Activity Control of an Enzyme Immobilized on a Capsule Membrane with Synthetic Bilayers*

YOSHIO OKAHATA,’ SATOSHI HACHIYA, and TAKAHIRO SEKI, Department of Polymer Science, Tokyo Institute of Technology, Ookayama, Meguro-ku, Tokyo

152, Japan

Recently, we prepared newly functional nylon capsules whose porous membranes were coated with synthetic bilayers.I4 The capsule is formed by physically strong, ultrathin membranes and the coating shows the characteristics of bilayer vesicles. For example, permeation of NaCl trapped in the inner aqueous phase was reversibly controlled by the phase transition of coating bilayers.

In this article, glucose oxidase (GOD) was immobilized on the capsule membrane together with synthetic bilayers, and the activity control of the immobilized GOD by coating bilayers on the capsule membrane was investigated. Although GOD is a water-soluble enzyme, GOD is selected for the immobilized enzyme because of its excellent thermostability. The enzyme activity is expected to be controlled by chang- ing the microenvironments around the enzyme or by direct alteration of its con- formation. We are aiming to prepare the simple, reconstituted model of biomembranes on the physically strong nylon capsule walls. A schematic illustration of the capsule is shown below.

GOD molecule

bilayer-forming amphiphiie

II :b c r

a Cl,H2S~IHiz.oCJ-NHC0.CH~N~CH)

C i z ~ n + W i z - C K h apsule membrane 1

EXPERIMENTAL

Material

Large nylon-2,12 capsule membranes (2.5 mm diameter, 1 pm thickness) and the bilayer-forming amphiphile 1 were prepared according to previous paper~.2.~” Glu- cose oxidase [GOD: fi-D-glucose oxygen l-oxidoreductase, E.C. 1.1.3.4; from Asper- gillus niger] was obtained from Amano Pharmaceutical Co. (Nagoya, Japan). Other reagents were commercially available or laboratory grades.

Immobilization of GOD on a Nylon Capsule Membrane

After being partially hydrolyzed in HCl aqueous solution, 20 pieces of nylon capsule membranes were reacted with glutaraldehyde (2.5% in 0.1M phosphate

Functional Capsule Membranes. XII. + To whom all correspondence should be addressed.

Journal of Polymer Science: Polymer Letters Edition, Vol. 22, 595-599 (1984) @ 1984 John Wiley & Sons, Inc. CCC 1360-6384/84/11059505$04.00

596 OKAHATA, HACHIYA, AND SEKI

buffer, pH 7) a t 25°C for 40 min. The capsules were washed with excess water and then soaked in GOD solution (0.5 mg/mL of 0.1M phosphate buffer, pH 7) at 25°C for 3 h with slow stirring. Unbound GOD was washed off and the capsule was then treated in 0.1% NaESH, solution at 25°C for 10 min. The GOD content on the capsule membrane was estimated to be 1.0 f 0.2 pg per capsule (25 f 2 pg), which means about 80% of the glutaraldehyde group on the capsule reacted with the enzyme.

The GOD capsule membrane was coated with bilayers of amphiphile 1 according to the previous method.2~408 The amphiphile content was estimated to be 5 f 1 pg per capsule. Thus, one capsule (ca. 0.025 mg) contains ca. 0.001 mg of GOD and 0.005 mg of coating bilayers.

Determination of the GOD Activity

The activity of GOD capsule membrane was determined a t various temperatures with an H20, electrode (Ishikawa Seisakusho Co., Tokyo, Japan), which detects the generated hydrogen peroxide by the oxidation of glucose with an enzyme. The GOD activity was defined as the unit of pmol glucose min-I mg GOD-’.

Permeability of Glucose Across the Capsule Membrane

The permeability of glucose trapped in the inner aqueous phase across the capsule membrane (GOD is not immobilized) was measured by detecting hydrogen peroxide increases in the outer medium with an HzOz electrode covered with a GODfixed cellulose film.

RESULTS AND DISCUSSION

Figure 1 shows the temperature dependence of the activity of GOD immobilized on the capsule membrane, together with the result of a free enzyme in the solution. The activity of the free GOD in the aqueous solution increased with the temperature

I t 0 5 1 , , ‘C

-_ 31 3 2 3 3 3 4 3 5 3 6 :

T-’ / lo3 K-’

1

Fig. 1. Arrhenius plots of the GOD activity in 0.1Mphosphate buffer (pH 7): (a) in aqueous solution, (b) immobilized on the capsule membrane, (c) immobilized on the capsule together with synthetic bilayers of 1. An arrow shows the phase transition temperature of coating bilayers of 1 obtained from DSC measurements.

ACTIVITY CONTROL OF IMMOBILIZED ENZYME 597

Fig. 2. Lineweaver-Bulk plots of GOD capsules at 25°C in phosphate buffer (pH 7): (a) the bilayercoated MX;-capsule, (b) the uncoated GOD capsule.

increasing up to 35'C, and decreased over 35'C probably because of thermal dena- turation of an enzyme. On the other hand, the Arrhenius plot of GOD capsule membrane showed a simple straight line increasing up to 50°C. Although the activity of the immobilized enzyme was reduced by a factor of 44, the optimum temperature range was expanded by fixing in the capsule membrane. The activity of GOD cap sules was not altered for the 2-3 months that they were stored in phosphate buffer solution a t 5'C.

When the GOD capsule membrane was coated with the bilayer-forming amphi- phile 1, the GOD activity was reduced 2-4 times over the whole temperature range. The Arrhenius plot gave an inflection near 25°C; the enzyme activity was largely reduced above 25°C relative to below 25'C.

Differential scanning calorimetry (DSC) of the bilayercoated GOD capsule mem- brane showed a sharp endothermic peak a t 22T, as well as the bilayercoated, GOD unfixed capsule. This indicates that coating bilayers have the phase transition from gel to liquid crystal, even in the presence of an enzyme. The inflection of the Ar- rhenius plot of the bilayercoated GOD capsule well consists with the phase tran- sition of coating bilayers (shown as an arrow in the figure). That is, the GOD activity is regulated by the physical state of coating bilayers; it is largely reduced in the fluid liquid crystalline state of bilayers above their T, compared with bilayers in the rigid gel state below T,

In order to clarify the effect of coating bilayers on the enzyme activity, the GOD activity was studied under the various glucose concentrations a t 25"C, where the enzyme activity was most affected with coating bilayers. Lineweaver-Bulk plots of bilayercoated or uncoated GOD capsules are shown in Figure 2, and apparent Michaelis constants (K,) and maximum velocities (V,,,,,) of GOD capsules were summarized in Table I. The K, value of the bilayercoated GOD capsule was 3.2

TABLE I Apparent K , and V,, of GOD Capsule Membranes at 25%

K m Vrn" ( M )

Uncoated 0.10 20 Bilayercoated 0.32 18

(pg glucose rnin-' mg GOD-')

598 OKAHATA, HACHIYA, AND SEKI

t -61 , , Tc 1 1 I

31 3 2 3 3 3 4 3 5 3 6

1-j K)’ K-’

‘C

Fig. 3. Arrhenius plots of the glucose permeation across capsule membranes (GOD is not immobilized): (a) the uncoated capsule, (b) the bilayercoated capsule. An arrow shows the phase transition temperature of coating bilayers.

times larger than that of the uncoated GOD capsule, although both GOD capsules gave almost the same V,, values. This clearly indicates that the large activity decrease in the fluid state of coating bilayers above T, is not explained by the denaturation of an enzyme by covering amphiphiles, but by decreasing the apparent affinity between GOD and glucose by coating bilayers. This is also confirmed by glucose permeation experiments across the bilayercoated capsule membrane.

The permeability of glucose trapped in the inner aqueous phase from the bilayer- coated capsule was also affected with the phase transition of coating bilayers. Figure 3 shows Arrhenius plots of permeation constants (P) of glucose across the uncoated and bilayercoated capsule membranes (GOD is not immobilized). In the case of the uncoated capsule, the straight plot of log P vs. T - was obtained. On the other hand, the Arrhenius plot of the bilayercoated capsule gave the inflection near T,of coating bilayers (shown as an arrow) and the permeability was also decreased above their T, compared with below T, Both the substrate approach to an enzyme and the glucose permeation decrease above the T, of coating bilayers; this phenomenon may be explained as follows. At temperatures below To the glucose permeation or the substrate approach may be relatively easy because of the defective pores in the rigid coating bilayers. Since these defects disappear in the fluid liquid crystalline state of bilayers, the glucose permeation and the substrate approach decrease above the T, of the bilayers.

The activity of enzymes in biomembranes or reconstituted liposomal membranes is reported to increase in the fluid bilayers above their T, relative to the rigid bilayers below T,*l1 It is interesting that the activity of the enzyme immobilized on the capsule membrane is reduced above the T, of the coating bilayers, although the detailed explanation is not yet confirmed. Although GOD is a water-soluble enzyme rather than a membranebound enzyme, the bilayercoated, GOD capsule membrane has an advantage for the reconstitution system of biological membranes; a large inner aqueous phase, and a physically strong capsule wall with bilayer properties. The generality and application of this study, using other membrane enzymes and coating bilayers, is currently in progress.

The authors wish to express their thanks to Ishikawa Seisakusho for the use of H202 elec- trodes and to Amano Pharmaceutical for the present of an enzyme (GOD).

ACTIVITY CONTROL OF IMMOBILIZED ENZYME 599

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Received February 20, 1984 Accepted May 2, 1984