NADH recycling by coimmobilized lactate dehydrogenase and glutamate dehydrogenase

NAD1/NADH recycling by coimmobilized lactate dehydrogenase and glutamate dehydrogenase Min Le and Gary E. Means Department of Biochemistry, Ohio State University, Columbus, OH U.S.A. Most biologically active proteins bind irreversibly to Amberlite XAD-7 in dilute buffer at approximately neutral pH. Small amounts of protein (i.e., ;4% w/w or less) are adsorbed at the periphery of the porous, spherical, beads where the protein is relatively accessible to external solvent and solvent components. Additional amounts of the same or different proteins up to ;35% (w/w) absorb to deeper layers of the same beads. The order in which two or more proteins are adsorbed can be used to arrange them in a series of concentric layers on Amberlite XAD-7. An NAD1/NADH recycling system for the synthesis of L-glutamate from ammonia, a-ketoglutarate, and lactate was prepared by coimmobilizing rabbit muscle lactate dehydrogenase (LDH) and bovine liver glutamate dehydrogenase (GDH) in different amounts and in different arrangements on Amberlite XAD-7. The overall efficiency of that immobilized system based on the rates of glutamate production varied with both the ratio of the two enzymes and their arrangement on the polymer beads. Those variations suggest that NADH formation was rate limiting under most conditions and demonstrated the importance of enzyme proximity. The immobilized two-enzyme system, reflecting the pH dependence of its constituent enzymes, had optimal activity at pH ;8.5 and retained up to 75% of its activity after one week of repeated use. © 1998 Elsevier Science Inc. Keywords: Multienzyme coimmobilization; controlled enzyme distribution; NAD1/NADH recycling

Introduction Many metabolic reactions in cells are catalyzed by multienzyme complexes. The product of one enzyme is the substrate of the next in such complexes and is channeled directly from one to the other without diffusion from the complex.1,2 For reactions requiring coenzymes such as NAD1/NADH, NADP1/NADPH, etc., the two forms of the coenzyme are continuously regenerated by the various enzymes so as to maintain the metabolic cycles. Systems with multiple enzymes coimmobilized on the same support have been designed based on the assumption that the advantages associated with natural existing multienzyme complexes, i.e., close enzyme proximity, short substrate transient times, and efficient coenzyme recycling will also apply to those immobilized systems.3 Immobilized multienzyme systems have been studied as models of cellular and organellar compartmentalization.4

Address reprint requests to Dr. G. E. Means, Ohio State University, Department of Biochemistry, 484 West 12th Avenue, Columbus, OH 43210. Received 28 July 1997; revised 2 January 1998; accepted 14 January 1998.

Enzyme and Microbial Technology 23:49 –57, 1998 © 1998 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010

They have been used for the multistep syntheses of biologically important molecules such as ATP and creatine phosphate5 and for recycling coenzymes such as NAD1/ NADH.6 Chemiluminometric sensors for a wide variety of biologically important molecules have also been developed utilizing immobilized multienzyme systems.7 While immobilized enzymes are used for many purposes, multienzyme systems are still largely in the laboratory development stage. In addition to the problems usually encountered with the immobilization of a single enzyme, there are several additional problems to be considered in the case of multiple enzymes. The amounts and ratios of the individual enzymes in a multienzyme system, for example, can be varied and should ideally be adjusted so as to achieve an optimal overall rate. To minimize transient times, enzymes catalyzing successive steps in such a process should also be arranged so as to be in close proximity to each other. These goals are difficult to achieve due to the relatively low and unpredictable efficiencies of most procedures for the immobilization of enzymes. Amberlite XAD-7, a nonionic, macroreticular, polyacrylate bead product, is commonly used to adsorb, concentrate, and remove small, relatively polar organic molecules from air, water, and biological samples,8 –10 but has also been

0141-0229/98/$19.00 PII S0141-0229(98)00010-6

Papers used to adsorb proteins11–14 and, in some cases, as a support for enzymes.12,15–20 Previous studies have shown it to adsorb large amounts of proteins (i.e., up to ;35 w/w%).20 With small amounts of protein (less than ;4 w/w%), the adsorption appears to be confined to a thin shell at or near the periphery of the porous beads.13,17,19 On the basis of these properties, we thought Amberlite XAD-7 might be an effective support for multienzyme systems. To demonstrate whether this might be the case, we first examined the adsorption behavior of 13 native proteins with large differences in their molecular weights and isoelectric points. We then adsorbed two proteins, cytochrome c and egg white lysozyme, in different orders and showed that the two proteins were immobilized in concentric layers under such conditions. Furthermore, the arrangement of the layers depended on their order of adsorption. We finally coimmobilized lactate dehydrogenase and glutamate dehydrogenase in various amounts and arrangements and determined their ability to recycle NAD1/NADH by following the formation of glutamate from a-ketoglutarate, ammonia, and lactate.

Materials and methods

Figure 1 Effect of enzyme arrangement on the efficiency of glutamate formation. Different arrangements of LDH and GDH were achieved by manipulating the sequence of adsorption as described in MATERIALS AND METHODS. The diagrams on the left show schematically the relative position of the enzyme and BSA layers on the beads. The thickness of the layers in the diagrams do not reflect the actual thickness of the layers in the beads. The results presented, based on the amount of glutamate produced in 2 h, were the average of three separate experiments and were normalized by setting the highest value to 100%

Materials Amberlite XAD-7 (wet mesh size 20 – 60, specific surface area 450 m2/g, average pore diameter ;90 Å) was obtained from Sigma Chemical Company (St. Louis, MO) and was washed two or three times with methanol, dried, and fractionated by passage through 180 and 250 mm sieves. The sieved beads were then washed for at least 8 h with refluxing methanol in a Soxhlet extractor and again dried. Chicken egg white lysozyme, cytochrome c, human serum albumin (HSA), bovine serum albumin (BSA) (fraction V), trypsin, equine myoglobin, horseradish peroxidase, yeast alcohol dehydrogenase, Crotalus adamanteus amino acid oxidase, porcine pepsinogen, Aspelgillus niger glucose oxidase, l-lactic dehydrogenase (LDH; EC 1.1.1.27, type XI from rabbit muscle), lglutamic dehydrogenase (GDH; EC 1.4.1:3, type III form bovine liver), cytochrome c, NAD1, NADH, a-ketoglutarate, pyruvate, phenylisothiocyanate, and picrylsulfonic acid were obtained from Sigma. dl-Lactic acid was from Fisher Scientific (Springfield, NY).

Adsorption and desorption The dried XAD-7 (100 mg in 8-ml screw cap vials) was soaked in 20 mm phosphate buffer pH 7 for at least 30 min and drained just prior to protein adsorption. Solutions of each protein (5 ml of 0.2 mg ml21 in 20 mm phosphate buffer pH 7) were added to the vials, sealed, and gently mixed by slow, continuous end-over-end rotation (;20 rpm) at 23°C. At various times, 40 ml aliquots of the solution were removed for determination of the protein concentration using a slightly modified BCA procedure.21 Protein concentrations, expressed as a percentage of the initial concentration, were then plotted versus incubation time and adsorption rates were determined from the initial slopes. Samples of Amberlite XAD-7 (100 mg) with 1% (w/w) of each absorbed protein were washed three times with 20 mm phosphate buffer pH 7 and drained. Another 2 ml of buffer was added and the vials were sealed and rotated at ;20 rpm and ;23°C. At various time intervals, samples of the supernatant were removed and their protein concentrations were determined as described above.

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Coimmobilization of lysozyme and cytochrome c Lysozyme and cytochrome c were adsorbed to 100 mg samples of presoaked Amberlite XAD-7 as follows. Lysozyme (10 mg in 5 ml of phosphate buffer pH 7) was added to one vial and gently mixed by slow, continuous end-over-end rotation (;20 rpm) at 23°C for 24 h. Under those conditions, lysozyme adsorbs rapidly (i.e., t 1/2 ; 10 min) and completely to the polymer beads. The resulting lysozyme-loaded beads were then washed with the buffer and drained. A solution containing 1 mg of cytochrome c in 5 ml of the same buffer was then added and the beads were again incubated for 24 h with slow rotation. In a second vial, 1 mg of cytochrome c was adsorbed by the same procedure to another 100 mg sample of Amberlite XAD-7 followed by 10 mg lysozyme. The two samples of lysozyme-cytochrome c beads were rinsed with buffer, drained, sliced with a razor blade on a glass slide, and air dried at room temperature. Photographs of the sliced and intact beads were taken using an Olympus C-35AD-4 camera attached to an Olympus SZH-ILLD inverted microscope.

Coimmobilization of LDH and GDH Solutions of LDH and GDH (1–3 mg each in 5 ml of 20 mm phosphate buffer containing 0.5 mm NAD1 pH 7) were added to a 100 mg sample of presoaked Amberlite XAD-7 beads and gently mixed by slow end-over-end rotation. At various times, 40 ml aliquots of the solution were removed to determine the protein concentration using a slightly modified BCA procedure.21 After complete adsorption of the enzyme usually after about 6 h the beads were rinsed several times with buffer and the other enzyme or BSA (1–3 mg in the same buffer) was added and allowed to adsorb for an additional 6 –12 h. In some cases, LDH was adsorbed first followed by GDH. In other cases, the order was reversed or BSA was adsorbed after the first and before the second enzyme, thereby creating a BSA layer between the two enzymes as illustrated in Figure 1.

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NAD1/NADH recycling by LDH/GDH-amberlite XAD-7:M. Le and G. E. Means Table 1 Adsorption of proteins to Amberlite XAD-7

Proteins Lysozyme Trypsin Cytochrome c Horseradish peroxidase Myoglobin Amino acid oxidase Alcohol dehydrogenase Human serum albumin Bovine serum albumin Lactate dehydrogenase Glutamate dehydrogenase Glucose oxidase Pepsinogen

MWa (Kd)

pIa

Adsorption rate3 3 102 (min21)

Percent adsorption (24 h)

14 24 13 40 16 140 150 68 68 140 332 150 41

10.5 10.1 10.0 7.2 6.9 5.2–8.4b 5.4 4.9 4.8 4.5–4.8c 4–5d 4.2 3.7

6.9 6 1.3 9.5 6 0.2 7.0 6 0.3 3.8 6 0.2 1.9 6 0.2 3.5 6 0.2 2.4 6 0.1 1.9 6 0.2 1.1 6 0.2 1.1 6 0.3 0.6 6 0.2 1.5 6 0.1 0.1 6 0.1

99.5 85.4 89.2 59.2 95.4 82.6 94.2 98.6 98.4 93.8 81.9 65.6 44.1

a

Reference 26. A mixture of at least eight isozymes 27 c Reference 24 d Reference 25 e Various proteins (1mg) were incubated with 100 mg Amberlite XAD-7 in 20 mM phosphate buffer pH 7.4. The protein concentration was determined at various times. Adsorption rates were calculated from plots of protein concentrations versus adsorption time (see details in MATERIALS AND METHODS). Data were the averages of three independent determinations b

Enzyme assays

pH dependence of immobilized LDH and GDH

Activities of immobilized LDH preparations were determined in 5 ml of 2.5 mm pyruvate, 1 mm NADH, and 20 mm phosphate pH 7.4, or in 5 ml of 100 mm d, l-lactate, 1 mm NAD1, and 50 mm phosphate at pH 7.7 in an 8-ml screwcapped vial under continuous end-over-end rotation at room temperature. At various times, 100 ml aliquots were removed, diluted ten-times, and their absorbancies at 340 nm were measured with a Hewlett Packard 8452A diode-array spectrophotometer. Activities of immobilized GDH were determined under similar conditions by following the conversion of NADH to NAD1 in the presence of 10 mm a-ketoglutarate, 1 mm NADH, and 50 mm ammonium phosphate at pH 7.7. Aliquots of reaction solutions were taken at various times, diluted ten-fold, and their absorbancies at 340 nm were determined.

Activities of the immobilized LDH, GDH, and LDH/GDH preparations at various pH values were determined by following absorbance changes at 340 nm versus time and by following glutamate formation in 50 mm phosphate pH 6.6 – 8.1, and 50 mm pyrophosphate pH 8.6 –9.1.

Activities of the coimmobilized LDH/GDH Samples of Amberlite XAD-7 beads (100 mg) with various amounts of immobilized LDH and GDH were incubated in 5 ml of 100 mm d, l-lactate, 10 mm a-ketoglutarate, 1 mm NAD1, and 50 mm ammonium phosphate pH 7.7 in 8-ml screwcapped vials. The vials were gently rotated end-over-end at room temperature. At various times, 100 ml aliquots were removed and applied to a Bio-Rad AG 50W-X12 cation exchange column (0.5 3 2.5 cm) equilibrated with 20 mm phosphate buffer pH 7.4. The column was washed with 1.4 ml of the same buffer, eluants were collected, and their glutamate contents were determined by assay with TNBS22 and, after derivatization with phenylisothiocyanate,23 by RPHPLC (4.6 3 250 mm column of Lichrosorb C-18) eluted with a 0 –18% acetonitrile gradient in 25 mm phosphate pH 6.6 at 1 ml min21. NAD1 and NADH levels in the same reaction solutions were also followed by taking similar aliquots and applying them directly without derivatization to the same HPLC column and eluting under the same conditions. Elutions of NAD1 and NADH were monitored at 215 nm and those of Glu derivatives (PTK-Glu) were at 254 nm.

Stability of immobilized LDH/GDH Stabilities of immobilized LDH/GDH preparations were determined as follows. Amberlite XAD-7 to which GDH and LDH had been adsorbed was incubated in 20 mm phosphate buffer pH 7.4 containing 0.5 mm NAD1 with or without various additives and their activities were determined from rates of glutamate formation. At various times, the beads were washed with fresh buffer, drained, the assay solution was added, and the ability to convert a-ketoglutarate into glutamate was determined. After assay, the beads were again washed with buffer, drained, and returned to the original storage conditions.

Results Adsorption and desorption Most proteins adsorb readily to Amberlite XAD-7 and most of those in this study were completely adsorbed in 24 h. The adsorption rates for 13 different proteins to 100 mg of Amberlite XAD-7 along with their molecular weights and isoelectric points are shown in Table 1. In the case of trypsin, some unadsorbed “protein” after 24 h probably reflected its partial autolysis during or prior to adsorption. Incomplete adsorption of horseradish peroxidase and glucose oxidase (i.e., only ;59 and 66% adsorbed in 24 h, respectively), the only glycoproteins included, and pepsinogen (i.e., ;44% in 24 h), the most anionic protein studied, on the other hand appeared to reflect weak and/or slow adsorption.

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Figure 3 Time courses for the adsorption of LDH and GDH to Amberlite XAD-7 beads. LDH (O), 1 mg in 5 ml of 20 mM phosphate buffer containing 0.5 mM NAD1 pH 7.4 was adsorbed to 100 mg of Amberlite XAD-7 for 24 h. The beads were washed with fresh buffer, drained, and 1 mg of GDH (h) in 5 ml of the same buffer was incubated with the beads for another 24 h

Coimmobilization of lysozyme and cytochrome c

Figure 2 The distribution of cytochrome c coimmobilized with lysozyme on Amberlite XAD-7. Two sliced and one intact bead of Amberlite XAD-7 with 1% (w/w) cytochrome c are shown. A red color confined to the outermost surface of the beads is seen as a slight darkening of the surface (A). A sliced and an intact bead from a similar preparation with 1% cytochrome c adsorbed after 10% (w/w) of lysozyme is shown against a dark background to reveal the white surface and an interior red ring after slicing (B). A sliced and two partially intact beads from a preparation with 1% (w/w) cytochrome c followed by 10% (w/w) lysozyme with the red color again confined to the surface (C)

When samples of various proteins adsorbed to Amberlite XAD-7 beads were suspended in 2 ml of phosphate buffer and rotated at 23°C, small amounts of protein were slowly released into the buffer. After six days, protein concentrations ranged from undetectable (i.e., , 1 mg ml21) to ;10 mg ml21. When identical Amberlite XAD-7 samples were stored under similar conditions without agitation, however, no “desorption” of protein was detectable. 52

To determine more clearly how proteins adsorb to Amberlite XAD-7, we examined the adsorption of two similar but readily distinguishable proteins, lysozyme, with no visible color, and cytochrome c, which is red. Figure 2 shows some Amberlite XAD-7 beads with adsorbed cytochrome c (A), with both lysozyme and cytochrome c adsorbed in that order (B), and with both lysozyme and cytochrome c in the reverse order (C). When only cytochrome c was adsorbed (Figure 2A), the intact beads were red and, when sliced, all of the red color was confined to a thin shell at the outer surface of the beads. When cytochrome c was adsorbed followed by lysozyme, the beads were again red and all of that red color was again confined to a thin shell at or very near the bead surface as shown in Figure 2C. The location of the adsorbed cytochrome c was not, therefore, materially affected by the subsequent adsorption of lysozyme. When lysozyme was adsorbed to Amberlite XAD-7 followed by cytochrome c, however, the beads were obviously a less intense red and, as can be seen in the sliced beads, all of that color was located inside the beads at a distance determined by the amount of previously adsorbed lysozyme (Figure 2B). Prior adsorption of lysozyme thus prevented adsorption of cytochrome c to the outer surface of the beads. The adsorption of one protein to a site or region of the beads thus appears to preclude subsequent binding of others to the same region and differences in the order of adsorption gives distinctly different distributions of proteins in the beads.

Coimmobilization of LDH and GDH Based on the results obtained with lysozyme and cytochrome c, we immobilized two enzymes, lactate dehydrogenase and glutamate dehydrogenase, on Amberlite XAD-7. Figure 3 shows a time course for the adsorption of 1 mg of LDH and for the subsequent adsorption of 1 mg of GDH to

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NAD1/NADH recycling by LDH/GDH-amberlite XAD-7:M. Le and G. E. Means

Figure 4 Activities of immobilized LDH and GDH. Amberlite XAD-7 with immobilized LDH and GDH were incubated with substrate solutions at room temperature under mild agitation. At various times, aliquots of the solution were taken to measure their absorbance at 340 nm. LDH activity using NADH and pyruvate as substrates (A). LDH activity using NAD1 and lactate as substrates (B). GDH activity using NADH, ammonium phosphate, and a-ketoglutarate as substrates (C)

the same beads. LDH adsorbed at a moderate rate and approached ;90% completion after 3 h. GDH, which has a twofold slower adsorption rate on fresh beads (Table 1), adsorbed even more slowly to those LDH-containing beads and approached ;85% adsorption after overnight incubation. Activities of immobilized LDH were determined using changes in absorbance at 340 nm to follow both the conversion of NAD1 into NADH and, in the reverse direction, from NADH to NAD1. Figure 4A shows a plot of NADH concentration versus reaction time for the reduction of pyruvate by 100 mg of LDH/GDH beads. The initial activity calculated from the plot was 0.29 mmol NADH converted to NAD1 min21 mg21 LDH. When d, l-lactate was used as the substrate, the initial conversion rate of NAD1 to NADH was 0.17 mmol min21 mg21 LDH, but as shown in Figure 4B, it did not go to completion. An equilibrium constant of 3.4 3 10212 m calculated from the apparent equilibrium concentrations was in good agreement with an earlier value obtained with soluble LDH.24

Figure 5 Glutamate formation using LDH/GDH coimmobilized on Amberlite XAD-7. Amberlite XAD-7 (100 mg) with 1 mg of immobilized LDH (E) and different amounts of GDH, or with different amounts of LDH and 1 mg of GDH (h), adsorbed in this order, were incubated for 2 h with 5 ml of 100 mM lactate, 10 mM a-ketoglutarate, 50 mM ammonium phosphate, and 1 mM NAD1 at pH 7.7. The solution was applied to an AG 50W-X12 cation exchange column to remove ammonium ions. Glutamate content was then determined by the TNBS assay

Decreases in absorption at 340 nm were used to follow the conversion of NADH to NAD1 during the reductive amination of ammonia and a-ketoglutarate to glutamate catalyzed by 100 mg of LDH/GDH beads as shown in Figure 4C. The enzyme activity calculated from that plot was 0.13 mmol NADH converted to NAD1 min21 mg21 GDH.

Glutamate formation by coimmobilized LDH/GDH The reactions catalyzed by the immobilized LDH/GDH system are illustrated in Scheme 1. The combined reactions required repetitive cycling of NAD1 and NADH and were followed by measuring the amount of glutamate formed. Initial reaction solutions contained 10 mm a-ketoglutarate, 50 mm ammonium phosphate, and 1 mm NAD1 and were initiated by adding 100 mm d,l-lactate. Figure 5 shows the amounts of glutamate produced in 2 h by varied amounts of LDH at a fixed level of GDH and

Scheme 1 Reactions catalized by coimmobilized lactate dehydrogenase (LDH) and glutamate dehydrogenase (GDH)

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Figure 6 pH dependencies of immobilized LDH, GDH, and LDH/GDH. Activities of LDH using pyruvate and NADH as substrates (h) and GDH using a-ketoglutarate, ammonium chloride, and NADH as substrates (‚) were determined in 50 mM phosphate buffer from pH 6.6 – 8.1 and in 50 mM pyrophosphate from pH 8.6 –9.1 as described in Figure 4. Activities of the coimmobilized LDH/GDH (F) were determined under the same conditions by following glutamate formation as described in Figure 5

by varied amounts of GDH at a fixed level of LDH. When the amount of immobilized GDH was fixed at 1% (w/w), glutamate production increased with increased amounts of LDH. When LDH was fixed at 1% (w/w) and the amount of GDH was increased, glutamate formation increased initially but leveled off at a GDH/LDH ratio of about two and, at higher levels, appeared to be governed entirely by the amount of immobilized LDH.

pH optima of immobilized LDH, GDH, and the LDH/GDH system Figure 6 shows the relative activities of LDH and GDH separately and of the LDH/GDH system immobilized on Amberlite XAD-7 versus pH. The optimum for the immobilized GDH was between pH 7– 8 whereas activities of LDH increased gradually from pH 6.5–9.2 similar in both cases to the soluble enzymes.24,26 Coimmobilized LDH/ GDH on Amberlite XAD-7 had an optimum around pH 8.6 and appeared to be a combination of the other two pH dependencies.

Stability of the LDH/GDH system Activities of the immobilized LDH/GDH were monitored for one week in the presence and absence of both b-mercaptoethanol and NaCl. In phosphate buffer pH 7 containing 0.5 mm NAD1, the activity decreased to about 30% in one week. Including b-mercaptoethanol or NaCl, however, increased the stability as shown in Figure 7, and their effects appeared to be additive, giving about 78% of the initial activity after 7 days when both b-ME and NaCl were present.

NAD1/NADH recycling by the LDH/GDH system Glutamate formation by the LDH/GDH system required repeated NAD1/NADH recycling and was accelerated by 54

Figure 7 Stability of immobilized LDH/GDH. Rates of glutamate formation by coimmobilized LDH/GDH on Amberlite XAD-7 beads were determined at various times. The beads were washed with buffer after each assay and stored in 20 mM phosphate buffer (E), or 20 mM phosphate buffer with 100 mM NaCl (h), 4 mM b-ME (‚), and 100 mM NaCl and 4 mM b-ME (ƒ)

both forms of that coenzyme. To determine steady-state levels of the two forms under operating conditions, aliquots of reaction solutions were taken at various times and subjected to RP-HPLC. As shown in Figure 8A, when NADH was used to start the reaction, most of that NADH was converted to NAD1 within 30 min to give an NAD1/ NADH ratio of at least 100/1 and then was maintained at approximately the same level for at least 24 h. Glutamate concentrations of the same solutions, determined by RP-HPLC after derivatization with PITC, however, gradually increased over the same time period. As shown in Figure 8B, glutamate contents increased steadily for at least 5 h long after the NAD1/NADH ratio ceased to change. That continued formation of glutamate and that the total glutamate formation far exceeded the initial amount of NADH showed that the reduced and oxidized forms of the coenzyme were regenerated and used repeatedly. Table 2 shows the number of NAD1/NADH cycles required to obtain the observed glutamate levels at three different initial NAD1/NADH concentrations. The high steady-state NAD1/NADH ratio suggested that the activity of LDH controlled the overall rate of reaction.

Effect of enzyme arrangement on the efficiency of glutamate formation To determine the importance of proximity on the efficiency of NAD1/NADH recycling in this case, we immobilized the two enzymes in different arrangements on the beads, sometimes with an intermediate layer of BSA, and determined their rates of glutamate formation. Five different arrangements of the two enzymes and BSA, as schematically shown in Figure 1, were examined. In each case, 0.5 mg LDH and 1 mg GDH were immobilized on 100 mg beads. The lowest overall catalytic efficiency was obtained when the two enzymes were immobilized separately on two identical samples of Amberlite XAD-7 and then mixed (L 1 G). The highest efficiency was obtained

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NAD1/NADH recycling by LDH/GDH-amberlite XAD-7:M. Le and G. E. Means slightly lower. The presence of an inert protein layer between the two enzymes (GBL and LBG) also decreased rates of glutamate formation for both arrangements. Coimmobilization of LDH and GDH in the same layer has not been included in the present study although such arrangements should be, theoretically, even more efficient. Due to the different adsorption rates of LDH and GDH (Table 1), simultaneous adsorption of the two enzymes would be expected to produce layers with a disproportionate amount of LDH which is the more rapidly adsorbed at the periphery and of GDH in the interior.

Discussion

Figure 8 HPLC analyses for NAD1/NADH levels and glutamate formation. Amberlite XAD-7 (100 mg) with 0.5 mg immobilized LDH and 1 mg of GDH was incubated in 5 ml of substrate solution as described in Figure 5 but with 1 mM NADH replacing NAD1. Aliquots of reaction solutions were taken at various times, their NADH (F) and NAD1 (■) levels were determined by C-18 RP-HPLC (top). A second series of aliquots were taken at various times, derivatized with PITC, and the amounts of PTHGlu (Œ) was determined by C-18 HPLC (bottom)

when the two enzymes were immobilized in adjacent layers on the same beads with GDH in the outermost layer (i.e., GL). When immobilized in the opposite order with LDH in the outer layer (i.e., LG), the activity was Table 2 Number of NAD1/NADH cycles at different initial coenzyme concentrations Initial [NAD1] (mM)

Cycles hr21a

0.1 0.5 1.0

103 156 166

a Cycles h21 5 Glu produced (mol h21) divided by LDH active sites (mol)

As shown in Table 1, a wide variety of proteins adsorb readily to Amberlite XAD-7. Only highly anionic proteins (e.g., pepsinogen) and some glycoproteins (i.e., horseradish peroxidase and glucose oxidase) were not readily adsorbed. The polarity of the carboxylate and oligosaccharide groups of those proteins presumably interfere with their adsorption to Amberlite XAD-7. At relatively low protein levels (;4% w/w and lower), adsorption rates varied for different proteins as shown in Table 1 and appeared to correlate primarily with their isoelectric points. Proteins with isoelectric points such as lysozyme and trypsin thus adsorbed much faster than those with low isoelectric points such as glutamate dehydrogenase and pepsinogen (Table 1). Molecular weights of the proteins included in this study varied from ;14,000 –332,000 D but with little apparent effect on adsorption behaviors. Similar adsorption rates were found, for example, for myoglobin (;16 Kd) and yeast alcohol dehydrogenase (;150 Kd), and also for BSA (;68 Kd) and LDH (;140 Kd) as shown in Table 1. One of the problems associated with coimmobilizing several enzymes is the random distribution of the individual enzymes in the support matrix. The manner by which proteins are attached to most supports allows for little or no control of their arrangement in or on the support, the proximity of one relative to the other, or even in their relative amounts. With Amberlite XAD-7, unlike most other supports, the amount of an immobilized enzyme can be easily varied; a second, third, or forth enzyme can be introduced; and the total amount of the individual enzymes, their relative amounts, and their locations in the support beads can be varied by the amount and the order in which they are immobilized. Cytochrome c, for example, can be adsorbed in a thin layer at the periphery of the beads, internally at various distances from the periphery, or more or less throughout the beads depending on the amount and order in which it is adsorbed along with lysozyme (Figure 2) or other proteins. In the case of LDH and GDH, which also adsorbed readily (Figure 3), they retained their catalytic activities (Figure 4), and pH dependencies similar to the soluble enzymes (Figure 6), and variations in the amounts and the order of their adsorption affected overall rates of lactate-dependent glutamate production. At a relatively low fixed level of GDH, for example, rates of glutamate formation increased more or less linearly with the amount of coimmobilized LDH (Figure 5), suggesting that the formation of NADH was rate-limiting under

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Papers those conditions. NADH concentrations were in fact low under the conditions generally employed. Even when added in large excess, NADH concentrations were rapidly reduced to low levels and then maintained, more or less, while large amounts of glutamate were produced (Figure 8). Repetitive NAD1/NADH recycling between the immobilized LDH and GDH was clearly necessary to account for the large amounts of glutamate produced after the first 30 min of reaction. Varied amounts of added NADH had only small effects on observed rates of glutamate production. The efficiency of NAD1/NADH recycling by different coimmobilized LDH/GDH preparations varied with the arrangement of LDH and GDH in the beads. The least efficient arrangement was that wherein the two enzymes were immobilized on different beads. In that case, NADH was generated by LDH-containing beads and used by GDH-containing beads. In addition to the catalytic steps, transport of NADH and NAD1 between beads was, apparently, a significant step and accounted for the relatively slow rates of glutamate production. When both LDH and GDH were immobilized on the same polymer beads, transport of NAD1 and NADH between the two enzymes can take place within the individual beads and should be much faster. As shown in Figure 1, rates of glutamate formation were significantly faster in such LDH/GDH beads. Different arrangements of these two enzymes in the beads, however, also affected their activities. Those wherein GDH was at the surface with an underlying layer of LDH, for example, were more active than the reverse order. The difference may be rationalized as due to greater leakage of NADH from the latter beads. NADH produced near the periphery of a bead can diffuse either more deeply into the bead or out of the bead whereas that produced internally cannot escape without first passing through the GDH layer. Introducing BSA between the two enzyme layers to increase the NAD1/NADH transport distance also decreased rates of glutamate formation. Even faster rates should be possible for beads wherein the two enzymes are coimmobilized in a single layer. Although such layers can be prepared by simultaneous adsorption of the two enzymes, differences in adsorption rates give nonuniform distributions of the two (or more) enzymes wherein the more rapidly adsorbed are more abundant at the periphery and the more slowly adsorbed are more abundant in the interior. Homogenous layers of two or more enzymes in a predetermined ratio should be possible by this means for those special cases where the enzymes have identical adsorption rates. In other cases, the preparation of a series of very thin concentric layers by stepwise adsorption of individual enzymes appears to be the only clear-cut way to control enzyme ratios. Amberlite XAD-7 has several important advantages as a support for enzyme immobilization. It requires no activation and undergoes no spontaneous deactivation process. The procedure is very simple and involves no additional chemicals nor any chemical alterations of the enzymes. Immobilization results from a spontaneous, irreversible, adsorption of enzymes to the polymer surface, is very efficient, usually quantitative for reasonable amounts (i.e., less than 4% w/w) of most enzymes, and usually has little or no obvious effect on their catalytic activities. It appears to be particularly well 56

suited as a support for multienzyme systems as the amounts, location, and arrangement of the enzymes can be easily controlled.

Acknowledgments We would like to thank Hao Zhang and E. J. Behrman for their advice during the course of this work and Amanda Simcox for the use of her inverted microscope.

References 1. 2. 3. 4.

5.

6. 7.

8.

9.

10. 11.

12. 13. 14. 15.

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