A biosensor stabilized by polyethylene glycol for the monitoring of hydrogen peroxide in organic solvent media

ELSEVIER

A biosensor stabilized by polyethylene glycol for the monitoring of hydrogen peroxide in organic solvent media Hyun Joe,* Young Je Yoo,* and Dewey D. Y. Ryut *Department of Chemical Engineering, Seoul National University, Seoul, Korea; and ‘Department of Chemical Engineering, University of California, Davis, California

Since many chemical/Biochemical reactions containing hydrogen peroxide are performed in organic solvent media, the development of a stabilized biosensor in organic solvent media is very crucial. A stable hydrogen peroxide sensor with a wide measurement range and a long life in organic solvent as well as aqueous solution was developed. To maintain the stability of the sensor in the organic solvent system, catalase was mixed with polyethylene glycol (PEG). The treatment could apparently enhance the stability of the enzyme activity. The induction of hydrogen bonding between enzyme and PEG was assumed to be the possible reason for the stabilization, and was also confirmed by infrared spectrophotometry and circular dichroism (CD). The stability of the enzyme depended upon the content and molecular weight of PEG. PEGS (MW 3,3504,000) with a mixing ratio of 0.2 g PEG to 2.8 x 104 catalase activity units showed the highest stability level. The biosensor developed in the present study, therefore, worked well even in 50% (v/v) dioxane solution for 2 days; 90% of the initial activity was maintained. The detection limit of the sensor was about 140 mM and the response time was 40 s in aqueous buffer and 60-90 s in the organic solvent.

Keywords: Biosensor; enzyme stabilization

hydrogen

peroxide;

catalase;

polyethylene

Introduction The detection and control of the hydrogen peroxide concentration are increasingly required in various industrial processes such as phenolic polymerization, textile bleaching, pulping, and metal purification, food sterilization (e.g., aseptic carton packing process, continuous milk pasteurization), contaminant removal, etc.‘,* Many analytical methods3” are available for the detection of hydrogen peroxide chemiluminescence, fluorimetric, am(e.g., calorimetric, perometric, etc.). Although these methods are known to be time consuming and very sensitive to other foreign species, the procedures are still being used. With the development of biotechnology, studies have been conducted to monitor the concentration of hydrogen peroxide by using on-line measuring techniques. In 1974,

Address reprint requests to Dr. Young Je Yoo, Deartment

of Chemical Engineering and Institute of Genetics and Molecular Biology, Seoul National University, Seoul 151-742, South Korea Received 25 February 1995; revised 10 August 1995; accepted 1 September, 1995.

Enzyme and Microbial Technology 19:50-56, 1996 Q 1996 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

glycol; polyvinyl

alcohol membrane;

organic

solvent;

Aizawa et al.’ developed the hydrogen peroxide sensor based on an amperometric dissolved-oxygen (DO) electrode and catalase entrapped in collagen membrane. Racek and Peter* devised a biosensor for the determination of hydrogen peroxide based on the catalase activity of human erythrocytes. Recently, a novel catalase-immobilized acoustic emission (AE) sensor was developed.9 An audible acoustic wave counting through a piezoelectric transducer was established for about 2-125 rrt~ hydrogen peroxide in aqueous solution. However, these developed biosensors need high operational stability in order to function under severe conditions such as high temperature or in an anhydrous organic solvent. Since it is becoming increasingly important to measure the substrate concentration in organic solvent systems, the development of a new biosensor system which has a long-term operational stability in organic media is very crucial. lo In our preliminary experiments,” a polyvinyl alcohol (PVA) membrane was used to overcome the substrate inhibition of catalase. The developed sensor had a wide linear detection range and favorable dynamic responses. However, it was impossible for the biosensor to work in organic sol-

0141-0229/96/$15.00 SSDI 0141-0229(95)00178-6

Biosensor vent because the activity of catalase was apt to deteriorate rapidly in an organic solvent system. Indeed, in the organic media, stabilization of the enzyme activity is very important and becomes the key point in many engineering aspects.“.13 To cope with this problem, we introduced polyethylene glyco1 (PEG) as a catalase stabilizer to construct a highly stabilized hydrogen peroxide biosensor working in an organic solvent system. In some cases, water-soluble polymers were used to make polymer-induced agglomerates and then these agglomerates could be possibly recovered in the form of stabilized polymer-enzyme complexes.‘4-‘6 PVA, polyethylene oxide (PEO), and their derivatives such as polyethyleneimine chloride and polyvinyl pyridine chloride were used for this purpose. In the present study, various molecular weights of PEG (3,350; 6,000; 8,000; and 33,500) were chosen as a catalase stabilizer to maintain the storage stability of the enzymes in the catalytic conversion unit.

Materials and methods Construction

of the biosensor system

In the construction of the biosensor system, catalase was premixed with PEG to maintain the stability of the sensor in an organic solvent. The nonporous hydrophilic PVA membrane was used as an enzyme protection barrier in order to prevent direct exposure of an organic solvent and inhibition of hydrogen peroxide. When hydrogen peroxide permeates the PVA membrane, the PEG-mixed catalase decomposes hydrogen peroxide and generates oxygen which is subsequently detected by a galvanic-type dissolvedoxygen (DO) electrode via oxygen permeable hydrophobic teflon (PTFE) membrane. The enzymes (2.8 x lo4 activity units) mixed with PEGS (MW = 3,350; 6,000; 8,000; and 33,000) were immobilized between the PVA and PTFE bilayer membrane outside of the galvanic type DO electrode. For the immobilization, five ml of sodium phosphate buffer (Na,HPO,/NaH,PO, pH 7.0.0.1 M) containing a catalase/polymer mixture (2.8 x lo4 U catalase 0.2 g-’ PEG) or catalase alone was uniformly sprayed onto the inner layer of the PVA membrane by using a microsyringe. Afterwards, the membrane was left at 4°C for about 4 h in a refrigerator and then the enzyme could be adsorbed on the membrane. The current generated from the DO probe was analyzed via 10 kfl variable resistance.

Materials Catalase (bovine liver purified powder; EC. 1.11.1.6, 2,800 U mg-’ solid) was purchased from Sigma Chemical (St. Louis, MO, U.S.). Hydrogen peroxide solution (30 wt%) and PEGS with the MWs 3,350; 6,000; and 33,000 were from Junsei Chemical (Tokyo, Japan). PEG with the MW of 8,000 was from Aldrich. 1,4dioxan (dioxane) was purchased from Avondale Laboratory (Banbury, England). The galvanic-type DO electrode (I.D. 0.8 cm) and polytetrafluoroethylene (PTFE) membrane were purchased from DKK Co. (Japan). The DO analyzer was from New Brunswick Scientific (Model DO-50).

Preparation

of cross-linked

PVA membrane

The used PVA membrane support was prepared by using the phase inversion techniques. ” PVA powder (MW 500 and 2,000) was gradually dissolved in deionized water at over 60°C and boiled for 1 h to make a 6.7 wt% PVA aqueous solution. After overnight cooling at 18°C the PVA-casting solution was degassed under the

for hydrogen

peroxide

in organic

solvent:

H. Joo et al.

vacuum condition. Afterwards, the PVA solution was cast onto the glass plate and one side (upper exposed side) of this membrane was subsequently cross-linked with formaldehyde in order to give high mechanical strength against swelling. Therefore, the developed membrane has a nonporous and asymmetric structure. The membrane thickness was measured in a wet state with a micrometer (0.2 mm thickness).

Enzyme assay The activity of catalase was measured using the spectrophotometric method. One unit is defined as the quantity of enzyme that decomposes 1.0 pmole H,O, per minute at H 7.0 and 25°C. The buffer used was sodium phosphate (NqHP x ,MaH,PO,, pH 7.0, 0.1 M). With the conventional assay procedure,i8 it was difficult to measure the decomposition rate of hydrogen peroxide due to the interference by generated oxygen. Hence, we used the modified assay method by adding oxygen zero-adjusting solution as previously reported.” The stock solution of catalase was prepared by adding 2.8 x lo4 U catalase in 10 ml 0.1 M sodium phosphate buffer, pH 7.0 at 25°C. This solution (1 ml) was pipetted into the reaction cuvettes which contained 2 pl of oxygen zero-adjusting solution and hydrogen peroxide solution. Absorbances at 240 nm were measured by an UV-VIS spectrophotometer (Kontron Co., UVICON 930).

Measurement

of retention activity

Aqueous-organic solvent mixtures were prepared by varying the volume fractions of 1&dioxan in 0.1 M sodium phosphate buffer, pH 7.0. In order to test the stability of catalase, the assay procedure outlined above was repeated after incubation of catalase for various volume fractions of dioxane solvent. Two experiments were performed to investigate the protecting effect of the membrane against organic solvent. One was the activity measurement of free enzyme and another was the measurement of the activity of catalase entrapped in a bilayer membrane (PVA and PTFE).

Permeability

test for PVA membrane

Another important parameter is the membrane permeability. Since permeability greatly influences the dynamic responses of the sensor, the membrane permeability (P,) was investigated. The Lewismeasuring device was prepared in type I9 diffusional-permeability our laboratory and operated by adequate stirring in each compartment. Because the membrane permeability is governed by the solute diffusion and membrane solubility (boundary-layer resistance), the variation of the permeability was tested for various dioxane concentrations. The simple dialysis-type equationzO was selected and modified to measure the specific permeability of the hydrogen peroxide. The following equation was used: p

= (ACdAC,)ViS, m

(A,)(r)

(1)

where P, is the specific permeability of the membrane to hydrogen peroxide and the effective permeable area (A,,,) was 0.503 cm.2 AC, is the concentration difference of both sides of the membranes. AC,=, is the initial concentration difference, Vi is the inside volume of the measuring chamber (same as the volume of electrode, 0.503 cm2), and 6, is the membrane thickness (0.02 cm).

Analysis of CD and IR spectrum To investigate the PEG-induced stabilization of catalase activity in the organic solvent, the whole region of the infrared spectrum was

Enzyme Microb. Technol.,

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Papers investigated by varying the concentration of PEG (0.0241 g ml-’ solution). The enzyme concentration was fixed to 5,600 U ml-’ and the same content of PEG (MW 3,350) and dioxane was referenced in the spectral double-beam system to eliminate the spectral effects of the PEG and dioxane. Circular dichroism (CD) measurements were taken at room temperature with a JASCO J-600 spectropolarimeter. The denaturation of catalase in the organic solvent was measured in the near and far UV region (200-300 nm) using a measuring quartz (0.1 cm light path). 0

in organic solvent

Biosensors using enzymes are often limited by their loss of catalytic activity. This is also true in the case of organic solvent systems. Since the changes in enzyme activity alter the output signals of the biosensor, enzyme-loaded biosensors cannot be used in organic solvent systems. Figure 1 shows the stabilities of catalase in 100 mu phosphate buffer pH 7.0 and 50% or 75% (v/v) dioxane-mixed phosphate buffer solution. In the case of free enzyme, the activity loss in 50% (v/v) dioxane solution after 3 h was nearly 80% of the initial activity. A more severe loss of catalase activity was observed when 75% (v/v) dioxane solution was applied. The activity of catalase began to decline after 15 min when the enzyme was exposed to an organic solvent. However, the activity of catalase deposited on the inner surface-layer of the PVA membrane was considerably higher than that of the free enzyme state at the same condition. The stability of the enzyme in the PVA membrane could be maintained to some degree (80% of the initial activity was observed after 3 h) but the membrane did not have complete protection against enzyme denaturation when it was exposed to the organic solvent mixture. Figure 2 shows the inadequate stability of the biosensor for the hydrogen peroxide mea-

0

60

120

180

Incubation time(min) Figure 1 Retention of catalase activity in the various volume fractions was of dioxane with aqueous buffer (0.1 M sodium phosphate, pH 7.0). The activity of enzymes was separately tested by using the free- and membrane-entrapped enzyme. Enzyme + PVA membrane in aqueous media, 0; free enzyme in aqueous media, a; enzyme + PVA membrane in 75% (v/v) diox; free enzyme in 50% ane, q; free enzyme in 75% (v/v) dioxane, (v/v) dioxane,!ZI

52

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Time(min)

Results and discussion Stability of biosensor

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60

0

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00

Time(min)

Figure 2 Comparison of biosensor stability in aqueous or organic solvent system (measuring concentration of hydrogen peroxide: 91 mM). The stability of the biosensor in aqueous phosphate buffer (a); and the stability of biosensor in 50% (v/v) dioxane solution (b). Both of the biosensors were not pretreated with enzyme stabilizer such as PEG

surement in 50% (v/v) dioxane-contained aqueous solution. Both sensors in the two experiments were constructed using the enzymes immobilized onto the PVA membrane. Although the stability of the sensor was successfully maintained for a long time in an aqueous buffer, the stability of the sensor in the organic solvent was not maintained for a long time (Figure 2b). The decline in the measured curves was mainly due to the denaturation of enzyme activity in the catalytic-sensing unitsI as evidenced by the results of Figure 1 and Figure 2b. Recently, some researchers have reported the effect of additives on enzyme stabilization. Many proteins including RNase, phage T4 lysozyme, oxygenase, Japanese peroxidase, etc. usually denatured in thermal or nonaqueous organic media could reverse back to the active conformations21,22Nozaki and Tanford have studied the evidence to show that this is valid for aqueous urea, ethylene glycol, and ethanol. Therefore, we tried to apply such principles to develop a stabilized biosensor system. Figure 3 shows the performance of the biosensor using various MWs of PEG as enzyme stabilizer in the 50% (v/v) dioxane solution. In this experiment, catalase was mixed with the various PEGS. The three curves in Figures 3a-3c showed highly stabilized sensing abilities even when the sensor was exposed to an organic solvent. As shown in Figure 3d, when a high MW of PEG (i.e., 33,500) was used, the initial activity drop was remarkable and the enzyme activity eventually decreased to 20% level of the initial activity. Figure 4 shows a good linear relationship for the developed biosensor both in aqueous and 50% (v/v) dioxanecontaining aqueous solution. The detection limit of the sensor was O-145 mu H,O, in aqueous solution and about O-151 mu H,O, in 50% (v/v) dioxane solution. The linear range in an aqueous solution was larger than that obtained by Wentzell et aL9 Figure 5 shows the dynamic responses to the developed biosensor. Output signals were recorded using a DO analyzer after forcing the step changes of hydrogen peroxide concentration (the points are indicated by arrows). In the 50% (v/v) dioxane solution, the sensor showed a more sluggish response than in the aqueous solution. The time to

Biosensor

for hydrogen

in organic

peroxide

H. Joo et al.

solvent:

i.;‘l: i:~~ a

10

20

30

40

50

60

0

10

20

Time(min) Q 6

0

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d

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g 6

40

'5 rj

20

s c;

0 0

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10

20

30

40

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4

2

0

Measuring time(min)

60

Figure 3 On-line measurement of biosensor and enzyme stability in 50% (v/v) dioxane solution. The enzymes were pretreated with: PEG 3,350 (a); PEG 6,000 (b); PEG 8,000 (cl; and PEG 33,000 (d) in the catalytic sensing unit. In all four cases, the concentration of hydrogen peroxide was 110 mM

Figure 5 Dynamic responses of hydrogen peroxide sensor in aqueous and 50% (v/v) dioxane solution. The response to reach the steady state level was about 40 s in aqueous solution (0) and about l-l.5 min in 50% (v/v) dioxane solution (0)

Analysis of PEG-induced reach the steady state level in aqueous solution was about 40 s, but in the organic solvent system, it was about 60-90 s. The response of the sensor in organic solvent was more sluggish than in aqueous solution. The effects are possibly explained by the property of the PVA membrane. Since the solubility of the membrane active layer affects the permeability and the PVA membrane has hydrophilic characteristics,2” the permeability decreased due to the low degree of hydration of the membrane active layer when the content of dioxane increased. Figure 6 represents the relationship between the permeability and volume fraction of organic solvent where the slopes represent the permeability of hydrogen peroxide through the membrane. The permeability is inversely proportional to the dioxane content in the solution.

IO

8

6

protein stabilization

The denaturation of catalase in the organic solvent was observed by circular dichroism (CD) spectropolarimetry. The CD data of polypeptide in the random coil, ol-helical, and p-conformations were investigated by some researchers. In this analysis, the methods of Gratzer and Cowbum were used to predict the conformation changes of catalase. Since catalase possesses six tryptophan and three cysteine moieties,26 the possible changes of large ltotatory chroism for these aromatic chromophores and the cystinyl chromophore were expected in the near UV region. The dichroism changes in this region represent the structural changes of some moieties related to tryptophan and cystinyl residues and were also expected. Figure 7 shows the variations of molar ellipticity of the

0.08

1

0.06

0 0

20

40

60

80

100

120

140

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20

160

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40

50

60

Time(min)

Concentration of hydrogen peroxide Figure 4 Measurement range of hydrogen peroxide using the sensor developed in the present study. The sensor has a favorable linear range of O-145 mM in aqueous solution (C!, regression coeff. = 0.996) and O-152 mM in 50% (v/v) dioxane solution (0, regression coeff. = 0.997)

Figure 6 Permeability of hydrogen peroxide through PVA membrane for various fractions of dioxane in an aqueous solution at 25°C. The measured liquid hydrogen peroxide permeabilities were: 0% (v/v) dioxane, permeaikity = 7.73 x 10m6cm2 s-‘, A; 50% (v/v) dioxane, permeability = 5.4 x IO-’ cm’ s-’ C!; and 90% (v/v) dioxane, permeability = 3.86 x lo-’ cm* s-l, 0

Enzyme

Microb.

Technol.,

1996,

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19, July

53

Papers

1. 2. 3. 4.

PEG O.lg PEG 0.26 PEG 0.3g

PEG 0.4g 6. PEG 0.6g

190

Wave

length(nm)

300

Figure 7 CD spectra of catalase in the near UV region in aqueous buffer or 50% (v/v) dioxane solution (catalase, 0.3 g solid protein ml-’ solution; beam path length, 0.1 cm). Catalase in aqueous phosphate buffer (pH 7.0, 0.1 M sodium phosphate), -; catalase in 50% (v/v) dioxane solution (after 5 min incubation), - - - - - -; and catalase in 50% (v/v) dioxane solution (after 45 min incubation), ......

native and denatured states of catalase. The solid curve represents the native state while both dashed or dotted curves represent the denatured state of catalase in phosphate buffer solution containing 50% (v/v) dioxane. In the far UV region (200-240 nm), the CD spectra of catalase in 0.1 M sodium phosphate buffer, pH 7.0 and spectra in 50% (v/v) dioxane solution showed all negative minimum. The changes in the peak shape and ellipticity values in Figure 7 indicate that the spread of the optical signals are mainly due to changes in the a-helix and P-chains in the organic solvent.27*28The addition of dioxane induced a decrease in magnitude of the negative band centered at 220 nm, but in the near UV region (250-300), the solvent did not induce any dichroism change. As a result, Figure 7 indicated that the denaturation of catalase in the organic solvent was mainly due to the conformational changes of a-helical or P-sheet polypeptide chains. Other expected changes including breakage of disulfide bonds and exposure of aromatic residues such as tryptophan were not observed. Although the catalase denaturation in the organic solvent was observed by CD analysis, there were still many ambiguities on the nature of forces that control the stabilization of the protein. The data for the positions and intensities of the IR spectra allow us to predict the changes of secondary structure and their interactions in the protein. Analyses of the stretching band of the protein amide I (primary C = 0 stretch) and protein amide II (N-H band, C-N stretch) were used for the interpretation of the secondary structure of catalase.27 For most of the proteins, it is generally known that peaks at 1617 cm-’ and 1685 cm-’ are characteristic of the P-sheet structure and peaks at 1635-1640 cm-’ are attributed either to an a-helix or P-parallel sheet. In the study, the presence of intensity vibrations at 1685 cm-’ with respect to the content of PEG (Figure 8) clearly reflects the existence of conformational changes in P-sheet structure and the optimal content of PEG are needed for the changes. However, the structural changes of the a-helix and random coil would be reflected by the band near 1650 cm-’ and at 1647-1670 cm-‘, but it was not observed in the IR study. These results were very similar to those obtained with CD spectra. 54

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1600

1800

nm

Figure 8 Infrared spectra of catalase for various contents of PEG (MW 3,350) in 50% (v/v) dioxane solution. (The concentration of catalase used was fixed at 2.8 x lo4 U/5 ml solution)

Figure 9 shows the strong intensity changes in the 3,400-3,600 cm-’ region which indicate the induction of hydrogen bonding.29 Because PEG has a oxymethylene link in a monomeric unit,30 the induction of hydrogen bonding is possible. The formation of hydrogen bonding in the catalytic antibody with the activated PEG was reported by Inada et ~1.~’ When the 0.2 g PEG was used with the 2.8 x lo4 U catalase (about 0.01 g solid protein), the intensity change of the secondary structure and the hydrogen bonding force reached the highest level. Nearly 96% of the initial activity remained after 3 h in the organic solvent. In Figure 8 and 9, the low level of PEG (co.2 g) did not induce strong hydrogen bonding and the activity of the enzyme was very poor

80

(1)PEG :0.1 g, (2)PEG :0.2g

10

v

1

,,//,,,,,,,,,,,,,,,,,,,/,,,,,

3600

3200

2600

2400

2000

1600

Wave number (cm -‘) Figure 9 Infrared spectra of catalase for various contents of PEG (MW 3,350): 0.1 g PEG per 2.8 x IO4 U-’ catalase (1). 0.2 g PEG per 2.8 x IO4 U-’ catalase in 5 ml 50% (v/v) dioxane solution (2). At the level of 0.2 g PEG, the spectral changes in the secondary structure for the region of 1800-1600 nm and the hydrogen bonding force (3600-3400 nm) have the highest value

Biosensor

for hydrogen

peroxide

in organic

solvent:

H. Joo et al.

(below 50% of the initial activity>. Therefore, strong hydrogen bonding seemed to be the reason for the increased stability of the catalase, and the results conclude that the optimal concentration of PEG exists for the strong hydrogen bonding induction. Vazquez er aL3* reported about the role of PEG as a cytochrome C stabilizer. They suggest that the PEG modifications are important to remove the distortions of the wetted state of the enzyme molecules in watermiscible organic solvent systems. Unfortunately, the results from their experiment were not in agreement with the proposed stabilization mechanism due to the high inactivations of cytochrome C by the localized high concentration of hydrogen peroxide associated with PEG modifications. However in our experiments (Figure 3a-3~) the signals more clearly showed PEG-induced catalase stabilization effects even at high concentrations of hydrogen peroxide since the PVA membrane acts as a barrier against the high hydrogen peroxide concentration. Other interactive mechanisms between PEG and proteins are possible. According to the proposed mechanism by Cleland and Wang, 33 PEG has the ability to interact with the hydrophobic portions of carbonic anhydrase (CAB) by both surface binding and exclusion. However in the case of catalase, unlike the protein CAB (MW 35,000), it might be impossible to maintain the conformation of the large catalase molecule (MW 270,000) by PEG-induced hydrophobic surface binding forces alone. In addition to the effect of the PEG mixing ratio to the enzyme, the choice of an adequate MW (i.e., molecular size) of PEG was also important for the construction of a stabilized hydrogen peroxide biosensor. When the high MW of PEG (33,000) was mixed with catalase in the sensing unit, the biosensor stability was very poor (Figure 3d). Ye et ~1.“~ studied the size effect of PEG on the thermal stability of glucose oxidase. In their experiments, enzymes with extremely low and high MWs of PEG (below 400 and above 20,000) had no protection even when the concentration of PEG increased. However, PEG with MWs between 3,350-8,000 demonstrated more protective effect at low concentrations.

can be applied for constructing more stabilized biosensors or stabilizing enzymes in organic solvent media.

Acknowledgments Dr. Soon Jae Park of LG Biotech Research Laboratory is thanked for his helpful CD analysis and suggestions. This research was in part supported by the Bioprocess Engineering Research Center.

References I.

Kirchner. J. R. Peroxides. In: Kirk-Othmer Encyclopedia ofchemiccd Technology, Vol. 13 (Mark, H. F., Othmer, D. 0.. Overberger, C. G., and Seaborg, G. T., Eds.). John Wiley Ilr Sons, 1987, 12-28

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Chatterjee, U., Kumer, A.. and Sanwal, G. G. Goat liver catalase immobilized on various solid supports. J. Ferment. Bioeng. 1990. 70,429430 Clapp, P. A., Evans, D. F., and Sheriff, T. P. Spectrophotometric determination of hydrogen peroxide after extraction with ethyl acetate. Anal. Chim. Acta. 1989, 218, 331-334 Malehom, C. L., Hinze, W. L.. and Riehl. T. E. Improved determination of hydrogen peroxide or lucigenin by measurement of lucigenin chemiluminescence in organized assemblies. Analyst 1986. 111,941-947 Lazrus, A. L., Gitlin, S. N.. Heikes, B. G., Kok, G. L.. Lind, J. A.. and Shetter, R. E. Automated fluorometric method for hydrogen peroxide in air. Anal. Chem. 1986, 58, 596597

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Racek, J., and Peter, R. Biosensor for determination of hydrogen peroxide based on catalase activity of human erythrocytes. Anal. Chim. Acfa. 1990, 239, 19-22

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Wade. A. P., Sibbald, D. B., Bailey. M. N., Belchamber, R. M., Bittman, B. S., McLean, J. A., and Wentzell, P. 1). Acoustic emission, Anal. Chem. 1991, 9,497A-507A

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Zaks, A., and Klibanov. A. M. Enzyme-catalyzed processes ganic solvents. Proc. Nafl. Acad. Sci. 1985, 82, 3 192-3196

Since the simple addition of PEG as an enzyme stabilizer solved the denaturation problem of catalase in the organic solvent system, the developed biosensor, therefore, showed high operational stability in the organic solvent. The initial activity (90%) remained after 2 days in the 50% (v/v) dioxane solution. The stabilization was possibly due to the induction of hydrogen bonding between enzyme and PEG as evidenced by IR analysis. PEG (MW 3,350-6,000) with the optimal mixing ratio (0.2 g PEG per 2.8 x lo4 U catalase, about 0.1 g solid protein) was best in stabilizing the enzyme. The stabilization method described here differs from that of other sensors constructed using conventional immobilization between catalytic enzyme and solid support. Even though the immobilized enzymes are relatively stable compared to the free enzymes in solution, the disadvantage of these sensors is the low stability of biocatalyst in severe conditions such as anhydrous organic media, high temperature, etc. Therefore, the results obtained in the present study

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Pina. C.. Clark, D., and Blanch, H. The activity of PEG-modified chymotrypsin in aqueous and organic media.. Biotechnol. Tech. 1990,5,333-338 Strege, M. A., Dubin, P. L., West, J. S.. and Flinta, C. D. Complexation between poly(dimethyl-diallyl-ammonium chloride) and globular proteins. In: Downsrrenm Processing and Biosepararion (Hamel. J. P. F., Hunter, J. B., and Sikdm, S. K.. eds.). American Chemical Society. Washington, D.C.. 1990. 158-169 Yamada, S.. Nakagawa, T., and Abo, T. Pervaporation of waterethanol with PVA-fluoropore composite membrane. In: Proc. Fourth Int. Con& Pervaporarion Proc. Chem. Ind. (Bakish, R.. Eds.). Bakish Material Corporation, Englewood, N.J. 1989, 64-74 Chance, B., and Maehly, A. C. Assay of catalase and peroxidases. In: Methods in Enzymology Vol. 2. (Colowick, S. P.. and Kaplan. N. 0.. Eds.). Academic Press, New York, 1963. 764-775

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