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Bioelectrochemistry and Bioenergetics, 27 (1992) 199-205 A section of J. Electroanal. Chem., and constituting Vol. 342 (1992) Elsevier Sequoia S.A., Lausanne

JEC BB 01438

Amino acids and urea sensors using parsley seeds as biocatalyst

Yuichi Sato, Tetsuya Makino and Koichi Kobayakawa

Department of Applied Chemistry, Faculty of Engineering, Kanagawa University, Rokkakubasi 3-27-I, Kanagawa-ku Yokohama 221 (Japan)

(Received 5 August 1991)

Abstract

Minced parsley seeds coupled with a potentiometric ammonia gas sensing electrode showed effective biocatalytic activity, i.e. responded to L-asparagine, L-glutamine, r_-serine and urea with a nernstian slope of 43-51 mV per decade of substrate concentration. Very good time stability with no significant loss of biocatalytic activity over 30 days was maintained. A celery seed immobilized ammonia electrode showed similar behaviour.

INTRODUCTION

Plant substances have been used as biocatalytic layers coupled with potentio- metric or amperometric techniques [1,2]. The specialized structures of plants, e.g. leaves [3,4], fruit [5], flowers [6,7] etc. offer particularly attractive properties as biocatalysts because structures related to growth, reproduction, and nutrient storage concentrate and stabilize highly selective biocatalytic activity. Their easy preparation and low cost are very attractive for the construction of biosensors. In a previous paper [8], we presented an amino acid and urea sensor using minced parsley leaves coupled with a potentiometric ammonia gas sensing electrode. Its principle is that a substrate such as L-glutamine in the solution passes through the membrane, diffuses into the plant tissue and decomposes to ammonia and other substances by the catalytic action of the enzyme contained in the parsley. The ammonia electrode then responds to the ammonia produced. When using parsley leaves, however, it is suspected that the activity of the enzyme in the leaves may change with a change in seasons and in sampling point, i.e. near the root, the tip of leaves, stem etc. If parsley seeds were used in place of leaves, such discrepancies

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would be avoided. In this study, therefore, parsley and celery seeds were tested for amino acid and urea sensing.

EXPERIMENTAL

An Orion model 9.5-10 ammonia gas electrode was used in the construction of the biosensors. Potentiometric measurements were carried out using a Corning Model SA 720 ion meter. The parsley (Petroselinum crispum (Miller) A.W. Hill) and celery (@urn gravedem L.) seeds used were commercially available (Sakata Seeds Corp.). After soaking in 0.1 mol/dm3 Tris-HCl buffer solution at pH 7.4 for 24 h, the seeds were minced using a razor blade or ground in an agate mortar with a pestle. Then, l-10 mg minced seeds of parsley were attached to the surface of the ammonia electrode, covered with a dialysis membrane and fixed using an O-ring. The immobilized-seed electrode was dipped in 0.1 mol/dm3 Tris-HCl buffer solution. To determine the response at this electrode, various types of amino acids (cited in Table 1) or urea solutions were added to the buffer solution and the potential change due to NH, liberated from the substrate by the catalytic action of the enzyme contained in parsley was followed. All chemicals used were of reagent grade (Wake Junyaku Co. except p-glutamine and o-asparagine, which were purchased from Daiichi Kagaku Co.). Amino acid solutions were prepared fresh each day with deionized water (18 MfI cm).

RESULTS AND DISCUSSION

At first the parsley seed electrode was tested for various amino acids. In Fig. 1, the potential-time curve obtained by titration of L-asparagine solution to the 25 ml

‘0.1 mol/dm3 Tris-HCl solution is shown. Using this figure, a typical L-asparagine calibration curve was obtained (Fig. 2). The response slope is 51 mV per concen- tration decade over its linear range which extends from 6 X 10m5 to 1 X 10m3 mol/dm3 t-asparagine. By increasing the amount of immobilized parsley seeds, the response slope increased and showed a maximum at 6 mg, then decreased again. The response time increased with increasing amount of seeds (Fig. 3). The pH dependence on the response slope was measured from pH 6.0 to 8.0 using 6 mg of parsley seeds at 30°C. Over pH 8.0, L-asparagine was hydrolysed. Between pH 6.5 and 8.0, the response slope maintained almost the same value (Fig. 4). The activity of this electrode immediately after immobilization of the minced seeds was not very high, but it attained a high value after two or three repeated measure- ments which was maintained for more than 30 days (Fig. 5). In these lifetime studies, the biosensor was stored at room temperature in the working buffer solution. The temperature dependence was measured between 20 and 40°C (Fig. 6). The slope at 30°C showed the highest value, indicating the enzyme is optimized at this temperature. Over 30°C the enzyme may be partly inactivated by heat denaturation. Celery seed also showed its highest value at 30°C for L-asparagine. At 45”C, r_-asparagine itself was hydrolysed. Theoretical values in Fig. 6 were

201

TABLE 1

Selectivity of immobilized parsley- and celery-seed electrodes in 0.1 M Tris-HCI buffer at pH 7.4, 30°C

Substrate Response (slope)/mV decade - ’

Parsley Parsley Celery seeds leaves a seeds

NH&I 58 L-Asparagine 51 o-Asparagine 12 L-Glutamine 51 o-Ghrtamine 19 L-Serine 43 o-Serine 0 L-Threonine 25 o-Threonine 0 L-Alanine 25 L-Histidine 20 L-Arginine 5 L-Glutamic acid 34 L-Varine 2 L-Phenylalanine 0 t-Ornithine 8 L-Citrulline 1 L-Glycine 13 L-Methionine 0 L-Lysine 2 L-Tryptophan 0 Formamide 0 Urea 47 Thiourea 0 NaNO, 0

58 48 26 48 38 13

10

6 5

10 5 4 2 3 1 0 0 0

0 47 0 0

49

49

20

20

40 29 8

40 20 18

1 0

13

30

a Ref. 8.

calculated from the nernstian slope. This immobilized-parsley-seed electrode also showed a good response at pH 7.4 for L-glutamine with a response slope of 51 mV per concentration decade from 7 x 10m5 to 8 x 10m4 mol/dm3, and a detection limit of 1 X lop5 mol/dm3; for L-serine with a slope of 43 mV from 7 X 10e5 to 6 x 10m4 mol/dm3, and a detection limit of 1 X 10m5 mol/dm3; and for urea with a slope of 47 mV from 7 X lo-’ to 1 x 10v3 mol/dm3, and a detection limit of 3 X 10e5 mol/dm 3. Some D-isomer amino acids were also tested (Table 1). Generally, L-isomers responded to this electrode with higher sensitivity. In particu- lar, r_-serine and r_-threonine responded with a slope of 43 and 25 mV, respectively, while their o-isomers did not. Such a response pattern suggests that the primary biocatalytic activity in parsley seeds involves the enzyme glutamine-(asparagine-) ase, E.C. 3.5.1.38 [71 and threonine dehydratase, E.C. 4.2.1.16. A principal feature of the former enzyme is that it catalyses the hydrolysis of the D-isomers of asparagine and glutamine with less activity than the L-isomers [9]. The latter

202

0.01~ L-Asparagine, 0.04ml added

O.OlM, O.lml

O.lM, 0.04ml

O- 0.5ml

-2o- L I I I

0 40 60 120 160’

nmlmh

Fig. 1. Potential-time curve obtained by titration of L-asparagine solution to the 25 ml 0.1 mol/dm3 Tris-HCl solution at pH 7.4, 30°C using a 6 mg immobilized minced parsley seed electrode.

enzyme responds to the L-isomers of serine and threonine, while it does not respond to their o-isomers [lo]. These features are in accord with our case. The experimental data also suggest parsley seeds contain urease.

For other amino acids, the slopes are listed in Table 1. Comparing parsley seeds and leaves, seeds generally have a larger slope value compared with those of leaves. An immobilized celery seed electrode was also tested for various amino

80

0

-20

t

t_ I 1 a

10’ 10’ 10” 10S2 Concentration of L-asporagine/moll”

Fig. 2. Calibration curves for L-asparagine using a 6 mg immobilized minced parsley seed electrode in 0.1 mol/dm3 Tris-HCI buffer at pH 7.4, 30°C.

203

4!

1 5 E9 .

H

l!

7

5-

0-

5-

Fig. 3. Relationship between amount of immobilized parsley seeds and slope (0) or response time (0) for L-asparagine in 0.1 mol/dm3 at pH 7.4, 30°C.

Fig. 4. Relationship between slope and pH for L-asparagine using a 6 mg immobilized minced parsley seed electrode at 30°C.

._ 6.0 7.0 a0

PH

Fig. 5. Time dependence of the slope of a 6 mg immobilized minced parsley seed electrode for t_-asparagine in 0.1 mol/dm3 Tris-HCI buffer at pH 7.4, 3O’C.

151 ’ I I 20

g/“c 40

Fig. 6. Relationship between slope and temperature for L-asparagine using a 6 mg immobilized minced parsley seed electrode in 0.1 mol/dm3 Tris-HCI buffer at pH 7.4. (0) Theoretical values, (*I experimental values.

acids and urea, and its behaviour was similar to that of the parsley seed electrode (Table 1).

Though the selectivity of these immobilized parsley and celery seed electrodes is not very good, they are usable as biosensors for L-asparagine, L-glutamine, L-serine and urea. This simple technique also offers a very useful method for enzyme screening in plant substances.

REFERENCES

1 G.A. Rechnitz, Science, 214 (1981) 287. 2 M.A. Arnold and G.A. Rechnitz in A.F.P. Turner, I. Karube and G. Wilson (Eds.), Biosensors,

Fundamentals and Applications, Oxford University Press, 1988, p. 30.

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3 N. Smit and GA. Rechnitz, Biotechnol. Lett., 6 (1984) 209. 4 S. Uchiyama, M. Tamata, Y. Tofuku and S. Suzuki, Anal. Chim. 208 (1988) 287. 5 J.S. Sidwell and G.A. Rechnitz, Biotechnol. Lett., 7 (1985) 419. 6 S. Uchiyama and G.A. Rechnitz, Anal. Lett., 20 (1987) 451. 7 S. Uchiyama and G.A. Rechnitz, J. Electroanal. Chem., 222 (1987) 343. 8 Y. Sato, K. Chikyu and K. Kobayakawa, Chem. Lett., (1989) 1305. 9 B. Maruo and N. Tamiya (Ed%), Koso Handbook, Asakurashoten, 1987, p. 593.

10 B. Maruo and N. Tamiya (Eds.), Koso Handbook, Asakurashoten, 1987, p. 686.