Pertanika 3(1),1-4 (1980)

Immobilised Glutamate Dehydrogenase: Possible UseIn Automated Analysis.

A. B.SALLEHfabatan Biokimia dan Mikrobiologi, Fakulti Sains dan Pengajian Alam Sekitar. Universiti Pertanian Malaysia

Serdang. Selangor. Malaysia.

Key words: Immobilised, Glutamate dehydrogenase

RINGKASAN

Glutama t dehydrogenase dilekat ke dalam saluran 'nylon' dan aktiviti enzim yang terlekat dikaji.Dua jenis molekul 'spacer'dan dua teknik penyambung digunakan untuk perlekatan enzim ini.

Aktiviti yang agak rendah diperolehi di dalam saluran tetapi bila saluran mengandungi enzim ini di-sambungkan ke dalam sistem pengaliran terus bagi menganalisa larutan piawaulI2 ammonia, aktivitinya men-cukupi untuk menghasilkan tindakan yang boleh diukur.

Kajian ini menunjukkan kemungkinan penggunaan 'immobilised' glutamat dehydrogenase di dalamkaedah analitik biasa.

SUMMARY

Glutamate dehydrogenase was immobilised onto nylon tube and the activity of the enzyme tubeinvestigated. Two different spacer molecules and two coupling techniques were utilised for immobilisingthe enzyme.

Relatively low activity was retained on the tube, but when the enzyme tube was incorporated into acontinuous flow system, and used to assay standard ammonia solution, there was adequate response formeasurement.

The study indicated the potential use ofimmobilised glutamate dehydrogenase in routine analyticaloperation.

INTRODUCTION

Present methods for the analysis of bloodammonia and urea still utilise chemical reagentsfor colour formation (Gutman and' Bergmeyer,1974; Szasz, 1974), which may render the methodsto problems of unspecific reactions, resulting ininaccuracy in estimating the levels of these com-pounds. The advantages of using enzymes inclinical analysis have been discussed (Bergmeyer,1965). Enzymatic methods for estimation ofammonia and urea have been developed (Kun andKearney, 1975, Gutman and Bergmeyer, 1975).The enzymatic reactions are as follows:

Urea + H2° urease) CO2 + 2NH3GDH

NH3 + 2-oxoglutarate + NADH~glutamate+ NAD + H2 0

A recent advance in enzymatic methods isthe use of immobilised enzymes, which can beincorporated into a continuous flow systemresulting in highly precise, efficient and economi-cal operations (Hornby and Noy, 1976). Ureasehas been successfully immobilised into a tubularreactor, and used in continuous mode operations,coupled to the Berthelot reaction for analysis ofurea (Filippusson et ai, 1972).

GDH glutamate dehydrogenase (L-glutamate : NAD(P) oxidoreductase (deaminating) (EC 1.4.1.3)DAE diarninoethane. A.F.U. = Arbitrary Fluorescent Unit.

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IMMOBILISED GLUTAMATE DEHYDROGENASE: POSSIBLE USE IN AUTOMATED ANALYSIS

TABLE I

Coupling data for the immobilisation of GDH onto nylon tube. The data are obtained from nylon-adipic tube.A volume of 0.8 ml enzyme solution was required to ftllim tube.

Activity (V{ml)Coupling technique tube activity

coupling post-coupling V{msolution solution

glutaraldehyde 2.04 1.12 0.12

imidate 2.04 1.69 0.17

% activitycoupled

45

17

%activityretained

5.9

8.3

of enzyme activity was retained on immobilisa-tion. The difference in the overall activity coupledand retained, using two coupling techniques,was apparent in all experiments.

Fig. 2. Calibration curves obtained using 1m and2m immobilised GDH tube. The tube wasincorporated on to the flow system shownin Fig. 1.

Fig. 2 and 3 show results when immobilisedGDH tubes were incorporated into a continuousflow system. Fig. 2 shows the calibration curvesobtained when aqueous ammonia standards wereassayed using 1 m- and 2 m- tube length. Fig. 3shows the response when tubes prepared by usingdifferent spacer molecules and coupling techniqueswere incorporated into the flow system. Adipicdihydrazide as spacer molecules seemed to pro-duce higher activity tubes. Irnidate coupling alsoresulted in better activity retention, as also shownin Table 1.

The pH profIle of the immobilised and solubleenzyme is shown in Fig. 4. There seemed to be noapparent variation in the pH optimum of theenzyme.

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Fig. 3. Calibration curves obtained using 2m tubewith different spacers and coupling tech-niques. GDH was attached to nylon-adipictube by glutaraldhyde (A) and imidate (C)coupling and to nylon-DAE tube byglutaraldehyde (B) and imidate (D) coupling.

DISCUSSION

The results show fairly low retention ofenzyme activity on immobilisation. The loss inactivity was not unexpected. The enzyme comprises subunits which may be dissociated into in-active subunits (Frieden, 1959). The immobilisa-tion procedure may contribute towards this dis-sociati.on. Covalent attachment usually produceslow activity retention. Glutaraldehyde randomlyre.acts with free amino groups and thus may reactwIth certain active site amino groups or aminogroups essential for comforrnational structure.Irnidate, though less efficient in protein coupling,actually retained higher enzymes activity.

The hydrophobicity of the support materialmay further enhance the enzyme denaturation,

A. B. SALLEH

REFERENCES

BERGl\IEYER, H. U. (1965): Methods of EnzymaticAnalysis. New York. Academic Press.

BROUGHTON, P. M. G., BUTTOLPH, M. A., GOWENLOCK,A. H., NEILL, D. W., SKENTELBERRY, R. G. (1969):Recorrunended scheme for the evaluation ofinstruments for automated analysis in clinicallaboratory. J. Clin. Path. 22, 278-284.

FILIPPUSSON, H., HORNBY, W. E., McDoNALD, A.,(1972): The use of irrunobilised derivatives ofurease and urate oxidase in automated analysis,FEBS Letters, 20, 291-293.

FORD, J. R., LAMBERT, A. H., COHEN, \V., CHAMBERS,R. P. (1972): Recirculation reactor system forkinetic studies of immobilised enzymes. Bio-technol. Bioeng. Symp., 3, 267-284.

FRIEDEN, C. (1959): Glutamate dehydrogenase, 1.Eftect of coenzyme on the sedimentation velocityand kinetic behaviour. J. Bioi. Chem, 234, 809-814,

GUTMANN, 1., BERGMEYER, H. U. (1974): Urea:"Determination of urea, Indicator reaction withphenol and hypochlorite" in Methods of EnzymaticAnalysis. H. U. Bergmeyer (Ed.) Vol. 4 p. 1791-1794. New York. Academic Press Inc.

HORNBY, W. E., Noy, G. A. (1976): "The Appli-cations of immobilised enzymes in automatedanalysis" in Methods in Enzymology. K. Mosbach(Ed.) Vol. 44, 633-646. New York. AcademicPress.

JULLIARD, J. H., GODlNOT, C., GAUTHERON, D. C-(1971): Some modifications of the kinetic proeperties of bovine liver glutamate dehydrogenasf(NAD(P)) covalently bound to a solid matrix o.collagen. FEBS Letters, 14(3), 185-188.

KUN, E., KEARNEY, E. D. (1974): "Ammonia" inMethods of .'Enzymatic Analysis. H. U. Bergmeyer(Ed). Vol. 4 p. 1802-1806. New York. AcademicPress,

MORRIS, D. K., C.\MI'BELL, J., HORNBY, W. E. (1975):A chemistry of immobilisation of enzymes onnylon. Biochem J. 147, 593-603.

SZASZ, G. (1974): "Urea, determination with Auto-matic analyser" in Methods of Enzymatic Allalysis.H. U. Bergmeyer (Ed). Vol. 4 p. 1798-1801.New York. Academic Press.

(Received 6 December 1979)

For its acceptance as a replacement for thesoluble counterpart, studies on the stability of theimmobilised GDH are essential. The whole analyti-cal system has to be optimised for accuracy andprecision as outlined by Broughton et al (1969).However, the present studies have shown thepossibility of utilising these immobilised GDH forsuch a purpose.

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