH 5.5 containing 0.5% GAD at a final concentration of 100 mg magnetite ml -~. This microparticle will be referred to as M-GAD, and its weight as the mass of magnetite entrapped in the core.

Spacer derivatization One hundred milligrams of M-GAD was suspended in 10 ml of a 0.2 M acetate buffer pH 5.5 and the spacer was added under fast stirring at a final concentration of 50 mM. After 1 h, the derivatized particles were washed twice with water and stored at 4°C in a 50 mM acetate buffer pH 5.5 at a final concentration of 20 mg ml -l. ADH, EDA, and HMDA were used as spacers and the particles will be respectively referred to as M-ADH, M-EDA, and M-HMDA.

Electron micrographs Particle micrographs were taken with a scanning electron microscope HITACHI $800 at the Centre de Microscopie Electronique de l'Universit6 Lyon 1. The samples were previously metallized with a gold-palladium alloy.

Enzyme immobilization on a low-cost magnetic support: B. R. Pieters and G. Bardeletti

Enzyme immobil&ation A 5-ml test tube containing 1 ml of enzyme solution in 50 mM acetate buffer and 5 mg of magnetic particles was agitated 2 h at 4°C on a rollermixer. After removal of the supernatant, the particles were washed twice at 4°C with 1 ml KCI 1 M in 0.2 M acetate buffer, I h and 20 min respectively. The loading (L) (mg/g), referred to as the weight of enzymes immobilized per gram of particles, was calculated by mass balance and the protein concentration in each fraction was measured with the BIORAD protein assay. The maximal loading capacity (MLC) of the particle was determined by immobilizing enzyme in excess.

Glycoenzyme oxidation GA and GOx were oxidized with periodate according to the procedure of Marek in 50 mM acetate buffer at pH 5 and 5.5, respectively. 17.18Increasing volumes of a 50 mM periodate solution, from 20 to 320 /~1, were added to 1 ml of 1 mg m1-1 enzyme solutions. These solutions were adjusted to 1.5 ml with the same buffer and reacted on a rollermixer 6 h at 4°C in the dark. Periodate was removed by passing the solution through PD-10 Sephadex columns (Pharmacia). For clarity, the oxidized enzymes will be designated by their abbreviation followed by the volume of periodate solution added (e.g. GA120 means l ml o f a 1 mg ml -j glucoamylase solution treated with 120/zl of a 50 mM periodate solution).

Carbohydrate crosslinking of glycoenzymes Immoblized GA and GOx were crosslinked according to the method of Kozulic. 2° Five milligrams of M-ADH-GA or M-ADH-GOx were gently agitated in 2 ml of 50 mM acetate buffer pH 5 at 4°C, and ADH was added at a final concentration of 12.5 mM. The crosslinking reaction was stopped after 1 h by magnetic removal of the particles.

Enzyme coimmobil&ation Monolayer method. GOx20 and GA120 were successively immobilized onto M-ADH with an intermediate washing of I h. The general procedure is similar to that reported in Enzyme immobilization.

Bilayer method. At first M-ADH was saturated with GA120. After the crosslinking of this first enzyme layer with ADH according to the procedure reported in Glycoenzyme crosslinking, GOx20 was immobilized, so forming a bilayer coimmobilized system.

Enzyme activity measurement with biosensor 21,22 The biosensor system consisted of a Gluc 1 electrode connected to a polarograph type PRGE manufactured by Solea-Tacussel (France), and the anodic current was recorded by a Sefram-Servotrace. The activity

test is based on the detection of H202, and for this purpose, the potential of the platinum anode was fixed at +650mV vs. a Ag/AgC1 reference electrode. For the GOx test using glucose as substrate, the H202 stoichiometrically produced with gluconate is representative of the enzyme activity. A disk cut from a dialysis membrane Spectrapor, MW cutoff 6,0008,000, was tightly pressed on the platinum tip of the transducer with a screw cap to allow selective permeation of H202. For the GA test, the dialysis membrane was replaced by a preactivated polyamide membrane PallImmunodyne of 0.45 /~m in pore diameter on which GOx was previously immobilized. 23 The glucose produced in the reaction phase by the GA diffuses through the membrane where it is stoichiometrically converted into gluconate and H202, the latter being detected by the sensor. Maltose was used as substrate for the kinetic studies on soluble and immobilized GA, and maltodextrin for the coimmobilized system. The tests were performed in a small reactor thermostated at 30°C and filled with 10 ml of a reaction medium containing 0.2 M acetate buffer and 0.1 M KCI, which was agitated magnetically for the soluble enzymes and by an overhead stirrer for the immobilized enzymes. Tests with GOx were performed on dissolved oxygen. The biosensor system measures the enzyme activity in terms of units per milliliter of reaction medium (U ml -j =/~mol rain -j ml J). For soluble enzymes, the division of this activity by the protein concentration in the medium gives the catalysis velocity V or the specific enzyme activity in units per milligram protein (U mg-b. For immobilized enzymes, its division by the particle concentration gives the enzyme activity reported to the particle weight V' (U g 1), and their specific activity V (U mg ~) is calculated by dividing V' by the loading L of the enzyme on the particle (rag g i). Since GOx and GA fit with Michaelis-Menten kinetics, the kinetic constants Krn (raM) and Vm (U mg ~) were calculated from the Lineweaver-Burk plot. For the immobilized enzymes, they always refer to the apparent constants. The remaining activities after oxidation and immobilization were respectively referred to as the ratios of the Vm of the oxidized (Vm0 and immobilized (Vm2) enzyme to that of the native enzyme

(Vm0). At first the objective was to carry out kinetic studies only on GOx, which were performed at pH 5.5 and 30°C, its optimal conditions. 24 Because the particle was found attractive in the perspective of a coimmobilized system, kinetic tests with GA were performed at 30°C and pH 5 as recommended by Cho and Bailey. 24

Results and discussion

Particle characterization Figure 1A and B shows an electron micrograph of a population of M-ADH particles. As for silanized magnetite, 7 they have an irregular shape and size due to the aggregation of synthetic magnetite crystals of

Enzyme Microb. Technol., 1992, vol. 14, May

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Papers accessibility of the substrate to the active site. The decrease in activity observed at higher periodate additions may be due to the oxidation of some amino acids normally masked by the carbohydrates 15or to the possible breakage of carbohydrate residues. Glycoenzymes are indeed stabilized by their carbohydrates, 26 and their thermal stability increases with an increase in their degree of glycosylation.27 Recently, the carbohydrate moieties have been enzymatically oxidized with galactose oxidase rather than chemically with periodate, but this procedure is more complicated and expensive to operate on a large scale. 28,29 We have pursued the study by finding out the influence of periodate concentration on the kinetic constants to see if the GOx exhibits an optimal activity at a definite periodate concentration for different substrate concentrations. The results were compared with those obtained with the GA. Figure 2 shows the influence of the volume of periodate added on the Km and Vm of GOx and GA. The highest Vm appears at 40 /A for GOx, which agrees with the results of Marek, 17but 120 ~1 are required for GA. Compared to the Vm of the native enzyme, it means an increase of 18% and 16%, respectively. GA is also more sensitive than GOx to periodate, since its Vm may decrease below that of the native enzyme. What is particularly surprising is that at its highest Vm the oxidized enzyme exhibits its highest Km, which

35

1.0 Figure I

Electron micrographs representing (A) a population of polyethyleneimine-coated particles crossslinked with glutaraldehyde and derivatized with adipic dihydrazide, and (B) a particle containing magnetite microcrystals of about 10 nm in diameter

about 500 nm in diameter during the coating step, which occurred despite the ultrasonic treatment and the high-speed stirring. Particle size ranges from 5 to 20/~m. Figure 1B illustrates this aggregation phenomenon of microcrystals, which is possibly enhanced by particle crosslinking during the derivatization step with the spacer. Nevertheless, these particles are solid and small enough to exhibit good mechanical properties, and their immobilization capacity is increased by their highly macroporous surface. If required, ultrafine and large particle clumps can be easily separated by magnetic filtration in High Gradient Magnetic Separator 25 or in a fluidized bed by washout and settling, respectively.

Kinetics o f soluble GOx and GA oxidized with periodate An increase in GOx activity after oxidation of its carbohydrates was observed by Zaborsky and Ogletree.15 Marek et al. optimized the periodate concentration and found an optimal activity for GOx4017 and a similar result for invertase. 18 They assumed that partial destruction of the carbohydrate moieties facilitates the

364

Enzyme Microb. Technol., 1992, vol. 14, May

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Enzyme immobilization on a low-cost magnetic support: B. R. Pieters and G. Bardeletti 210

active site of the oxidized GA compared to that of the native one.

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Table 1 Influence of substrates on the kinetic constants of GA and GA120 K,~ (raM)

Maltose Maltodextrin Soluble starch

Vm (U mg -1)

GA

GA120

GA

GA120

0.60 0.15 0.30

0.98 0.25 0.42

3.75 20.80 24.40

4.2 22.2 25.0

Optimization of the spacer. Marek et al. 17immobilized oxidized GOx on macroporous glycidyl metacrylate particles by direct coupling and through their carbohydrate moieties after derivatization of the particle with EDA and HMDA. They obtained better activities with the latter system, 38% higher with EDA and 40% with HMDA. However, the amine bound between these spacers and the oxidized enzyme is relatively unstable and its reduction with sodium borohydride is necessary. The addition of this step in the procedure and the expensive cost of this reagent is economically disadvantageous. O'Shannessy and Hoffman 19 have recently circumvented this problem by using ADH as a spacer. ADH is attractive because of its length and its greater hydrophilicity than HMDA. It forms a stable hydrazone bound with oxidized glycoenzymes, which does not require further reduction with borohydride. This system has also been reported efficient for galacrose oxidase. 2s.29 The optimization tests were performed with GOx40 and GA80 immobilized on M-GAD previously derivatized with the three spacers at their maximal loading capacity. Figure 4 shows that ADH gives the highest remaining activity for GA80, the length being less critical for GOx when compared with EDA due to the better accessibility of a smaller substrate. However, HMDA does not give better results than EDA, probably due to the high hydrophobicity of this spacer which provokes particle flocculation. All the following experiments were carried out with M-ADH. pH optimum. The pH optima of GA and GOx are 4.5 and 5.5, respectively, z4 As reported in Figure 5, their

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means that the greater the substrate accessibility to the active site, the lower its affinity for the substrate. It is nevertheless dangerous to generalize this observation for all the glycoenzymes, since Marek ~8found a Km for the oxidized invertase lower than that of the native one. Figure 3 shows that at different substrate concentrations, the optimal volume of periodate can slightly change, and that the differences in activity are more pronounced at very high substrate concentrations. The kinetic constants of GA and GA120 for different substrates are reported in Table 1. The remaining activities of GA120 are respectively 112%, 107%, and 102.5% with maltose, maltodextrin, and soluble starch as substrates. We conclude that the shorter the chain of the sugar polymer, the greater its accessibility to the

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Enzyme Microb. Technol., 1992, vol. 14, May

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oxidation and their immobilization onto M-ADH does not entail pH shift. After immobilization, no broadening pH optimum was observed, and the activity of M-ADH-GA120 even decreased above its optimal pH.

Enzyme Microb. Technol., 1992, vol. 14, May

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Effect of enzyme loading. A maximal loading capacity (MLC) of 20 and 26 mg g i has been found for GOx and GA immobilized onto M-ADH. Such a difference was foreseeable, since GOx has a higher molecular weight than GA (186,000 vs. 70,000). Figure £ shows enzyme activity variations after immobilization V (U mg -j) and of the enzyme activity reported to the particle weight V' (U g-J) in relation to the loading of the particle L (rag g ]). For M-ADHGOx80 (Figure 9A), V decreases when the loading increases and V' rapidly reaches its maximal value, which means that GOx80 is subjected to steric hin-

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Optimization of the periodate concentration. GOx and GA oxidized with increasing volumes of periodate were immobilized onto M-ADH below their MLC, at 8 and 15 mg g-J respectively, for reasons explained in the next paragraph. As shown in Figure 6A and B, their kinetic constants exhibit a similar evolution as those found for the soluble enzyme with a broader Vm optimum for GOx. Km values slightly increase for GA but drastically decrease for GOx, which means that immobilized GOx has a higher affinity for glucose than soluble enzyme. The lower K,~ reported for GOxS0 makes this enzyme more attractive than GOx40, since they exhibit a similar Vm after immobilization. The best remaining activities were 29% for GOx80 and 88% for GA120 (Figure 7). Figure 8 shows that the optimal volume of periodate varies only at low substrate concentrations. The kinetic constants of immobilized GA120 for different substrates are reported in Table 2. The remaining activities of M-ADH-GA120 are respectively 88%, 70%, and 55% with maltose, maltodextrin, and soluble starch as substrates.

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Enzyme immobilization on a low-cost magnetic support: B. R. Pieters and G. Bardeletti through their carbohydrates onto M - A D H are compared in Table 3. The second method is particularly attractive for GA, since it increases its remaining activity from 43% to 88%, although that of GOx is improved from 22% to 29%. Both procedures drastically decrease the Km of GOx, whereas that of GA also decreases after direct coupling but increases after immobilization through carbohydrate groups. Another major advantage of enzyme immobilization through their carbohydrates is that the enzyme does not significantly adsorb onto the particle when added

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Vm (U mg ~)

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drance. This phenomenon is not observed for M-ADH-GA120 (Figure 9B), where V is constant and V' increases linearly, a steric effect probably due to the larger size of GOx. Therefore, as shown in Figure 9A, its immobilization should be undertaken at a maximal loading of 8 mg g J, where V' starts decreasing drastically. This loading was chosen for the previous experiments, but it must be noticed that a loading of 2 mg g-~ increases the remaining activity from 30% to 50%.

Comparison of the kinetic constants of GOx and GA immobilized by direct coupling or through their carbohydrates The kinetic constants of native GOx and GA immobilized through their amino groups by direct coupling onto M-GAD and of GOx80 and GA120 immobilized

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Table 3 Comparison of the kinetic constants of GOx and GA immobilized through their amino groups by direct coupling on M-GDA or through their carbohydrates on M-ADH. Their loading is 8 and 15 mg g 1, respectively, and maltose is used as substrate for GA

GOx native M-GDA-GOx M-ADH-GOx80 GA native M-GDA-GA M-ADH-GA120

Km (mM)

Vm (U mg 1)

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178.0 39.0 52.5 3.75 1.60 3.30

Enzyme Microb. Technol., 1992, vol. 14, May

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Papers below the MLC. This avoids e n z y m e loss in the supernatant and in the washing solutions, and is probably due to the highly reactive surface properties of these enzymes.

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It has been reported above that GOx immobilized onto M-ADH suffers from steric hindrance when the particle loading increases, especially above 8 mg g-J which is less than the half the MLC. Therefore it was interesting to see the possibility of coimmobilizing GA on the reactive groups left on the particle (enzymes working in sequence) on condition that it does not decrease the Vm of GOx. This system has previously reported on permeable membrane for product compartmentalization study. 3° Here in this coimmobilized system GOx is working at very low substrate concentration; another advantage is that the e n z y m e exhibits a higher remaining activity than the soluble one due to its lower Km. Based upon practical experience, GOx20 was selected for its lowest Km (Figure 6A) and maitodextrin was chosen as substrate. Both enzymes were coimmobilized onto the particle, together in a monolayer and separately in a bilayer. Monolayer immobilization. GOx20 was at first immo-

bilized onto M - A D H at different ioadings and the reactive groups left on the particle were saturated with GA120. As reported in Table 4, a loading of 4 mg g-i seems optimal in terms of remaining activity. Table 5 shows the kinetic constants of GOx20 at the same loading without coimmobilized GA120 and those obtained earlier for soluble and immobilized GOx20. It can be seen from both tables that the saturation of the reactive sites with GA120 only slightly decreases the Vm of GOx20 and also significantly decreases its Kin. It is also interesting to notice that when the loading of

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5 Influence of the loading on the kinetic constants of GOx20 immobilized on M-ADH compared to those of the soluble enzyme

Table

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Enzyme Microb. Technol., 1992, vol. 14, May

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0.2 0.1

0.0

,

I

,

1 1/Glucose

Table

(9

3

I

2 (mM "1)

Figure 11 Lineweaver-Burk plots for GOx20 coimmobilized with GA120 onto M-ADH with glucose ([i]) and maltodextrin (A) as initial substrates. Experimental conditions were as indicated in Figure 10

GOx increases, not only the V m decreases, as deduced from Figure 9A, but also the Kin, which means that the GOx affinity for the substrate increases when the enzyme proximity increases. The kinetics of GOx20 coimmobilized onto M-ADH with GA120 are shown in Figure 10. The initial maltodextrin concentration was 9 g 1-~, and every time a sample was taken in the reactor the glucose concentration was measured and related to the GOx activity calculated at this time. The specific activity of GOx20 increases rapidly but progressively tends to a maximum. Figure 11 shows the L i n e w e a v e r - B u r k plots of the coimmobilized GOx20 with glucose and maltodextrin as initial substrates. The deviation from linearity obtained with maltodextrin means that the glucose

Enzyme immobilization on a low-cost magnetic support: B. R. Pieters and G. Bardeletti concentration measured in the reactor is not representative of the higher glucose concentration present in the microenvironment of the particle due to the direct conversion by the GOx of the glucose produced by the GA. However, after 30 rain this deviation tends to rejoin the linear Lineweaver-Burk plot obtained with glucose as initial substrate, which means that the glucose gradient between the micro- and the macroenvironment tends to disappear. This monolayer system possibly reaches its equilibrium point at the connection of both curves, but problems of dissolved oxygen did not allow us to carry on the kinetic study further in order to prove this hypothesis.

Bilayer immobilization. With the aim of increasing this microenvironment effect, GOx20 was immobilized on M-ADH previously saturated with GA120. Both enzymes were coupled through their respective oxidized carbohydrates after derivatization of the first layer with ADH. The idea was that in such a bilayer system the glucose produced on the first layer would inevitably come into contact with GOx forming the second layer before diffusing in the reaction phase. Moreover, this system was expected to reduce the steric hindrance of the GOx due to the greater length of the spacer. Despite the experimental interest of this bilayer system, the Vm of GOx20 was not improved (Table 6).

Effect of carbohydrate crosslinking after immobilization. Kozulic et al. 2° recently reported that crosslinking of oxidized glycoenzymes with ADH increases their stability. Table 7 compares the kinetic constants of GOx20 and GA120 immobilized onto M-ADH with or without posttreatment with ADH. In both cases the Km and Vm values increase. The enzyme activity, calculated from these constants, is improved by this

Table 6 Comparison of the kinetic constants of GOx20 coimmobilized with GA120 on M-ADH according to the monolayer and the bilayer system (LGOx20 = 4 m g gM1)

Monolayer Bilayer

Kr. (mM)

Vm (U mg 1)

12.6 13.6

55.5 52.5

Table 7 Comparison of the kinetic constants of GOx20 coimmobilized with GA120 and of immobilized GA120 without or with posttreatment with ADH

treatment if the glucose concentration is higher than 16 mM for GOx20 and the maltose concentration higher than 5 mM for GA120. Stability tests are still underway, but these enzymes do not seem to be stabilized. This is probably due to the low concentration of periodate or the high concentration of ADH used, which yields derivatized, rather than crosslinked, enzyme molecules. Optimization of both parameters should be carried out.

Conclusion Immobilization of glycoenzymes through their carbohydrates onto PEI-coated magnetite crosslinked with GAD and derivatized with ADH has been successful for GOx and GA. The method yields higher residual activities than those found after direct coupling onto the crosslinked particle, especially for GA. This system has the following major advantages: 1. The particles are cheap and easy to handle, exhibit good mechanical and surface properties, and can be rapidly separated in highly viscous media by simply applying a magnetic field despite their micronic size. 2. The efficiency of the separation technique allows complete recovery of the particles. No enzyme activity has been detected after magnetic separation of the particles from the reaction phase. 3. The oxidation of the carbohydrate moieties of these glycoenzymes increases their Vm at a defined periodate concentration varying for each enzyme. 4. The ADH used as spacer gives higher residual enzyme activity after immobilization and more stable bonds than EDA or HMDA. 5. The immobilization of glycoenzymes through their carbohydrates prevents binding of their active site, multisite attachment, and their adsorption onto the particle due to high reactivity. The particle loading does not affect the kinetic constants of the glycoenzyme in a similar way. Those of GA are left unchanged, whereas those of GOx start decreasing significantly above a third of the maximal loading capacity. The immobilization of GA on the reactive groups left on the particle has been optimized and does not significantly affect the kinetic constants of GOx. Finally, the crosslinking of the oxidized carbohydrates left after immobilization with ADH has been found to increase the enzyme activity and to allow the immobilization of a second layer of glycoenzymes. Since these magnetic particles are particularly attractive for GA, their use in a fluidized bed reactor for the hydrolysis of maltodextrin is achieved. 31

Acknowledgments M-ADH-GOx20/GA120 M-ADH-GOx20/GA120 + ADH M-ADH-GA120 M-ADH-GA120 + ADH

Km(mM)

Vm(U mg -1)

12.60 14.40 0.72 0.92

55.5 59.0 14.5 15.0

The authors thank the European Communities which have financed this research within the Bridge program and Mr. P. R. Coulet, Head of the Laboratoire de G6nie Enzymatique, who has kindly welcomed the grantholder, Mr. B. R. Pieters, and has provided him facilities to pursue his research. E n z y m e M i c r o b . T e c h n o l . , 1992, v o l . 14, M a y

369

Papers Abbreviations adipic dihydrazide ethylenediamine EDA glucoamylase GA glutaraldehyde GAD glucose oxidase GOx HMDA hexamethylenediamine loading (mg g-I) L maximal loading capacity (mg g J) MLC polyethyleneimine-coated magnetite M M derivatized with (+ name of the spacer) Mpolyethyleneimine PEI specific enzyme activity (U mg -I) V enzyme activity relative to the particle weight V' ( U g J) Vm of the native enzyme (U mg -~) Vrn0 Vm of the oxidized enzyme (U mg ~) Vml Vm of the immobilized enzyme (U mg -~) Vm2

ADH

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