BIOTECHNOLOGY AND BIOENGINEERING,
`VOL. XVIII, PAGES 669-684 (1976)
`
`Effects of Immobilization on the Kinetics of
`
`Enzyme-Catalyzed Reactions.
`
`I. Glucose
`
`Oxidase in a Recirculation Reactor System
`
`K. B. RAMACHANDRAN and D. D. PERLMUTTER, Department
`of Chemical and Biochemical Engineering, University of Pennsylvania,
`Philadelphia, Pennsylvania 19174
`
`Summary
`
`Glucose oxidase from Aspergillus niger was immobilized on nonporous glass
`beads by covalent bonding and its kinetics were studied in a packed-column
`recycle reactor. The optimum pH of the immobilized enzyme was the same as
`that of soluble enzyme; however, immobilized glucose oxidase showed a sharper
`pH-activity profile than that of the soluble enzyme. The kinetic behavior of
`immobilized glucose oxidase at optimum pH and 25°C was similar to that of the
`soluble enzyme, but the immobilized material showed increased temperature
`sensitivity.
`Immobilized glucose oxidase showed no loss in activity on storage
`at 4°C for nearly ten weeks. On continuous use for 60 hr, the immobilized enzyme
`showed about a 40% loss in activity but no change in the kinetic constant.
`
`INTRODUCTION
`
`in-
`With the recent development of immobilization techniques,
`teresting potential applications of enzymes as catalysts have been
`proposed in fields as diverse as medicine, sewage treatment, and
`industrial processing. Since enzymes depend upon specific three-
`dimensional conformation of their molecules for activity, any physical
`influence of the matrix or chemical modification of the enzyme might
`alter its properties.
`Indeed, a number of recent publications1“‘ have
`reported altered properties of enzymes after immobilization. How-
`ever, it has not always been taken into consideration that the ap-
`parent change in the chemical properties are not entirely due to the
`physical
`influence of the matrix or chemical modification of the
`enzyme. External and internal diffusion effects can considerably
`alter the Michaelis-Menten constant, the activity, and the thermal
`sensitivity. For engineering purposes, better understanding is
`needed of immobilized enzyme kinetics and the factors that influence
`669
`
`© 1976 by John Wiley & Sons, Inc.
`
`Page 1 of 16
`Page 1of16
`
`BD EXHIBIT 1028
`
`BD EXHIBIT 1028
`
`

`
`670
`
`RAMACHANDRAN AND PERLMUTTER
`
`In this study glucose oxidase was im-
`the rate of the reaction.
`mobilized on nonporous glass beads by covalent bonding and its
`kinetics studied under well—defined reactor geometry and flow con-
`ditions. Glass was chosen as the support material because of its
`strength and incompressibility.
`
`BACKGROUND
`
`The kinetics of the homogeneous glucose oxidase reaction have
`been widely studied7‘” with 5-1) glucose, at 25°C, and pH = 5.5.
`The mechanism is generally given as
`
`Eo+G—»..,:_Eo—ai»E,+P
`
`E, + 02—"‘—> E» + H202
`
`(1)
`
`(2)
`
`where E0, E ,, and E0 - G stand for the oxidized and reduced forms
`of the enzyme and the enzyme complex, respectively. Based upon
`this mechanism the reaction rate at steady state can be expressed as
`
`kmEr[02l[Gl
`” = “““—;.c”“‘"‘“"”“z“t~’
`ma] +
`[021+ “ [G1
`kox
`
`kred
`
`(3)
`
`[cox = k4 and
`where ET is the enzyme concentration, Ice“ = 192,
`land = (klkg/k._1 + kg) or (klkz/k_1), if the assumption is made of
`rapid equilibrium.
`For a given amount of enzyme and at fixed glucose concentration
`eq. (3) can be reduced to
`
`where
`
`and
`
`VH1 axio 2]
`= J
`[02] + KM
`
`”
`
`kca.tkred[Gl
`K = —-—~——s—-—
`M
`koxkredigl + kcatkox
`
`Vm .__
`
`kred[Gl + kcat
`
`4
`
`( )
`
`(5
`
`)
`
`(6)
`
`Previous kinetic studies of glucose oxidase, immobilized by various
`techniques, have indicated that immobilization can affect the proper-
`ties of the enzyme. For example, Weibel and Bright” immobilized
`
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`Page 2 of 16
`
`

`
`IMMOBILIZED GLUCOSE OXIDASE
`
`671
`
`
`
`Rccycle stream
`
`Fig. 1. Flowsheet for a. recycle reactor system.
`
`glucose oxidase on porous glass beads by covalent bonding and found
`that the apparent bimoiecular constant was increased by a factor of
`14. For gel entrapped glucose oxidase, Hinberg et al.” observed an
`increase in the value of the bimolecular constant by a factor of 2,
`but Miyamura and Suzuki“ found that the values of the kinetic
`constants approached those of soluble enzyme as the particle size
`was decreased. Glucose oxidase crosslinked on a cellophane mem-
`brane” and covalently coupled on a nickel oxide screen” showed no
`change in the value of kinetic constants.
`In the same studies the
`optimum pH of the enzyme also did not change, however, in both
`cases the sensitivity of the enzyme to changes in pH was increased.
`Immobilized glucose oxidase showed decreased temperature sensi-
`tivity” and increased storage stability.”
`The chemical engineering literature” describes the advantages of
`a packed-bed reactor linked to an external recirculation system.
`In such a continuous flow recirculation reactor system part of the
`effluent stream is returned and mixed with the feed stream, as
`schematically shown in Figure 1. A mass balance on the substrate
`at the mixing point gives:
`
`[S]a.. = ”
`
`(7)
`
`As the ( R/F) ratio is increased sufficiently, the concentration changes
`within the reactor decrease to the point where the reactor is called
`“differential,” i.e.,
`the reaction may be considered to occur at a
`constant average concentration level. The overall conversion must
`however be significant enough to be detectable by the available
`measurement techniques. Under these circumstances the reaction
`rate can be calculated from
`
`__ F([S]o — isiout)
`”‘ —%”W:_
`
`(8)
`
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`Page 3 of 16
`
`

`
`672
`
`RAMACHANDRAN AND PERLMUTTER
`
`where W is the catalyst weight. Equation (8) can also be written
`in the modified form
`
`0 = F[O2],g(1 — X)
`l
`W
`
`(9)
`
`is the saturated oxygen concentration. Values for
`where [O2]s
`different temperatures are reported in the literature."
`Since the glucose concentrations used in this study were very much
`higher than those of oxygen, they can be assumed to be constant
`throughout the reactor and equal to the inlet value. From eq. (7)
`the inlet oxygen concentration to the reactor can be written as
`
`_ [021 (F + RX)
`[O2iIn —
`F + R
`
`and the average oxygen concentration is given by
`
`[02] =
`
`2
`
`(11)
`
`Equations (9)—(11) provide the numerical values needed in the
`evaluation of the rate expression eq. (3). Kinetic constants in this
`rate expression were calculated by using Rosenbrock’s search tech-
`nique.“
`
`EXPERIMENTAL
`
`Materials
`
`Nonporous glass beads (40-60 mesh) used for covalent coupling
`of glucose oxidase were obtained from Ana Laboratories Incorporated,
`New Haven, Connecticut. The enzyme preparation (analytical
`grade from Aspergillus niger) used for immobilization was obtained
`from Sigma Chemical Company, St. Louis, Missouri and further
`purified. D-Glucose solutions of different concentrations were pre-
`pared by using “Baker analyzed” reagents purchased from J. J.
`Baker Chemical Company, Phillipsburg, N. J. The buffer solution
`used was 0.1M sodium acetate and the pH was adjusted to the
`desired level by adding acetic acid. Since the enzyme preparation
`contained trace amounts of catalase, 0.1mM KCN was added to
`suppress its activity. EDTA in the amount of 0.5mM was also
`added to the glucose solution to protect the enzyme from metal ions
`which may deactivate it. Sodium acetate, KCN and EDTA used
`were analytical grade materials (J. J. Baker). Compressed air used
`
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`Page 4 of 16
`
`

`
`IMMOBILIZED GLUCOSE OXIDASE
`
`673
`
`for saturating the glucose solution was obtained from Linde Gas
`Company. All solutions were prepared from distilled—deionized
`water.
`
`Methods
`
`The nonporous glass beads were prepared by first adding 100 ml
`of water to 50 g of beads and then slowly adding 100 ml of 50%
`hydrofluoric acid, allowing the contents of the beaker to cool between
`additions- The mixture was allowed to react for 1 hr and then 10N
`
`NaOH was added, enough to cover the beads. The slurry was heated
`to 80°C for 1 hr, washed with distilled water, and dried overnight
`in an oven at 80°C. The dry beads were immersed in a 2% solution
`of 3-aminopropyltriethoxy silane in acetone. Excess liquid was
`decanted and the beads were allowed to stand in an oven at 45°C
`
`for 24 hr. The alkylamine glass was refluxed for 24 hr in 200 ml of
`chloroform containing 10 ml of triethylamine and 20 g of nitro-
`benzoyl chloride. The beads were washed with chloroform and
`
`ethyl alcohol and dried in an oven at 60°C for 12 hr. The arylamine
`glass was reduced by refluxing in 200 ml of 5% (w/v)
`sodium
`dithionite in water for 1 hr. The beads were washed with water
`
`and benzene and dried at 60°C.
`
`For diazotization and coupling the glass was slurried in 50 ml of
`2N HCl and placed in an ice bath in a dessicator connected to a
`vacuum source. When cool, 2.5 g of sodium nitrate was added to
`the slurry; the reaction was allowed to proceed under vacuum for
`20 min. The beads were then quickly but thoroughly washed with
`ice-cold 1% (w/V) sulfamic acid, until no more bubbling was seen.
`A 0.1M Tris—Cl solution (pH=8.7) was used for a last rinse. Excess
`liquid from the top of the beads was removed by decantation to
`prevent dilution of the enzyme solution. Glucose oxidase that had
`been column purified and concentrated was diluted by 1:10 and
`10 ml was added to the glass beads. The reaction was allowed to
`proceed for 1 hr. The beads were then washed with Tris—Cl buffer
`thoroughly to remove the loosely bound enzyme and the supernatant
`containing unreacted enzyme was saved. Glucose oxidase beads
`were stored at pH=6.5 in the cold.
`
`Recycle Reactor
`
`The essential features of the recycle system consist of the reactor,
`the feed preparation and product analysis parts, and the measure-
`ment and control devices. Glucose feed solution is maintained
`
`within :i:0.2°C by a Blue M Electric Co. Model MR—324OA-1 con-
`
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`Page 5 of 16
`
`

`
`674
`
`RAMACI-IANDRAN AND PERLMUTTER
`
`It is saturated with air by bubbling through
`stant temperature bath.
`four gas dispersion rods. A positive pressure Micro Pump, model
`12-00-316 supplies the feed to the reactor, at rates measured by
`rotameters. The heat added to the solution by the agitation of the
`pump is removed by passing the reactants through stainless steel
`coils maintained inside the constant temperature bath.
`The reactor consists of a glass tube of 2.5 cm diameter and 30 cm
`length, surrounded by a Plexiglas Water jacket. Spacers on both
`sides are used to adjust the bed height to any desirable level. The
`glass beads are supported inside the column by nylon screens, one of
`which also serves as a fluid distributor. Water from the constant
`
`temperature bath is circulated through the water jacket to maintain
`uniform reactor temperature.
`A part of the reactor effluent stream is metered and recycled.
`The rest of the stream is passed through a specially built holder for
`the dissolved oxygen electrode. The holder is provided with magnet-
`driven agitation to maintain a minimal velocity past the faces of the
`electrodes and the oxygen level in the reactor product is measured
`by using a polarographic electrode Model YSI 5331 Yellow Spring
`oxygen analyzer. The oxygen probe is connected to a Model YSI 53
`biological oxygen monitor, which measures the dissolved oxygen level
`as a percentage of saturation value and supplies a signal for a con-
`tinuous record. The reactor is also equipped with a bypass line for
`calibrating the oxygen probe.
`The temperatures in the water bath, and at the entrance and exit
`of the reactor are monitored by thermocouples. All tubings and
`tube fittings are made of either stainless steel or polyethylene.
`Further details of equipment and procedure are recorded elsewhere.”
`
`RESULTS AND DISCUSSION
`
`Two different preliminary experiments were run to evaluate the
`effects of external mass transfer on the chemical reaction rate to be
`
`In the first
`studied on the nonporous 50-60 mesh glass beads.
`experiment, conversions from a plug flow reactor were compared at
`given space time (W/F) but at three different catalyst weights.
`Figure 2 shows the results obtained. Since the data for all three
`catalyst weights overlap, conversion is evidently not aifected by
`flow velocity and it can be concluded that external film resistance
`is negligible.
`lll the second experiment, reaction rates from a differential recycle
`reactor were compared at a given feed rate (F) but at dlfferent ‘00’f»9~1
`
`(cid:51)(cid:68)(cid:74)(cid:72)(cid:3)(cid:25)(cid:3)(cid:82)(cid:73)(cid:3)(cid:20)(cid:25)
`Page 6 of 16
`
`

`
`IMMOBILIZED GLUCOSE OXIDASE
`
`675
`
`o
`
`W = 4-84 gnu
`
`A W-=7-43cm
`
`V
`
`In IE6! gm
`
`lOOmM
`-
`[5]
`pH 5.5. r=25°c
`
`'0
`
`8
`
`
`
`O0
`
`
`
`Percentconversion09oxygen MJ-O-0
`
`0-2
`
`0-4
`
`0-6
`
`0-8
`
`1-0
`
`|'2
`
`I- 4
`
`I-6
`
`specs Nmo , one min/ml
`
`Fig. 2. Dependence of oxygen conversion on space time.
`
`flow rates (F + R). Data obtained at four different feed rates from
`8.4 to 41.1 cc/min and over a range of recycle rates from 360 to 1640
`cc/min showed that the reaction rate is independent of total flow.
`It can therefore be concluded as before that the external mass transfer
`
`resistance is negligible above a total flow rate of 360 cc/min, cor-
`responding to a linear velocity of 1 cm/sec.
`In all subsequent experi-
`ments the reactor was operated well above this flow Velocity.
`Using the correlation of Wilson and Geankoplis” for mass transfer
`in packed beds, surface concentrations of oxygen and gluconic acid
`were calculated, corresponding to each of the experimental condi-
`tions.
`In all the cases, surface concentration of oxygen was found
`to be only slightly different from that of the bulk concentration
`(<<1%), confirming that external mass transfer effects are negligible
`for this system. The surface concentration of gluconic acid was
`similarly found to be nearly the same as that of the bulk, indicating
`that the microenvironment near the glass was not different from
`that of the bulk.
`
`Experiments were conducted with different amounts of immobilized
`enzyme to test whether axial dispersion effects and end effects are
`significant. Reaction rates were measured at 25°C and 10mM
`glucose concentration at different oxygen concentrations and over a
`threefold range of bed weight. The data were fitted to eq. (4) and
`the parameters Vmax and KM were estimated. The kinetic constants
`
`(cid:51)(cid:68)(cid:74)(cid:72)(cid:3)(cid:26)(cid:3)(cid:82)(cid:73)(cid:3)(cid:20)(cid:25)
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`
`

`
`676
`
`RAMACHANDRAN AND PERLMUTTER
`
`N0
`
`T= 25°c,pH=5-5
`[e]= I0mM
`
`Us
`
`
`
`(wxv,,,.,)xlo‘,moles/min 5
`
` 0
`
`IO
`
`20
`Cololyrl weighi . gm:
`
`30
`
`40
`
`Fig. 3. Effect of bed Weight on the reaction rate.
`
`are independent of bed weight, indicating that dispersive effects are
`not significant. Figure 3 shows the same data, but plotted in a form
`that would produce a straight line passing through the origin, if axial
`dispersion and end effects were negligible. The results clearly
`support such a conclusion.
`
`pH Profiles
`
`The effect of pH upon the activities of the enzyme in solution and
`in the immobilized form was studied at a glucose concentration of
`100mM. At this concentration level, the values of Vmax estimated
`by applying eq. (4) are essentially the true maximum velocities when
`oxygen and glucose are in great excess. The results for both soluble
`and immobilized enzymes are compared in Figure 4 for pH between
`3 and 8 on normalized scales.
`
`It can be seen from Figure 4 that the optimum pH = 5.5 of the
`immobilized enzyme is the same as that of the enzyme in solution,
`however, the immobilized enzyme seems to be more susceptible to
`the changes in pH of the bulk solution. Below a pH of 3 the im-
`mobolized glucose oxidase completely and irreversibly lost activity.
`The results of this Work are consistent With the findings of Broun
`et al.16.for glucose oxidase crosslinked on cellophane and also with the
`findings of Weetall and Hersh" for glucose oxidase covalently linked
`on nickel oxide screens. These effects have been attributed to
`
`insoluble carrier or unidentified chemical
`charges on the Water
`modification, but there exists no experimental Verification for these
`speculations.
`
`(cid:51)(cid:68)(cid:74)(cid:72)(cid:3)(cid:27)(cid:3)(cid:82)(cid:73)(cid:3)(cid:20)(cid:25)
`Page 8 of 16
`
`

`
`IMMOBILIZED GLUCOSE OXIDASE
`
`677
`
`T = 25‘t:, I= 0-IM
`
`
`
`[G]=lOOmM
`.
`lnmobmud Ethic work
`A Sollflo
`—-—~— lnlnobilizodon NiO(Rof|7)
`
`‘ii
`'\\\
`-
`
`_
`
`I
`
`5-
`.2
`‘g
`E3
`EIt
`
`02 3
`
`2
`
`2
`
`Fig. 4. pH—Activity profiles of soluble and immobilized glucose oxidase.
`
`Efiect of Substrate Concentrations
`
`At 25°C and at pH = 5.5 a series of runs were made to evaluate
`the effects of glucose and oxygen concentrations on the kinetics of
`immobilized glucose oxidase. Reaction rates were measured at
`different oxygen concentrations for each of seven glucose levels
`between 5 and 100mM. The experimental results are shown in
`Figure 5 together with curves computed from eq.
`(3) by inserting
`best estimates of the various parameters. The estimated parameter
`values and their respective 95% confidence interval values are given
`in Table I along with the comparable literature results for the free
`enzyme and for glucose oxidase immobilized on porous glass.
`Applying the standard statistical t—test for significant differences
`between means, a comparison of the kinetic constants for soluble and
`immobilized glucose oxidase shows no evidence that the former was
`affected by immobilization.
`It can be concluded that there is very
`
`TABLE I
`
`Comparison of Kinetic Parameters for Glucose Oxidase
`
`System
`
`kcat/kox X 103
`(mol /liter)
`
`heat/kred
`(mol /liter)
`
`Ref.
`
`Soluble enzyme
`Bound to porous glass
`Bound to nonporous
`glass
`
`0.51
`0.50
`0.59 :l: 0.177
`
`0.071
`0.005
`0.08 :l: 0.020
`
`12
`13
`This Work
`
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`
`

`
`678
`
`RAMACHANDRAN AND PERLMUTTER
`
`Ts 25°c, pH=5-5
`
`—- Model (Equaiion 3)
`
`'
`
`I
`
`0-05
`
`on
`[02] at IO’. mole:/liter
`
`015
`
`0-2
`
`I-25
`
`I-0
`
`0-75
`
`0-5
`
`025
`
`o
`
`0
`
`5O
`.5
`s\
`
`3 2
`
`"<_5X
`>
`
`Fig. 5. Effect of substrate concentration on the reaction rate at 25°C.
`
`little or no interaction between the carrier and the active enzyme
`sites at this optimum pH = 5.5. However, it should be noted that
`the kinetic constant could be significantly different at other pHs,
`as suggested by the trends shown in Figure 4. The results obtained
`from this study are not in agreement with the results obtained by
`Weibel and Bright,” who observed a 14-fold increase in the value
`
`(cid:51)(cid:68)(cid:74)(cid:72)(cid:3)(cid:20)(cid:19)(cid:3)(cid:82)(cid:73)(cid:3)(cid:20)(cid:25)
`Page 10 of16
`
`

`
`IMMOBILIZED GLUCOSE OXIDASE
`
`679
`
`loud. However, when the
`of the apparent bimolecular constant,
`data of Weibel and Bright were corrected for external and internal
`diffusion effects,“ the resulting constant was reduced by an order
`of magnitude. Moreover, when the data of the present work were
`analyzed by the inverse-plot method used by Weibel and Bright,
`it was found that the results could differ from the nonlinear parameter
`estimates by another factor of two.”
`An order of magnitude estimate of the surface concentration of the
`enzyme can be calculated from the value of kc,.tE;p for the immobilized
`enzyme. The value of kg“ for the enzyme is readily available since
`it did not change upon immobilization, but an estimate is needed
`of surface area per gram of glass beads. Assuming the average
`diameter to be 0.2 mm for 40-60 mesh beads and the glass density
`to be 2.2 g/cc,
`the surface concentration was calculated to be
`7.2 X 10*” mol/cm’.
`In actual preparation the surface concentra-
`tion will be smaller than this, because the surface area of textured
`glass beads is higher than that of uniform spheres. To evaluate this
`result it is of interest to compare it to the surface concentration level
`that corresponds to monolayer coverage. Using a molecular di-
`ameter for the enzyme of 10"’ cm, monolayer coverage on 0.2 mm
`uniform spheres corresponds to a surface concentration of 13 X 10*”
`mol/cmz, a figure which is twice as high as the surface concentration
`estimated for the enzyme preparation used in this study.
`It may be
`concluded, therefore, that approximately 50% (or less) of the glass
`surface is involved in the enzyme catalysis.
`
`Efeet of Temperature
`
`Reaction rates were measured at four different temperatures and
`the data for each temperature were fitted to eq. (3) to obtain the
`parameter estimates (with 95% confidence interval) shown in Table
`II. Since k.,“ET is the maximum velocity at excess concentration
`of glucose and oxygen, the rates are normalized for presentation as
`the temperature—activity profile in Figure 6. The corresponding
`profiles for soluble glucose oxidase and glucose oxidase immobilized
`on nickel oxide screen” are also shown.
`It can be seen from the
`
`figure that glucose oxidase immobilized on nonporous glass beads
`shows a sharper temperature—activity profile than those of glucose
`oxidase in solution or immobilized on a nickel oxide screen.
`
`The decreased temperature sensitivity reported by Weetall and
`Hersh” may also be attributed to external diffusion efiects. When
`such effects are significant, the enzyme molecules are not all eflici-
`ently used. As a result, denaturation due to increase in temperature
`
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`
`

`
`680
`
`RAMACHANDRAN AND PERLMUTTER
`
`-—-— Glucou oxiduu uolueion
`—-— immoumzou on mo l ‘M '7
`-9.. Immobilized on non-porouu glass
`In this work.
`pH'5-5. I80-IM
`
`acfiviiy
`36Maximum
`
`I5
`
`20
`
`35
`30
`6 25
`Temperature .°C
`
`40
`
`45
`
`Fig. 6. Effect of temperature on the activity of immobilized glucose oxidase.
`
`TABLE II
`
`Kinetic Constants for Immobilized Glucose Oxidase at Various Temperatures
`
`°C
`
`15
`20
`25
`30
`
`koat.ET X 106
`(mol/g/min)
`
`1.6 :i: 0.43
`2.3 :1: 0.36
`5.9 i 2.47
`10 i 0.12
`
`kcat/kred
`(moi/liter)
`
`0.051 :i: 0.0026
`0.036 :i: 0.0017
`0.080 :i: 0.0199
`0.006 :t 0.0002
`
`(kunt/kox) X
`(mol/liter)
`
`0.41 :I: 0.024
`0.27 i 0.015
`0.59 :1: 0.177
`0.11 2|: 0.003
`
`will not aflect the overall reaction rate since the latter is in any case
`mass transfer
`limited. One may therefore,
`in agreement with
`Ollis,25 anticipate a flatter
`temperature-activity profile and an
`increased optimum temperature when diffusional restrictions are
`present. A similar increase in the sensitivity of the temperature-
`activity profile upon immobilization was also observed for glucose
`oxidase crosslinked on cellophane membranes” and invertase co-
`valently coupled to porous glass.“
`
`Stability
`
`Two runs which were made ten weeks apart with 10mM glucose
`solution at 25°C gave the following values for Vmax (With 95%
`confidence interval) :
`
`Run no.
`
`Vmax X 10“ (moi/min g)
`
`1
`
`42
`
`0.59 :l: 0.036
`
`0.63 i 0.067
`
`(cid:51)(cid:68)(cid:74)(cid:72)(cid:3)(cid:20)(cid:21)(cid:3)(cid:82)(cid:73)(cid:3)(cid:20)(cid:25)
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`IMMOBILIZED GLUCOSE OXIDASE
`
`681
`
`It can be concluded that the immobilized enzyme lost no activity
`upon storage for nearly ten weeks at 4°C.
`The same batch of immobilized enzymes was used continuously
`for 60 hr with 10mM glucose at 25°C to obtain information on
`operational stability. At the end of each 15 hr, the oxygen level
`was varied inside the reactor to determine the kinetic constants
`
`KM and Vmx in eq. (4). The results, in Figure 7, show that K M
`does not substantially change from run to run, indicating that the
`three-dimensional structure of the enzyme was not altered due to
`fluid motion near the glass surface.
`It can be seen from Figure 8,
`however, that the immobilized enzyme lost 40% of its original activity
`after 60 hr. Since KM did not change upon continuous use for
`60 hr, the change in Vmax must be due to a change in ET, the con-
`centration of active enzyme. The loss in activity is due to de-
`naturation rather than physical leaching, for if physical leaching had
`been responsible, the enzyme would have lost activity on storage.
`When the deactivation data were fitted to a first order model, the
`magnitude of
`the deactivation constant
`(with 95% confidence
`interval) was found to be (7.6 :+: 3.09) X 10‘3hr*1, corresponding to
`a half-life of 91.0 hr.
`
`Efiects of Dzferent Preparations
`
`Glucose oxidase was immobilized in two different batches to test
`
`whether both batches would give the same kinetic constants. Re-
`sults obtained for 6.5mM glucose at 25°C and pH=5.5 buffer are
`
`I-2
`
`|.o
`
`T -25'c. DH=5~5
`
`[G] IIOIIIH
`
`K“"|
`
`5<C3OE
`
`'0
`
`0
`
`I5
`
`30
`
`45
`
`60
`
`Time , Hrs
`
`Fig. 7. Continuous operation of immobilized glucose oxidase, effect of KM.
`
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`Page 13 of16
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`

`
`682
`
`RAMACHANDRAN AND PERLMUTTER
`
`5
`
`1 - 2.-.'c . pH=5-5
`
`[3]-aomu O O
`Percentinitialactivity A o
`
`O 0
`
`
`
`
`
`0
`
`I5
`
`30
`
`45
`
`60
`
`Time . Hrs
`
`Fig. 8. Continuous operation of immobilized glucose oxidase, effect on Vmx.
`
`given in Table III. The KM values for both batches are the same to
`within their 95% confidence intervals, however, the Vmx for batch I
`is three times greater than that for batch II. That KM did not
`change from batch to batch indicates that the binding mechanism
`for immobilization did not change. The difference in Vmax is due to
`different amounts of enzyme immobilized on the glass surface.
`
`Kinetic Constants for Different Immobilized Enzyme Preparations
`
`TABLE III
`
`Batch no.
`
`I
`H
`
`KM X 105
`
`(mol/liter)
`
`5.2 i 2.87
`5.7 :i: 2.41
`
`Vmu X 107
`
`(rnol/min/g)
`
`5.3 :t 1.04
`1.8 2}: 0.25
`
`Nomenclature
`
`E1
`F
`[G]
`I
`kl, k_1, kg
`la;
`km
`loo,
`
`total immobilized enzyme concentration, mo]/g
`feed rate, cc/min
`average concentration of glucose, mol/liter
`ionic strength of buffer, mol/liter
`rate constants defined by eq (1)
`rate constant defined by eq. (2)
`rate constant for the dissociation of enzyme glucose complex, min"
`rate constant for the reaction between oxygen and reduced form of
`enzyme, liter/mol/min
`
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`Page 14 of 16
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`

`
`IMMOBILIZED GLUCOSE OXIDASE
`
`683
`
`kn-ed
`
`KM
`[02]
`[O2];,,
`[0213
`R
`[S] in
`[S10
`[Slout
`T
`:2
`Vnm
`
`W
`X
`
`apparent bimolecular rate constant for the interaction of substrate
`and the oxidized form of enzyme, liter/mo]/min
`Michaelis—Menten constant, mo]/liter
`average oxygen concentration, mol/liter
`oxygen concentration at the inlet of the reactor, mo]/liter
`saturated oxygen concentration, mol/liter
`recycle rate, cc/min
`substrate concentration at the reactor inlet, mol/liter
`substrate concentration in the make-up feed, mol/liter
`substrate concentration at the outlet stream, mol/liter
`temperature, °C
`reaction rate, moi/min/g
`maximum velocity at a fixed concentration of glucose and excess
`concentration of oxygen, mol/min/g
`catalyst Weight, g
`conversion
`
`This research was supported by a grant from the NSF—RANN program.
`
`References
`
`. A. K. Sharp, G. Kay, and M. D. Lilly, Biotechnol. Bioeng., II, 363 (1969).
`L. Goldstein, M. Pecht, S. Blumberg, D. Atlas, and Y. Levin, Biochemistry,
`9. 2322 (1970).
`3. L. Goldstein, Y. Levin, and E. Katchalski, Biochemistry, 3, 1913 (1964).
`4. W. F. Line, A. Kwong, and H. H. Weetall, Biochim. Biophys. Acta, 242,
`194 (1971).
`5. H. H. Weetall and G. Baum, Biotechnol. Bioeng., 12, 399 (1970).
`6. P. J. Robinson, P. Dunnill, and M. D. Lilly, Biochim. Biophys. Acta, 242,
`659 (1971).
`7. Q. H. Gibson, B. E. P. Swoboda, and V. Massey, J. Biol. C'hem., 239, 3927
`(1964).
`8. H. J. Bright and G. H. Gibson, J. Biol. Chem., 242, 994 (1967).
`9. S. Nakamura and Y. Ogura, J. Biol. Chem., 63, 3 (1968).
`10. F. R. Duke, M. K. Weibel, D. S. Page, V. C. Bulgrin, and J. Luthy,
`J. Am. Chem. Soc., 91, 3904 (1969).
`11. H. J. Bright and M. Appleby, J. Bioi. C’hem., 244, 3625 (1969).
`12. M. K. Weibel and H. J. Bright, J. Biol. Chem., 246, 2274 (1971).
`13. M. K. Weibel and H. J. Bright, Biochem. J., 124, 801 (1971).
`14.
`I. Hinberg, A. Kapoulus, R. Korus, and K. O’Driscoll, Biotechnol. Bioeng.,
`I6, 159 (1974).
`15. M. Miyamura and S. Suzuki, Nippon Kagaku Kaishi, 7, 1274 (1972).
`16. G. Broun, E. Selegny, S. Avrameas, and D. Thomas, Biochim. Biophys.
`Acta, 185, 258 (1969).
`17. H. H. Weetall and L. S. Hersh, Biochim. Biophys. Acta, 206, 54 (I970).
`18. H. H. Weetall, Biochim. Biophys. Acta, 212, 1 (1970).
`19. O. Levenspiel, Chemical Reaction Engineering, Wiley, New York, 1972.
`20. J. Robinson and J. M. Cooper, Analyt. Biochem., 33, 390 (1970).
`21. H. H. Rosenbrock, Computer J ., 3, 175 (1960).
`
`(cid:51)(cid:68)(cid:74)(cid:72)(cid:3)(cid:20)(cid:24)(cid:3)(cid:82)(cid:73)(cid:3)(cid:20)(cid:25)
`Page 15 of 16
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`684
`
`RAMACHANDRAN AND PERLMUTTER
`
`22. K. B. Ramachandran, Ph.D. Thesis, University of Pennsylvania, Phila-
`delphia, 1975.
`23. E. J. Wilson and G. J. Geankoplis, Ind. Eng. Chem. Fundam., 5, 9 (1966).
`24. P. Steiner, MS. Thesis, University of Pennsylvania, Philadelphia, 1973.
`25. D. F. Ollis, Biotechnol. Bioeng., 14, 871 (1972).
`26. R. D. Mason, and H. H. Weetall, Biotechnol. B'i0eng., I4, 637 (1792).
`
`Accepted for Publication January 23, 1976
`
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