Characterization of the biochemical behavior of glucose oxidase entrapped in a polypyrrole film

Characterization of the Biochemical Behavior of Glucose Oxidase Entrapped in a Polypyrrole Film

Guy Fortier* and Daniel Belanger Groupe de recherche en enzymoloqie fond?men<ale et appliquhe (GREFA), Dkpartement de Chimie, Universite du Quebec a MontrkaJ C.P. 8888, Succ.A, Montrhal, Qukbec, Canada, H3C 3P8

Received March 19, 1990Mccepted October 23, 1990

This article reports the characterization of the biochemical behavior of glucose oxidase entrapped in polypyrrole. The immobilization of glucose oxidase in a polypyrrole film was performed by entrapment during the electropolyrnerization of pyrrole at a platinum electrode poised at 0.65 V vs. SCE in aqueous solution in a one-compartment electrochemical cell. Thin films of polypyrrole (0.11 pm) were obtained and the entrapped enzyme obeyed Michaelis kinetics, indicating no diffusional constraints of the substrate. Our results indi- cate that the entrapped glucose oxidase is more resistant to denaturation conditions such as alkaline pH and temperature (50 and SOT) than the soluble form of the enzyme. The autoinactivation constant for the entrapped enzyme was also determined in presence of 0.25M of glucose and was 6.19 x min-’, i.e., corresponding to a half-life value of 20 h. The results reported here show clearly that polypyrrole matrix has a strong stabilizing effect on the stucture and on the activity of glucose oxidase. Key words: Polypyrrole glucose oxidase immobilization autoinactivation - thermodesactivation stability

INTRODUCTION

Glucose oxidase (E.C. No. 1.1.3.4) is a widely use enzyme as well for quantitative determination of glucose concentration than for glucose transformation in potential industrial processes. However, the glucose oxidase can be inactivated by hydrogen peroxide formed during the transformation of glucose to gluconic a ~ i d . ’ ~ , ’ ~ , ~ ~ The glucose oxidase can be also autoinactivated during continuous utilization by a mechanism not yet well un- derstood. The enzyme-substrate complex seems more susceptible to a~toinactivation~ than the enzyme itself. Recently, indirect evidences have suggested that the autoinactivation can, in part, result from the chemical reaction of the phosphate groups, brought by FAD, re- sulting in the formation of a bridge between two amino acids in the backbone of the en~yrne.’~

Success in stabilization of the glucose oxidase activity against autoinactivation and hydrogen peroxide inactivation will find direct application in the production of the gluconic acid which is an important food addi- tive.17 A stable immobilized glucose oxidase system will offer considerable advantages over the traditional fer-

*To whom all correspondence should be addressed.

Biotechnology and Bioengineering, Vol. 37, Pp. 854-858 (1991) 0 1991 John Wiley & Sons, Inc.

mentation process which is nonsterile, requires long fer- mentation time, use glucose for cell metabolism and releases by-products. Glucose oxidase finds other industrial applications in the beer industries2’ and in egg transformation .32

The coimmobilization of the glucose oxidase with scavenger such as catalase’ or the immobilization of the enzyme alone onto a platinum electrode, poised at a potential of +0.7 V vs. SCE, were carried out to limit the action of hydrogen peroxide” but not the autoinactivation of the enzyme. The substitution of oxygen by quinoidal acceptor^^,^,^^^'^ or by ferrocene derivatives,’ acting as final electron acceptor, have reduced the rate of the autoinactivation of the enzyme but introduced soluble chemical substances in the process. Today, part of the research is aimed at increasing the stability of the glucose oxidase activity by using new immobilization techniques and by using the advantage of electrooxidation of hydrogene peroxide.

New avenue in enzyme immobilization has originated from the electrochemistry field dedicated to polymer- modified electrode. More specifically, it is based on the physical entrapment of an enzyme during the electropolymerization process of soluble monomeric units of p y r r ~ l e , ~ ~ - l ~ , ~ ~ N-methylpyrrole,’ indole:’ or aniline.33 These monomers can be polymerized and deposited at the surface of various conducting substrates when the electrode potential is made sufficiently positive to oxi- dize the monomers.’0,” Conducting substrate such as platinum acted as solid support during the polymer formation and it also allowed, if poised at an appropriate potential, the electrochemical oxidation of hydrogen

It is well known that the electrooxidation of pyrrole in aqueous solution produces adherent polymer coatings at an electrode surface’ and simultaneously incorpo- rates the enzyme present in the electrodeposition solu-

The film thickness of polypyrrole is easily tion*13-’5,37 controlled by the amount of charge passed during the electropolymerization procedure.” Recently, it was demonstrated that the incorporation of platinum mi- croparticles in the polypyrrole film increased the rate of hydrogen peroxide electro~xidation.~ In this respect, polypyrrole offers excellent perspective as immobiliza-

peroxide.2,13-15,31,33.37

CCC 0006-3592/91/090854-05$0400

tion matrix for glucose oxidase that can circumvent problem related to inactivation.

In the present work, the effect on the catalytic current of the GOD concentration in the electrodeposition solution is studied. Also, we report the effects of the polypyrrole film on the biochemical behavior of the glucose oxidase in term of pH stability and in term of thermoinactivation kinetic. Finally, the autoinactivation rate of the immobilized glucose oxidase in a polypyrrole film is reported when the enzyme film is used in a microbioreactor electrochemically assisted under a continuous flow of unbuffered glucose solution.

MATERIALS AND METHODS

Materials

Glucose oxidase (GOD), type VII-S (E.C. No. 1.1.3.4), was purchased from Sigma Chemical (St. Louis, MO) and the pyrrole (PP) from Aldrich (St. Louis, MO). The pyrrole was distilled daily. The glucose solution was prepared in deionized water, filtered through a 0.22-pm filter (Millipore, USA).

Methods

PPIGOD Film Preparation

The platinum substrate was prepared as previously de- ~cribed.’~ Electrochemical polymerization of pyrrole was performed in a one-compartment cell at room temperature on a platinum disk (A = 0.28 cm’) with a platinum flag as auxiliary electrode. A saturated calomel electrode (SCE) was used as the reference electrode. The electrodeposition solution was composed of 0.3M pyrrole and various amounts of GOD in a final volume of 2 mL of 10 mM degassed KC1 solution, pH 6. The polypyrrole/glucose oxidase (PP/GOD) films were grown potentiostatically at +0.65 V. The film was washed for 3 min in phosphate buffered salt solution (PBS) containing 125 mM NaCL2.7 mM KCl, and 10 mM phosphate buffer, pH 7.5, der mild stirring. Before use, cyclic voltammetry wa performed in 0.1M aqueous KC1 to electrochemically characterize the polypyrrole films.4 The coated electrode was stored in phosphate buffer solution at 4°C when not used. The total charge (mC/cm2) is evaluated from the area under the anodic current- time trace recorded during the formation of the film. The film thickness is then estimated by assuming that 45 mC/cm’ of charge yielded a 0.1-pm-thick film.”

Assay of Activity for Soluble and Entrapped GOD

The glucose oxidase activity is related to the amount of hydrogen peroxide generated in presence of 20 mM glucose in 0.1M phosphate buffer solution, pH 7. The hydrogen peroxide produced by the enzymatic reaction is quantified by measuring the current corresponding to

its oxidation at the surface of a platinum electrode held at the potential of +0.7 V.” The solution was kept under gentle stirring at 200 rpm using a microprocessor- controlled stirring plate. After the current background was stabilized, the glucose stock solution was added and the current-time response was recorded. The catalytic steady-state current was usually reached after about 15 s.

Determination of Kinetic and Deactivation Parameters

The apparent kinetic parameters, KmaSp and Z,,,, were obtained by iterative nonlinear curve fitting of the raw values of the catalytic current at steady state generated versus the glucose concentrat i~n.~~ The regression fit- ted a modified Michaelis-Menten equation’:

where Z, is the observed catalytic current and I,,, is the maximal catalytic current. In addition, mass transport limitation of substrate and co~ubs t r a t e ’~ .~~ can be detected qualitatively by deviation of the linearity on a Lineweaver-Burk

The thermoinactivation constants for soluble and immobilized glucose oxidase were determined by nonlinear curve fitting using a double exponential model corresponding to a two stage deactivation process?l The soluble or the immobilized enzyme was incubated at 50 and 60°C and the activity was periodically evaluated at 25°C.

Determination of Autoinactivation Constant of the Entrapped GOD

The enzyme electrode was immersed in a 25-mL vessel containing distilled water at 25°C. Continuous addition of 0.25M unbuffered aqueous solution of glucose, pH 6.5, was flowed at a rate of 0.18 mL min-’ in the vessel using a peristaltic pump. The current generated was recorded over a period of 40 h. The autoinactivation constant (k,) was calculated from a first-order decay model7 from the plot of residual activity versus react ion time.

Instrumentation

All electrochemical experiments were carried out in conventional one-compartment cell. Potentials were applied to the cell with a bipotentiostat (Pine Instru- ments, Inc., model RDE4). All potentials were mea- sured and reported versus a saturated calomel electrode (SCE). Current time responses and voltammograms were recorded on a XYY‘ recorder (Kipp & Zonen, model BD91) equipped with a time base module. For autoinactivation studies, a recorder YT model SE120 from ABB Metrawatt was used.

FORTIER AND BELANGER: POLYPYRROLE AS A MATRIX 855

RESULTS AND DISCUSSION

Influence of GOD Amounts in the Deposition Media on the Catalytic Current

The kinetic curves obtained with different amounts of enzyme in the electrodeposition media are presented in Figure 1. The GOD concentration in the electrodeposition solution varied from 25 to 500 U/mL and the catalytic currents were recorded with glucose concentration ranging from 1 to 95 mM. A decrease in the catalytic current is observed when the concentration of glucose oxidase was raised from 25 to 500 U/mL. This could be related to a local decrease of glucose and oxygen concentrations at the interface of polypyrrole film/ platinum. These decreases resulted from an increase of glucose consumption at the interface of polypyrrole film/bulk solution due to higher loading of GOD in the film. Thus, the hydrogen peroxide mainly diffuses to the stirred bulk solution and cannot be detected at the surface of the platinum electrode. The decrease of the amperometric response can also be brought about by a slight increase of film thickness as the enzyme concentration in the deposition solution is in~reased.’~ Thus, we propose that these two factors are responsible for the observed behavior.

The amount of glucose oxidase incorporated in the film is difficult to estimate by using the Imax value. In the present case, the I,,, value represents only the amount of hydrogen peroxide electrooxidized at the platinum electrode and it is not representative of all the hydrogen peroxide formed in the film (see Fig. 1). The determination of the hydrogen peroxide formed in the film is complex and its evaluation requires taking into account the diffusion of hydrogen peroxide, the enzyme amount in the film, the film thickness, and the stirring speed of the solution. Experiments are currently

5.0 I

h 2 4,o: v Y

3,0{ a

.Y 2,o:

1,o:

L

V

c h m - u

0.0 0 20 40 60 80 100

Glucose (mM)

Figure 1. Effect of glucose oxidase concentration in the electropolymerization solution on the polypyrrole film activity. The glucose substrate varied from 1 to 95 mM and the catalytic current was evaluated at steady state at a potential of +0.7 V in a phosphate buffer, pH 7, under stirring (200 rpm) provided by a stirring plate. Enzyme concentration were 25 U/mL (€!-); 65 U/mL (-B-); 250 U/mL (-0-); and 500 U/mL(-0-).

in progress in our laboratories using rotating ring-disk electrode to evaluated the contribution of these parameters on the current generated. It was reported previously that the amount of GOD incorporated in a polypyrrole film increased linearly with the amount of GOD in the electrodeposition ~ o l u t i o n . ~ ” ~ ~ The values for I,,, and the apparent K , for various amount of enzyme in the electrodeposition solution for a 0.11-pm-thick film are given in Table I.

At the thickness of 0.11 pm, no deviation from the linearity on Lineweaver-Burk type plots is observed (results not shown) for all GOD concentrations indicating that limitation by internal diffusion of co-substrate and substrate is not o ~ c u r r i n g . ’ ~ , ~ ~ In Table I, the apparent Michaelis-Menten constants (KmaPp) are also sum- marized. The kinetic parameters were calculated from Figure 1 using a nonlinear regre~sion.’~ The Kmapp was independent of the enzyme amount incorporated in the film and the immobilized enzyme obeyed Michaelis- Menten kinetics. The average value of the Kmapp for the entrapped GOD is 31 mM and is similar to the litera- ture value of 33 mM35 for the soluble enzyme in air saturated solution. This suggests that the film does not impose more diffusional constraints on oxygen and on glucose than those existing in the bulk solution.

Effect of pH on the Catalytic Activity of Entrapped GOD

The pH dependence of the relative activity of the GOD entrapped in polypyrrole film was compared with the pH dependence of the soluble GOD and is depicted in Figure 2. There is no apparent shift of the optimal pH (5.5-6.0) following the immobilization of the enzyme as compared to the relative activity of soluble glucose oxidase. The immobilization procedure does not increase the acidic pH stability of the enzyme. On the other hand, at basic pH, the immobilized enzyme shows a fivefold increase in the stability compared to the soluble enzyme. At pH 8.0, the immobilized enzyme re- tained 50% of its relative activity compared to 10% for the soluble GOD. The reason for this increase at basic pH is unclear. Since polypyrrole is a cationic polymer in its oxidized state, a shift of the pH profile to lower pH is expected.18

Table I. Effect of the concentration of the GOD in the electropolymerization solution on apparent Michaelis constant (Km, app ) and maximal catalytic current (Imax) in the 0.11-pm-thick polypyrrole film.

[GODIso~ution K m , app. Imax

(U/mL) (mM ? std. dev.) (pA/cm2)

25 29.5 ? 0.9 21.0 65 33.0 ? 1.0 17.8

250 29.6 & 1.3 12.8 500 31.0 ? 2.1 9.8

856 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 37 APRIL 1991

- 80:

.= 60:

g 40:

h

* u m

* .-

.- * m 2 20: f 2,O 3,O 4,O 5,0 6,O 7,O 8,0 9,0

PH Figure 2. pH dependence of glucose oxidase enzymatic activity. The relative activity was evaluated against 20 mM glucose: soluble GOD (-0-) and entrapped GOD (-0-).

Thermodeactivation of Entrapped GOD

Comparative studies of thermodeactivation of the soluble and the entrapped GOD at temperature of 50 and 60°C are presented in Figure 3. Both forms of the enzyme were incubated at the mentioned temperatures and their residual activities were periodically evaluated in term of catalytic current generated in presence of 20 mM of glucose at room temperature. The curves of Figure 3 were analyzed as mentioned in the Materials and Methods section. The k , and k2 decay constants are given in Table 11. These constants represent the rate of transition of the enzyme structure to intermediate forms based on a two stage process.'l

E l E 2 -% E 3 (4)

where E l is the native form of the enzyme; E 2 is an active intermediate form; and E 3 is the denaturated form of the enzyme.

At 50"C, the soluble enzyme loses 25% of its activity in 150 rnin in comparison to a loss of 10% for the entrapped enzyme during the same period of time. At 60"C, the difference in the residual activity of the soluble and the immobilized enzyme is larger. The soluble enzyme loses 70% of its activity in less than 30 min in comparison to a decrease of 25% for the entrapped enzyme. At 60"C, the activity of the entrapped enzyme decreases to 50% of its initial activity only after a period of 200 min. The polypyrrole matrix shows a stabilizing and a protective effect by factors of 2 and 5 based on half-life values for the free and entrapped glucose oxidase at 50 and 60"C, respectively, as described in Table 11.

- i\\' 6"

0 50 100 150 200

Time (rnin) Figure 3. Thermodeactivation kinetics of soluble glucose oxidase (-0-,-M-) and entrapped glucose oxidase in polypyrrole film (-O-,-U-) at 50°C (-0-,-0-) and 60°C (-M-,-O-), respectively.

The rate of transition from El to E 2 forms for the entrapped enzyme is 9 x low4 min-' which is lower than the value of 15 x min-' retrieved for the soluble enzyme at 50°C. This difference is accentuated at 60°C (see Table 11). On the other hand, the second constant (k2) representing the rate of transition from the second active form of glucose oxidase to an inactivated form is higher for the entrapped enzyme than for the soluble form. The results on the rate of thermoinactivation sug- gest that the first transition step is the rate determining step for the denaturation of the immobilized enzyme in contrast to what is observed for the soluble enzyme.

Autoinactivation of Entrapped GOD in Presence of Constant Glucose Amount

The autoinactivation of entrapped glucose oxidase is an important parameter when the aim of immobilization was to extend the life of the biocatalyst in the bioreactor for continuous production of gluconic acid. In this ex- periment (Fig. 4), the concentration of glucose was held constant by continuous supply of unbuffered aqueous glucose solution 0.2544 in the 25-mL cell at a flow rate of 0.18 mL/min. In these conditions, the autoinactivation constant (ki) is 6.19 (kO.11) x min-'. The inactivation of the enzyme follows first-order decay kinetics as suggested previously.' The half-life for entrapped glucose oxidase in polypyrrole at 25°C in presence of 0.25M of glucose is 1120 min based on a triplicata experiments. This value compares very well with the value extracted from Figure 5 of ref. 7. It is shown that

Table 11. trapped GOD incubated at 50 and 60°C in the 0.11-pm-thick polypyrrole film.

Deactivation constants ( k , and k Z ) and half-life ( t l , z ) values for soluble and en-

Soluble GOD Polypyrrole/GOD

Temperature ki k2 t112 ki k2 t112

("C) (lo4 min-') (lo4 min-I) (min) (lo4 min-I) (lo4 min-') (min)

50 15 513 360 9 743 700 60 576 60 22 18 243 100

FORTIER AND BELANGER: POLYPYRROLE AS A MATRIX 857

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204 " " " " " " " " " " " " ' 0 500 1000 1500 2000 2500

Time (min)

Figure 4. Autoinactivation kinetic of glucose oxidase entrapped in polypyrrole at 25°C. The glucose solution was prepared in water at a concentration of 0.25M and was flowed in the vessel at a rate of 0.18 mL min-'. The film was 0.17 pm thick and was formed in the presence of 100 U/mL GOD in the deposition media.

the half-life of glucose oxidase chemically immobilized at the surface of a glassy carbon electrode is about 600 min for similar autoinactivation experimental conditions. Thus, the use of polypyrrole as immobilization matrix for glucose oxidase gives a twofold decrease of the autoinactivation rate in presence of high glucose concentration as compared to autoinactivation rates obtained for the soluble and chemically immobilized glucose o x i d a ~ e . ~ ~ ' , ~ ~ , ~ ' , ~ ~

CONCLUSIONS

The polypyrrole polymer used for glucose oxidase entrapped shows excellent characteristics and it compares advantageously to others GOD immobilization proce- dures. This procedure is easy and fast to perform and can be applied to the fabrication of a bioreactor electro- catalytically assisted for gluconic acid production as well as for a glucose biosensor.

The protective effect of the polymer for glucose oxidase against autoinactivation and thermodeactivation can be related in part, to the rigidity of the polymer itself as well as a close moulding of the structure of the enzyme with the polypyrrole during the electropolymerization process. Research is actually underway in our laboratory to increase the amount of enzyme entrapped with the aim of development of an highly pro- ductive bioreactor for gluconic acid. Also, recent development in the chemistry of polypyrrole have per- mitted the covalent binding of a redox mediator to the polymer structure that will allow new perspectives in anaerobic bioreactor design that might reduce the autoinactivation of glucose oxidase and also the inactivation of the enzyme by hydrogen

This work was supported by the Natural Sciences and Engi- neering Research Council of Canada, the "Action struc- turante" program of the QuCbec Government and the Universitk du Qukbec h MontrCal.

858 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 37, APRIL 1991

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Characterization of the biochemical behavior of glucose oxidase entrapped in a polypyrrole film