APPLICATIONS AND KINETICS OF IMMOBILIZED ENZYMES AND COUPLED ENZYME REACTIONS

APPLICATIONS AND KINETICS OF IMMOBILIZED ENZYMES AND COUPLED ENZYME REACTIONS Elena Dominguez!*, Gyö gy Ma ko-Va ga?, Bä bel Hahn-Häge dal' and Lo Go on?. ‘Depa men o Applied Mic obiology, * Depa men o Analy ical Chemis y, Uni e si y o Lund, P. O. Box 124, S-221 00 Lund, Sweden. SUMMARY Immobiliza ion o enzymes esul s in mo e adequa e eagen s o be used analy ically. A e immobiliza- ion, he kine ic pa ame e s o he enzymes a e modi ied and no hing can be p edic ed abou he ac i i y o he he e ogeneous sys em. In coupled enzyme eac ions wi h co-immobilized enzymes, his is e en mo e impo an owing o he kine ic dependence on each consecu i e eac ion. The de e mina ion o e hanol and ace aldehyde is conside ed in his pape , using wo di e en coupled enzyme sys ems. Some impo an pa ame e s, as he enzyme cha ged, he a io o each enzyme in he sequence and he immobiliza ion yield, a e conside ed in e ms o con e sion e iciency inally de e mining he sensi i i y o he analysis. INTRODUCTION Enzymes a e used in chemical analysis because o hei high deg ee o selec i i y and hei ca aly ic abili y o speed up eac ion a es. The numbe o compounds ha can be enzyma ically analyzed is li ui ed by he physical o chemical p ope ies o he subs a es and p oduc s. Enzyma ic eac ions which equi e co ac o s open he possibili y o moni o ing he eac ion ia he ans o med co ac o . In cases whe e none o he subs a es o p oduc s a e measu able, i is o en possible o de e mine one o hese componen s by coupled sequen ial o compe i i e enzyme eac ion [1]. This conside ably inc eases he numbe o analy es ha can be measu ed enzyma ically. Fu he mo e, by he use o coupled enzyme eac ions addi ional ad an ages can be gained: i) compounds in ol ed in un a o able equilib ia may also be e icien ly con e ed by shi ing he equilib ium cons an wi h he subsequen eac ion, esul ing in a sys em he modynamically a o able and wi h an expanded linea esponse ange; ii) he selec i i y o he analysis may be imp o ed and he e o e he accu acy, diminishing he isk o in e e ing subs a es; iii) inhibi o y p oduc s may be con inously emo ed and iiii) he sensi i i y is inc eased i any o he specimen (subs a es, p oduc s o co ac o s) en e s in a cyclic enzyme sequence wi h he possibili y o signal ampli ica ion and eco e ing o co ac o s. Endogenous coupled enzyme eac ions occu ing in subcellula ac ions and mic oo ganisms ha e been used in bioca aly ic elec odes [2]. These endogenous mul ienzyme sys ems mimic na u e and d i e he *On lea e om he Depa men o Pha macy, Nu i ion and Food Analysis, Uni e si y o Alcalé de Hena es (Mad id), Spain. 226 E. Dominguez e al. kine ic pa ame e s in he way o inc easing he eac ion luxes [3] and consequen ly o e ’na u al” he mo- dynamically a o able sys ems o be used analy ically. Mo e o en, he analys es ablishes he coupled enzyme eac ions acco ding o he analy es in he sample and uses highly ac i e, pu i ied and comme cially a ailable enzymes. The ac i i y in each successi e s ep should be kep highe han in he p eceding s ep [4], in o de o d i e he sys em owa ds he p oduc side and o achie e comple e con e sion in he o e all sys em. In soluble sys ems, his is ela i ely easy o con ol, bo h empi ical and heo e ically, a e knowing he kine ic pa ame e s (K,,, ,,,, and a e cons an ) o he ee enzymes. The ela i e ac i e enzyme concen a ions in he sys em can hen be es ablished. F equen ly i may occu , unlike “na u al” coupled sys ems, ha op imum condi ions (pH, T, ionic s eng h, ac i a o s) o each enzyme a e no he same and a comp omise be ween hem has o be made. The use o enzymes as analy ical eagen s in low sys em [5] is nowadays a equen p ac ice, mos ly in he o m o immobilized enzymes [6] owing o he ad an ages gained a e immobiliza ion [7]. By co- immobilizing he enzymes on he same suppo , a close coupling will be achie ed be ween he eac ion si es esul ing in a highe con e sion e iciency han by mixing enzymes sepa a ely immobilized [8]. Co- immobilized enzyme eac o s (CIMERs) ha e been desc ibed o he simul aneous de e mina ion o di e en analy es in low injec ion analysis (FIA) [9-11]. Unlike soluble sys ems, he use o co-immobilized enzymes in coupled eac ions o e s some a iables which make he e iciency o he sys em unp edic able. Conce ning he de e mina ion o e hanol and ace al- dehyde and using wo di e en coupled enzyme eac ions, some a iables in co-immobilized sys ems a e con- side ed. COUPLED ENZYME REACTIONS FOR THE DETERMINATION OF ETHANOL AND ACETALDEHYDE The mos common enzyma ic de e mina ion o e hanol is based on he use o alcohol dehyd ogenase (ADH, EC 1.1.1.1) [12] in he p esence o NAD*, see eac ion (1). ADH e hanol + NAD* —«g————_ ace aldehyde + NADH + H* (1) Because o he un a o able equilib ium o his eac ion Ke= 8.0 10°M (phospha e bu e pH 7.0; 20°C), ace aldehyde has o be emo ed in o de o shi he eac ion owa ds he p oduc side. Aldehyde dehyd oge- nase (AIDH, EC 1.2.1.5) oxidizes ace aldehyde o ace ic acid in he p esence o NAD* in an i e e sible eac ion ( eac . 2) which becomes he he modynamic d i ing o ce o he o e all sys em, AIDH ace aldehyde + NAD* + H,O ace a e + NADH + H* (2) Mo eo e , by using his indica o eac ion he sensi i i y o he analysis inc eases (one mole o e hanol gi es wo moleso NADH) and simul aneously allows he de e mina ion o aldehydes. A second al e na i e is based on he use o alcohol oxidase (AOD, EC 1.1.3.13), ca alase (CAT, EC 1.11.1.6) and AIDH. The addi ion o ca alase emo es hyd ogen pe oxide which may oxidize he aldehyde and eco e s he molecula oxygen which en e s in a cyclic sys em: CAT ne H,O +0, _— AOD —_, e hanol ace aldehyde NAD* oO ill AIDH ace ic acid + H* NADH 1) Enzyme cha ged pe g am o suppo . Fou di e en amoun s o AIDH we e added o comme cially glu a aldehyde ac i a ed silica suppo (Se a, Si 500, po e diame e 500 A and pa icle size 30 Lim). The loaded suppo s we e packed in 50 l eac o s and s udied in he FIA mode by injec ions o 50 ul o 2mM Applica ions o Immobilized Enzymes and Coupled Enzyme Reac ions 227 p opionaldehyde in o he ca ie s eam consis ing o 2mM NAD*, 4mM 2-me cap oe hanol, and 0.15 M KCl in 0.1 M py ophospha e bu e (pH 8.5). The low a e was 0.4 ml min". Fig. 1 shows ha a high amoun s o enzyme, he esponse eaches a maximum le el. A simila esponse pa e n has been ound wi h enzyme elec odes [1]. 150 2 z = 100 = oO = 3 Cc = = 50 < = © ao o T T T T T = o 500 1000 1500 2000 2500 3000 AIDH (IU cha ged / g suppo ) Fig. 1. E ec o di e en amoun s o AIDH cha ged pe g am o suppo on he ac i i y o he esul an immobilized enzyme o he con e sion o p opionaldehyde. 2) S udy o he in luence o wo di e en enzyma ic a ios on he con e sion o e hanol and ace alde- hyde. 2.1 ADH/AIDH coupled enzyme sys em. 250 IU o ADH and 2500IU o AIDH pe g am o suppo we e co-immobilized. 9500 IU o ADH and 300 IU o AIDH we e also cha ged pe g am o silica suppo . The con e sion e iciency was s udied in he FIA mode wi h he co-immobilized enzymes packed in 50 1 eac o olume. 25 il injec ions o 125 1M e hanol o 125 UM ace aldehyde we e made in o he ca ie s eam con aining 2 mM NAD*, 0.15 M KCl in 0.1 M phospha e bu e a pH 7.0. The con e sion e iciency is es ima ed in ela ion o injec ions o s oichiome ic amoun s o NADH. ® 50 50 = UO z 40 40 Oo a= 30 30 ie w 520 20 oO ng 10 - D S O-+ T oo T T DO; T 5 O T T TTTT T 207704700708 ,109.12 14 00.02 04 06 0:8. 1:0. To s FLOW RATE (ml/min) FLOW RATE (ml/min) Fig. 2. Va ia ion o he con e sion e iciency o (m) ace aldehyde and (1X) e hanol wi h he low a e o he ca ie and wi h di e en ADH/AIDH a ios; (A) 250/2500 and (B) 9500/300. Nei he o hese co-immobiliza ions esul ed in a good con e sion o e hanol. The ac ha he ini ial solu ions (be o e immobiliza ion) con aining bo h enzymes p esen ed ac i i y o e hanol (measu ed a ze o 228 E. Dominguez e al. o de eac ion), and he % o immobiliza ion we e in all cases highe han 88 % indica e changes in he kine ic pa ame e s and/o an inc eased inac i a ion a e cons an . I has been shown [13] o immobilized ho se li e ADH ha he s abili y o he immobilized p epa a ion depends on he quan i y o bound enzyme. 2.2 AOD/CAT/AIDH coupled enzyme sys em. 10000 IU o AOD and 1000 IU o AIDH we e co- immobilized pe g am o CPG-10 (po e diame e 500 A, pa icle size 37-74 im) ia glu a aldehyde as epo ed p e iously [11]. 1000 IU o AOD and 2500 IU o AIDH we e also co-immobilized unde he same condi ions. In bo h cases 160000 IU o ca alase we e also cha ged pe g am o he suppo . The expe imen al condi ions we e he same as ha men ioned abo e o he ADH/AIDH sys em wi h he sole di e ence ha he ca ie solu ion was sa u a ed wi h O,. 100 100 5 z 80 80 wu UO 60 60 uw oO Ww z 40 40 oO 2 20 20 Bi 3 oo Oo o 5 S 0 T T T T T ie: T TI 0 1 T T T T ¥ T 1 82.020400: 08.1.0. 1.2..21.40%.6702902.06808 1012.14 16 FLOW RATE (ml/min) FLOW RATE (ml/min) Fig. 3. Va ia ion o he con e sion e iciency o (m) ace aldehyde and (0) e hanol wi h he low a e o he ca ie and wi h di e en AOD/AIDH a ios; (A) 10000/1000 and (B) 1000/2500. These esul s clea ly show he impo ance o he enzyme a io in he e iciency o a coupled enzyme sys em. Wi h a su plus o AOD (Fig. 3A) he e is ampli ica ion o e hanol due o he cyclic egene a ion o oO 2 Financial suppo om he Na ional Ene gy Adminis a ion (STEV) and he Swedish Boa d o Thecnical De elopmen (STU) is g ea ully acknowledged. REFERENCES [1] F.W.Schelle ,R. Rennebe g andF. Schube , inK. Mosbach (ed.), Me hods inEnzymology, Vol. 137, Pa D, Academic P ess, New Yo k 1988, pp. 29-43. [2] I. Ka ube in A. P. F. Tu ne , I. Ka ube and G. S. Wilson (eds.) , Biosenso s, Ox o d Uni e si y P ess, New Yo k 1987, pp. 13- 59. [3] G. Pe e sson, Eu . J. Biochem., 184 (1989) 561-566. [4] P. W. Ca and L. D. Bowe s, in Immobilized Enzymes in Analy ical and Clinical Chemis y, John Wiley, New Yo k 1980, pp. 95-108. [5] L.D. Bowe s, in I. S. 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