Electrochemical methods for estimation of the ferment activity of a concentrate mixture of polyphenol oxidase

509

Bioelectrocirenristry and Bioenergetics, 27 ‘,I9921 509-512 A section of J. Efectroarraf. Chem., and constituting Vol. 342 (1992) Elsevier Sequoia !%A., Lausanne

JEC Short

BB 01476 communication

Electrochemical methods for estimation of the ferment activity of a concentrate mixture of polyphenol oxidase M. Cvetkovska

and T. Grchev

*‘Cyril and Methodius”

V. Bogdanovskaya A.N. Fntrnkirl (Received

hstitrrtc

29 October

University,

Faculty

and M.R.

of Technology

and Mctahrgy,

Skopje

(Yugoslavia)

Tarasevich

of Elcctroclmnistry,

Academy

of Sciences

of the USSR,

Moscow

(Russia)

1991)

Ferments belong to a very important group of biologically active materials which can be used to develop new electroanalytical methods. It has been found that tyrosinase (monophenol mono-oxidase; EC 1.14.181) possesses cresolase and catecholase activity 113. This ferment can be isolated from various materiaIs and its activity can be determined by different methods [2]. It has been shown 133 that tyrosinase extracted from potatoes and adsorbed on a carbon surface increases the rate of the electrochemical reduction of oxygen from neutral aqueous solutions. When the adsorbed ferment is present on the electrode surface the oxygen reduction yields water molecules as a reaction product and the open-circuit potential is very close to the equilibrium potential. A direct correlation was found between the activity of the ferment and its electrocatalytic activity. Another method of estimating ferment activity is based on monitoring oxygen consumption (at a constant potential) during the oxidation of phenol. In this communication the ferment activity of the tyrosinase concentrate isolated from mushroom waste was determined using both methods. The polyphenoloxidase concentrate, which had an activity of 4000 U ml-’ at 25°C and contained about 10% of the ferment, was obtained from the Alkaloid Company (Skopje, Yugoslavia). Three different types of working electrodes were used: a graphite disc of diameter 5 mm in a Teflon hoider, a bundle of carbon fibres (SIGRI-HM) placed in a glass tube and sealed with epoxy resin and a gold wire of diameter 0.4 mm and

510

w

-0,3

Fig. 1. Voltammograms for oxygen 6.5) and (2) with adsorbed ferment.

-0.6

E/ Vvs. SCE

reduction on pyrographite dE/df = 3 mV s-‘.

from (I) phosphate

buffer

solution

(pH

length 24 mm. E3efore ferment adsorption the electrodes were activated electrochemically (at 2.0 and -0.2 V vs. saturated calomel electrode (SCE) for carbon electrodes and from 0 to 1.2 V for gold), immersed in the ferment solution for 20 min and washed with water and buffer solution. The quantity of ferment adsorbed was estimated using double-layer capacity measurements (at 1 kHz and 1 mV) before and after adsorption according to the method described previously 141. The areas of the electrode surfaces were estimated using the voltammograms of the redox couple Fe(lI)/Fe(III) cyanide at several sweep rates ilOmV s-*> with D = 0.62 X 10m5 cm* s-l [5]. Voltammograms for the electrochemical reduction of oxygen were recorded at a sweep rate of 3 mV s-l. As can be seen from Table 1, after the electrodes are treated in the solution (containing ferment) the double-layer capacity of the electrode-solution interface decreases slightly. This is probabIy due to the adsorption of protein (present in the ferment solution) at the electrode surface and/or the porous structure of the adsorbed biopolymer. The adsorbed ferment has its most significant catalytic effect on electrochemical oxygen reduction on pyrographite (Fig. 1). The oxygen reduction current is considerably increased in all potential regions. These rest&s are in very good agreement with those obtained for the activity of tyrosinase from potatoes 131. In the second method proposed for estimating ferment activity the membrane was prepared by immobilizing a mixture of ferment concentrate in gelatine on

TABLE I Double-layer capacity values for several electrodes in phosphate buffer solution (pH 6.9 simuttaneously with voltammograms in a given potential region (d E/dl = 20 mV S-I) Electrode Pyrographite Carbon fibre Gold

Potential region/V

Cmin/pF

Without enzyme

With enzyme

0.5 to -0.5 0.5 to -0.5 0 to 1.2

0.50 5.50 14.00

0.45 5.20 12.30

taken

crne2

cuprephane foil. The membrane was then positioned over the platinum disc (working) and Ag/AgC1 (reference) electrodes, and the electroreduction current of oxygen (at -0.6 V) was recorded. As can be seen from Fig. 2, in the case of the oxygen-saturated soIution a constant value of the fimiting current is obtained. A subsequent addition of phenol to the solution provoked a sharp decrease in the current, but when the solution in the ceI1 is. repIaced with a fresh buffer solution the previous value of the reduction current is regained (Fig. 2). In the presence of phenol the current depends on the phenol concentration in the soIution and the amount of the immobilized ferment on the membrane.

0

5

20

G/min

Fig. 2. Amperomctric curve (at - 0.6 V/SCE) for oxygen reduction on platinum from phosphate buffer sofution (pH 6.5) and in the presence of immobilized ferment on the membrane.

512 REFERENCES 1 A.M. Mayer, Phytochemistry, 26 (19ti7) 11. 2 M.N. Keyes and FE. Semersky. Arch. Biochem. Biophys., 148 (19721 256. 3 VA. Bogdanovskaya, E.F. Gavrilovn, M.R. Tarasevich and D.I. Stom, Elektrokhimiya, 16 (1980) 1596. 4 M.M. Lohrengel, K. Shubert and J.W. Schultze, Werkst, Korros., 32 (1981) 13. 5 R.N. Adams, Electrochemistry at Solid Electrodes, Marcel Dekker, New York, 1969, p. 220.