Enzyme Electrode for Specific Determiuation of L-Lysine

J. L. ROMETTE, J. S . YANG, Luboratoire de Technologie Enzymatique, Universite' de Technologie de Compi&ne, B. P. 233, 60206 Compiegne,

France, H. KUSAKABE, Research Laboratories, Yamasa Shoyu Co, Ltd., Choshi, Chiba-Ken 288, Japan and D. THOMAS, Luboratoire de

Technologie Enzymatique, Universitk de Technologie de Compic?gne, B. P. 233, 60206 Compiegne, France

Snmmarv L-Lysine a-oxidase from Trichodenna viride Y244-2 is immobilized in a gelatin support and

fixed on a pOz sensor. The enzyme electrode obtained is used in a continuous flow system in order to measure the concentration of L-lysine in a fermentor. The sample oxygen-content depen- dance of the signal is minimized because of the enzyme support properties. The enzyme elec- trode response is set for lysine concentration from 0.2mM to 4mM. The specificity of lysine is tested with other amino acids. The enzyme membrane for lysine electrode can be used 3000 times or stored six months with good stability.

INTRODUCTION

Research on the control of fermentation processes suffers from a serious lack of continuous chemical analyses which describe the actual situation in the fermentor. Most of the low-molecular-weight organic compounds in the fermentor, nutrients as well as the products, can function as a substrate for an enzyme and thus, theoretically, be available for analysis with an enzyme electrode. The analytical principle of the enzyme electrodes and the potential use of it as an analytical tool for fermentation control have been discussed.'

Enzyme electrodes, which generally consist of an electrochemical sensor with an immobilized enzyme in close contact, have several advantages over other methods of analysis. Enzymes frequently are very substrate-specific. Chemical immobilization generally stabilizes the enzyme, enables repeated measurements, and offers the possibility of continuous analysis which is a ma- jor advantage in any process control. In addition, there is the possibility of sterilization of these enzyme electrode.

Enzyme electrodes for amino acids have been developed by coupling an en- zyme to the appropriate ion-selective ele~trode.~-~ The use of gas-sensing membrane electrodes has led to the development of better enzyme elec- trodes5-12 which are free from ionic and protein interferences. However, the

Biotechnology and Bioengineering, Vol. XXV, Pp. 2557-2566 (1983) 0 1983 John Wdey & Sons, Inc. CCC 0006-3592/83/11W7-10$02.00

2558 ROMETTE ET AL.

applications of such enzyme electrodes in the rapid and continuous deter- mination of amino acids is limited by the time consumed to reach the steady state of electrode response. An attempt is made here to reduce the response time of the enzyme electrode by considering dynamic response.

The L-lysine a-oxidase has been studied by Kusakabe et a1.13 The oxidase catalyses the oxidation of L-lysine as follows:

L-lysine + O2 -I- H20 - a-keto-eaminocaproate 4- NH3 + H202

f H20

A1-piperideine-2-carboxylate

Cyclization of a-keto-eaminocaproate to the intramolecular dehydrated form, A1-piperideine-2-carboxylate, proceeds spontaneously. The consump- tion of 0 2 is detected by the p 0 2 sensor. In this article, experiments have been conducted for determination of L-lysine using an electrode coupled with L-lysine a-oxidase. The enzyme was immobilized directly on the selective gas membrane of the p02 sensor by copolymerization with gelatin, using the bifunctional agent glutaraldehyde. Electrode optimization and analytical characteristics were determined and the enzyme electrode was used in an automatic continuous flow system for measurement of lysine.

APPARATUS

The p02 sensor used was a Radiometer electrode model E.5046. The selec- tive gas membrane was modified from the commercially available model. An Apple 11-plus computer system is applied to the automation of the enzyme electrode (Fig. 1). It was composed of an Apple I1 microcomputer (Apple Computer, Inc., Cupertino, CA) with 48K RAM of main memory. The inter-

Hobby Computer miv. unit

Fig. 1. Schematic diagram of the analyzer.

ENZYME ELECTRODE FOR LYSINE 2559

nal peripheral bus of the computer enables addition of a variety of peripheral units. We have added a disk controller card, parallel interface card for the printer, and p02 interface card. The p02 interface card (U.T.C. Electronic Dpt, CompPgne, France) is mounted to the computer through a ribbon cable and contains circuit boards for signal conditioning and buffering, an analog to digital data treatment card based upon the Date1 ADC EK 12 bit, a p02 analyzer-a 4-bit logical system.

The flow block system (Fig. 2) is connected to the pOz analyzer. It is com- posed of a measurement cell (Fig. 3) connected to the pump through elec- tromagnetic valves, which controlled the circuit.

REAGENTS

The L-lysine a-oxidase (from Trichoderma viride Y244-2) was obtained from Yamasa Shoyu (Choshi, Chiba, Japan). Lysine and all other amino acids were obtained from Sigma Chemical Company, St. Louis, MO). The 240 Blooms bone gelatin was from Rousselot Chemical Company (France). The glutaraldehyde (1.25% aqueous solution) was from Merck Company (W. Germany). The polypropylene selective gas membrane was from Bollore Com- pany (France). All other chemicals were reagent grade.

ELECTRODE PREPARATION

The methodology for obtaining active film on polypropylene membrane has been previously described14 and optimized for the oxidase enzyme.15 The selective hydrophobic film used was a polypropylene membrane with a thickness of 6 pm. In order to get good mechanical properties, the active layer containing the enzyme was coated on the selective film by pouring 1 mL of a solution of enzyme and gelatin on 35 cm2 of the film. A 5% solution of 240

M MC P vs V B VA W S B A

P~ICROCOMPUTER

NEASUREMENT CELL

BUFFER VALVE AIR VALVE NASTE SAMTLE BUFFER AIR

PUMP SAMPLE VALVE

- Fig. 2. Fluidic block system.

2560 ROMETT'E ET AL.

A1 pOg SENSOR B1 DETAILS OF THE ENZYME ELECTRODE T I P

d a b

C'

a- body o f sensor

b- Jacket

e- j o i n t

Q-

d- i i C-

f -

C i

k-

n-

r-

. jo in t

e l e c t r o l y t e

s e l e c t i v e f i l m

j o i n t o f the measurement c e l l

ac t ive -se lec t ive b i l a y e r

thermostated j a c k e t

body of the c e l l

screw for f i x i n g the enzyme

e lec t rode ins ide the measurement

c e l l

Fig. 3. Details of the measurement cell.

blooms gelatin was prepared in 0.02M phosphate buffer (pH 6.8) and solubilized overnight at 40°C.

After solubiliiation, enzyme was added to the gelatin solution. The solution spread on the polypropylene was dried at 25°C for 4 h and then immersed in 2.5% glutaraldehyde solution (in distilled water) for 3 min. By fixing this bilayer on the top of the p02 sensor, the enzyme electrode was obtained.

ELECTRODE MEASUREMENTS

The sensor bearing the selective-active composite membrane was settled in a cell allowing flow measurements. The electrode was exposed sequentially to air rinsing, to a sample containing lysine, and to glycine sodium buffer rins-

2561 ENZYME ELECTRODE FOR LYSINE

Fig. 4. Concentration of oxygen for different media: gelatin solution-albumin solution- substrate solution.

ing. Air rinsing had the effect of saturating the membrane with oxygen. The concentration of oxygen in the active layer was roughly 20 times higher than in water for the same partial pressure (Fig. 4).16 During the measurement step, the oxygen contained inside the enzyme support was consumed. In order to feed the enzyme support with oxygen in the quickest time, a rinsing step with air was performed. Buffer rinsing removed the remaining lysine out of the membrane by diffusion.

Under software control (Fig. S), the air valve was opened until a plateau was obtained for the electrode signal. Then the air valve was shut and sample valve opened. Within seconds after the admission of sample in the measurement cell, the electrode signal is sampled. The liquid rinsing step started at a given time after the beginning of the measurement. After a given time air rinsing resumed, and so on.

During the 02 consumption step, the electrode signal was sampled by the microcomputer (30 measurement data points during 9 s). An approximation of this signal by a third-order polynomial was ~erf0rmed.l~ This makes it

Fig. 5. Response of the enzyme electrode for different lysine concentrations in automatic mode and under software control.

2562 ROMETTE ET AL.

d ;t 1

mM O ! . . . . !I . ' '

L-lyul-

Fig. 6. centrations.

Calibration curve. Slope of the signal at the inflexion point versus L-lysine con-

possible to calculate the slope at the inflection point which constitutes the in- formation about the sample substrate concentration. The calibration curve was obtained by plotting the slope (dp02/dt) at the inflection point of the electrode signal against lysine concentration (Fig. 6).

ELECTRODE OPTIMIZATION

pH and Ionic Strength Effect

The pH dependence of the electrode signal is shown in the Fig. 7. The pH optimum is found to be around 9. Kusakabe et a1.13 have determined the pH optimum of the enzyme in solution as being from 7.5 to 8.0.

The shift of the pH optimum observed can be explained by two phenomena.

1 I 1

7 9 n PH

Fig. 7. The pH dependence of the sensor response.

ENZYME ELECTRODE FOR LYSINE

!$ lo d

2563

ImM Lynne O r;: PH9

I

Fig. 9. Temperature dependence of the sensor response.

2564 ROMETTE ET AL.

& 30 IU

20 IU

8 IU b

I 5 IU

3 IU

0

u) - 0"

1 IU

I I

6 8 mM

-

& 30 IU

20 IU

8 IU b

I 5 IU

3 IU

0

u) - 0"

1 IU

I I

6 8 mM L- Lysine concentration

Fig. 10. Effect of the amount of enzyme on the calibration curve.

be 100% of the original activity level when the enzyme was stored 30 min at 55°C. This stability at high temperature has permitted to test the enzyme electrodes

in afermentation control using thermophilic bacteria. Figure 9 gives the signal of the electrode obtained with 0.5mM of lysine for different temperatures.

Effect of the Amount of Enzyme

Figure 10 shows how the amount of enzyme spread on the surface of the pOz sensor affects the response. The widest range of linearity (down to 2mM) was obtained with 8 International Units (IU) per 35 em2 of membrane. With ex- cess of enzyme, the electrode response in this range was not improved.

Hmbadtunr

Fig. 11. Stability of an enzyme membrane.

ENZYME ELECTRODE FOR LYSINE 2565

ANALYTICAL CHARACTERISTICS

Stability

The stability of the lysine electrode was characterized by both its storage and operational conditions. When stored at 5°C in buffer containing 10-4M of sodium azide, the enzyme membrane remained stable for at least six months. The operational stabilility was examined by repeatedly measuring the electrode response (a measurement every two minutes). The test realized with a 10 IU enzyme membrane and with different lysine concentrations (Fig. 11).

The results of Fig. 11 show that the enzyme seems to be inactivated by the reaction. One molecule of enzyme can transform only a limited number of molecules of substrate. For 1mM of lysine concentration, 200 measurements can be done; for O S m M of lysine concentration, 400 measurements are per- formed. This type of inactivation is very similar to those observed for the glucose oxidase enzyme reaction.18 It seems to be a constant for FAD oxidase enzyme. Three thousand measurements were assayed successfully. Remem- ber that dynamic state response is being considered here. Thus, the time dur- ing which the enzyme membrane is in contact with a high concentration of lysine (sample) is short (ca. 8 s/min) giving to the electrode a good stability by minimizing the denaturation phenomena because of work.

a

6

b4 0 ln -

2

0

i L-Lysine oxydase 8 U

I 1

5 10 MM L-LysinelL-Orithine

I I 1

0 50 1 0 0 MM L- Phenyl alanhe/L-Arginine

Fh. 12. Selectivity of the L-lysine electrode; calibration curves for different amino acids.

2566 ROMETTE ET AL.

Specificity

Kusakabe et aI.l3 have studied the specificity of the enzyme to different substrates. In our case, the possibilities of finding interfering compounds have been reduced to three amino acids: L-arginine, L-phenylalanine, and L-ornithine. Figure 12 gives the results obtained when a sample of lysine or other compound is measured.

Accuracy

Reproducibility is better than 5% when the electrode is used fully auto- matically. The stirring of the solution did not change the value of the electrode response when the sample is introduced inside the measurement cell.

CONCLUSIONS

The results presented indicate that the L-lysine enzyme electrode allows the control of the fermentor process. A full automatization of the system can be performed by using a microcomputer to regulate the measurement cycle to collect and analyze the kinetic data from the electrode. An important prob- lem remains: the automatic sampling of fermentation broth under sterile conditions.

References

1. S. 0. Enfors and N. Molin, Process Biochem., 13(2), 9 (1978). 2. G. G. Guilbault and E. Hrabankova, Anal. Chem., 42, 1779 (1970). 3. T. A. Neubecker and G. A. Rechnitz, Anal. Lett., 5, 653 (1972). 4. S. L. Tong and G. A. Rechnitz, Anal. Lett., 9, 1 (1976). 5. A. M. Beqonneau, D. Thomas and G. Broun, Pathologie-Biologie, 22,497 (1974). 6. C. Calvot, A. M. Berjonneau, G. Gellf, and D. Thomas, FEBS Lett., 59, 258 (1975). 7. G. G. Guilbault and F. R. Shu, Anal. Chem., 44,2161 (1972). 8. B. K. Ahn, S.-K. Wolfson Jr., and S. J. Yao, Bioelectrochem. Bioeng., 2, 142 (1975). 9. K. W. Fung, S. S. Kuan, H. Y. Sung, and G. G. Guilbault, Anal. Chem., 51,2319 (1979).

10. W. C. White and G. G. Guilbault, Anal. Chem., 50, 1481 (1978). 11. C. L. Di Paolantonia, M. A. Arnold, and G. A. Rechnitz, Anal. Chim. Acta, 128, 121

12. R. R. Walters, P. A. Johnson, and R. P. Buck, Anal. Chem., 52, 16M (1980). 13. H. Kusakabe, K. Kodama, A. Kuninaka, H. Yoshino, H. Misono, and K. Soda, J . Biol.

14. J. L. Romette, B. Froment, and D. Thomas, Clin. Chim. Acta, 95, 249 (1979). 15. J. L. Romette, Ph.D. thesis, University of Compikgne, CompiZgne, France, 1980. 16. J. P. Quenesson, Ph.D. thesis, University of Compikgne, Compikgne, France, 1979. 17. J. P. Kernevez, L. Konate, and J. L. Romette, Biotechnol. Bioeng., to appear. 18. C. Bourdillon, T. Vaughan, and D. Thomas, Enzyme Microb. Technol., 4, 175 (1982).

(1981).

Chem., 255, 976 (1980).

Accepted for Publication March 29, 1983