Nonisothermal Heterogeneous Reaction in a Denaturable Immobilized Enzyme Catalyst

INTRODUCTION
In recent years, considerable attention has been focused on heterogeneous catalytic reaction in immobilized enzyme catalysts.'-9 Lee and Tsao,I Ollis,* Rony,3 Sundaram et al.,d and Vieth et al.5 examined the effectiveness factor of immobilized enzyme catalysts of different geometries. In these works, a simplified Michaelis-Menten rate equation of zero or first order has been employed so that a closed form of effectiveness factor in terms of the Thiele Modulus and other parameters could be obtained. In reality, such a simplified Michaelis-Menten rate equation constitutes only a special case of its general form which may not be able to display the true reaction characteristics of the immobilized enzyme catalysts. Fink et a1.,6 Miyamoto et al.,? and Moo-Young and Kobayashis investigated the effectiveness factor by using the complete form of the Michaelis-Menten rate equation. Horvath and Engasserg considered the effectiveness factor in a pellicular immobilized enzyme catalyst. A common assumption made in all the above works is that the enzymatic reaction is carried out under isothermal condition and with a constant enzyme activity during the whole reaction. This may not be true in a number of real circumstances because of the exothermic nature of enzymatic reactions.*0-I6 For a nonisothermal reaction, temperature plays an extremely important role in evaluating the effectiveness factor of an immobilized enzyme catalyst because the activity of enzyme is rather sensitive to the reaction temperature. It has been widely recognized that above a certain level of temperature, the enzyme activity decreases significantly.10-' This phenomenon, known as the thermal inactivation or denaturation, occurs frequently in many enzymatic reactions. The decreasing enzyme activity tends to place the entire enzymatic reaction in a transient state rather than a steady state as is generally considered. Thus far no work has ever taken thermal inactivation into consideration in the investigation of the effectiveness factor of an immobilized enzyme catalyst. The purpose of this work is to point out that it is important to consider the transient state when investigating the heterogeneous reactions in an immobilized enzyme catalyst.

TRANSIENT MATHEMATIC MODEL

Consider the following enzymatic reaction ki k

+ S F= ES -A E + P

k-i 1237 @ 1975 by John Wiley & Sons, Inc.

1238 BIOTECHNOLOGY AND BIOENGINEERING VOL. XVII (1975) The unsteady state material and energy balances are given by

&,? = k,
bt brz

(? + ?.> +

k$E( -AH) exp

(- ">

(3)

c be S k, ROT in which the enzyme concentration, E, is given by

-bE

= -k.E exp

(- g)

bt (4)

The initial and boundary conditions for the above equations are: t = o ; S = 0, T = To, E = Eo bS bT

br be
f

=o;-=o,-=o

By imposing appropriate assumptions, eqs. (2) through (7) can be reduced to all the cases investigated by the previous authors.1-9
00

0.6
0

04

0.2 CII1 30 60 90 I20 Fig. 1. Effect of mass transfer Nusselt number on the dimensionless substrate

concentration with a, = 1.0, K, = 0.35, p2 = 117.5,@, = 18.5, K. = 1.1 X 1046, a2 = 0.5, h = 0.5 and (Nu),, = 0.5.
COMMUNICATIONS TO THE EDITOR 1239 It can be noted that it is more difficult to solve eqs. (2), (3), and (4) than to solve the corresponding material balance equation for the steady state because of the highly nonlinear reaction terms involved in these equations. However, eqs. (2) and (3) can be numerically solved by the implicit Crank-Nicolson finite difference method," and the fourth order Runge-Kutta method" can be employed to integrate eq. (4). Figures 1, 2, and 3, respectively, show the transient variations of the dimensionless substrate concentration, enzyme activity, and dimensionless temperature at the catalyst center (dashed lines) and the catalyst surface (solid line) for a specific example. For steady state enzymatic reaction, the effectiveness factor is defined as the ratio of actual reaction rate to the hypothetic reaction rate in the absence of internal diffusion resistance.lsJ9 According to the definition, the effectiveness factor can be written as
I

04
Df

8
04

0.2
C 0 30 60 90 120

r Fig. 2. Effect of mass transfer Nusselt number on the enzyme activity.

1240 BIOTECHNOLOGY AND BIOENGINEERING VOL. XVII (1975)

1.07r
0 30 60 90 120

Fig. 3. Effect of mass transfer Nusselt number on the dimensionless temperature. For the steady state reaction, the concentration gradient at the catalyst surface is an invariant; however, for the unsteady state, it is time-dependent. The substrate which has already diffused into the particle does not represent the actual reaction rate because part of the substrate is accumulated inside the particle. Therefore, eq. (8) can not be considered as the effectiveness factor for the unsteady state case and does not have specific meaning except for representing the variation of substrate concentration gradient at the catalyst surface.
Nomenclature
a

C
CP

D E

Eo
AEi AEz
h

surface area per unit volume of catalyst particle dimensionless substrate concentration, S/So heat capacity of fluid effective diffusivity enzyme concentration initial enzyme concentration activation energy for enzymatic reaction activation energy for enzyme inactivation Thiele modulus, rg dkoEo/DSo COMMUNICATIONS TO THE EDITOR 1241 hr heat transfer coefficient ( -AH) heat generation by enzymatic reaction turnover number inactivation coefficient of enzyme mass transfer coefficient Michaelis-Menten coefficient frequency factor thermal conductivity dimensionless inactivation coefficient of enzyme, k,ro2/ D dimensionless Michaelis-Menten coefficient, k,/So

heat transfer Nusselt number, htro/kt mass transfer Nusselt number, kLaro/D r radial coordinate ro radius of catalyst particle R R, gas constant S substrate concentration

so
t time T temperature To initial temperature Greek Letters
a1

a2

81

82 P fluid density e dimensionless temperature, TITO

'T

dimensionless time, t D/ro2

+ enzyme activity, E/EO

7 .: dimensionless

radial coordinate, r/ro substrate concentration outside the catalyst ratio of Schmidt number to Prandtl number, kl/pCpD dimensionless heat generation parameter, ( - AH)SO/~C,TO dimensionless activation energy for enzymatic reaction, AEI/R,To dimensionless activation energy for enzyme inactivation, A&/R,l'u dimensionless parameter defined by eq. (8)
[Repr intedf rom the Journal of the Amer i canC hemi calS <i c iet l ' .99,2: ]6(61 977) .1 Copvright 1977 bv the American (-'hemical Societv and reprint,ed bv permission of the copvright owner

Large-Scale Enzymatic Synthesis with Cofactor RegenerationG: lucose6 -Phosphatel Sir.' Ma ny i mp ortan t reactionsi n enzyme-c atalyz edb iosynth esis consumec ofactorsin stoichiometriqcu antitiesT. he cost2o f the mostc ommonlyr equiredc ofactorsh asd iscouragetdh e use of thesee nzymaticr eactionsf or the synthesiso f organic compoundosn any scaleg reatert hana fractiono f a mole.3'a We havep reviouslyp roposeda schemefo r thee nzymaticr egenerationo f ATP from ADP or AMP, ando utlinedi ts possibleu sei n large-scalceo factor-requirinsgy nthesisH.5e rew e demonstratteh ep racticalityo f thiss chemeb y thep reparation of glucose6 -phosphate(G -6-P) from glucoseo n a mole scale. A representativree actionw as carriedo ut in a 5-L flask modifiedt o accepta pH electrodeT. he flaskw asc hargedw ith 1200m L of solut ion( pH 6.6)c ontainingg lucose(1 .4m ol ) , ATP (10 mmol), MgCl2 (98 mmol), EDTA (4.8 mmol), and dithiothreito(l I 8 mmol).P olyacrylamidgee lp articles(2 0-50 pm in diameter)c ontainingc ovalentlyim mobilizedh exokinase (ATP: D-hexose-6-phosphotransfeEr.a Cs.e 2,. 7. l .l, 1200U .) and acetatek inase( ATP: acetatep hosphotransferasEe. ,C . 2.7.2.1,I 100 U. ) weres uspendeidn this solut ion.D6 iammoniuma cetylp hosphate(A cP, 0.7 M) wasa ddedc ontinuouslyo ver4 8 h at 40 mL/h to them agneticallsyt irredr eactronm ixture.TT he solutionw asm aintainedb etweenp H 6.6 and6 .9 by additiono f 4 M potassiumc arbonates olutionu sing an automaticp H controller.T8 he reactionw asc onducteda t 25 "C, and the reactionm ixturea nd reagents olutionsw ere deoxygenatebde foreu sea nd maintainedu ndera rgon.A fter 50 h of operation( I .36m olo f AcP added)e, nzymatica ssaye indicatedth at 1.09m ol of G-6-Ph ad beenf ormed:i ts final concent rat iowna s0 .31M . The polyacrylamidgee lp ar t icles werea llowedto settlea, ndt hes olutionw asd ecantedl.n organic phosphat(e0 .27m ol,e stimatedb y thed ifferenceb etweenth e AcP addeda ndt heG -6-Pf ormed)w asp recipitatebdy addition of a stoichiomet.rqicu antityo f Ba(OH)2a ndr emovedb y filtration.

G -6-Pw ast henp recipitatebdy additiono f 1.2m ol of Ba(OH)2:t he resul t ings ol id( 502g ) contained9 2o/Bo a G6-P- lH2O( 0.89 mol ) by enzymat ica ssay.eT his quant i ty correspondtso a 65%y ieldb asedo n AcP added.T he activities of hexokinasaen da cetatek inasew erer ecovereidn theg el in 93 and 75 o/oyi eld,r espectivelyT.h e turnovern umberf or ATP duringt her eactionw as) 100;n o effortw asm adet o recover it. Three points concerning experimental details deserve ment ionF. i rst ,t hei ni t ialq uant i t ieosf ATP andM g( l l ) were chosens ucht hatt hec oncent rat ioonf MgATP andM gADP wouldb e well abovet he Michaelisc onstantsfo r the soluble enzymes, le0v ena f terd i lut ionb y theA cP solut ionS. econd, the reactionp roceedesda tisfactorilwy ith AcP having) 80o/o purity. If the purity fell below8 0o/oc,o rnplexatioann d precipi tat iono f Mg( l l ) by thep hosphatiem pur i t iesm adei t di f f icul t to maintaina dequateco ncent rat ionosf MgADP and MgATP in solut iona, ndt roublesomteo isolateB a G-6-Pi n highp urity.T hird, it wasu sefutl o carryo ut ther eactions ot hat addi t iono f AcP to thes olut ionw aso veralrl ate- l imi t inga nd AcP wasn everp resenitn the reactionm ixturei n high concent rat ionsto, minimizes pontaneouhsy drolysiosf AcP wi th concomitanrte leaseo f phosphate. Compar isono f this preparat iono f G-6-P wi th exist ing chemical tot r enzymat iclm2 ethodsi l lust ratetsh e potent ial of ATP-requiringe nzymatics ynthesisfo r the regioselective modificationo f unprotectedw, ater-solublep,o lyfunctional substratesS.i ncet heh exokinasehsa veb roads ubstratesp ecif ici ty, rst his sequencseh ouldb e di rect lya ppl icableto the preparationo f phosphateosf a numbero f others ugars( e.9., fructosem, annosed, eoxy-o-glucosgel,u cosamineI)n. broader terms,t hisc onversioens tablishethsa t it rsp racticalt o couple enzymaticA TP regeneratiowni th ATP-requiringe nzymatic synthesitso achievela rge-scaoler ganict r ansformat ionRse. actionsw hichr equirer egeneratioonf ATP from AMP area lso accessibules ingt hisr eact ions equenceb,y addinga denylate kinase(A MP:ATP phosphot ransferaEs.e C, . 2.7. 1.3\t o cat alyzet hec onversioonf AMP andA TP to ADP;5w ew ill providee xampleso f this typeo f reactions equencien the immeI 977

;].T
G-6-P

OH

( ATP \ cH,]co2\ ADP

oo ll ll -ocHrcoPi
\ - - - tJ
AcP \L cH,:t-:0 \ *'r' H3P0,

Journal of the American Chemical Society / 99:7 / March 30, diatef uture.T he goods tabilityo f the immobilizede nzymes, andt he easeo f theirr ecoverys, uggesttsh at theses ynthesiasn d regeneratiosnc hemessh ouldh aveb roada pplicabilityin preparativeo rganicc hemistryI. 2 Referencesa nd Notes

(1) Supportedb y the NationalS cienceF oundation(R ANN),G rantN o. Gl 34284. (2) Estinntedc osts,$ /nrole:A TP,2 ,000:N AD+,2 ,500;N ADH.1 8,000;N ADP+, 60,000;N ADPH2, 50,000. (3) J. B. Jones,D . Perlmana, ndC . J. Sih,E d.," Applicationso f Biochemical Systernsin OrganicC hemistryP, arts1 and2 ", Wiley,N ew York, N.Y., '1976. (4) C. J. Suckl inga ndK . E. Suckl ingC, hem.S oc.B ev. ,3,3 87 (1974) . (5) C. R.G ardneer t al.i n "EnzymeE ngineerin2g", ,K.E. foe andL . B.W ingard, Ed.,P lenumP ress,N ew York,N .Y.,1 974,p 209;G . M. Whitesidese t al., ibid., 9 217. (6) Enzymesw ere immobilizedb y additionto a solutionc ontaininga polymerizingm ixtureo f acrylamideN, ,M-methylenebisacrylamiadned, t he l* hydroxysuccinimidaec livee stero f methacrylica cid, 10s beforeg el formation. The procedure used is a modification of that described (G. M. Whitesidese t al., MethodsE nzymol.i,n press).l mmobilizationy ieldsw ere 35o/o lor hexokinase, and 40o/o for acetate kinase. The enzymes were commerciapl reparation(sS igma)a, nd were usedw ithoutp urificationt:h eir specifica ctivities(p molm in-1m g-1)were:h exokinas(ef romy east)4, 20; acetatek inase( fromE . colr)f ollowinga ctivationw ithd ithiothreito3l,0 0. (7) Diammonium acetyl phosphate was prepared in a separate step by reaction of ketenew itha nhydroups hosphorica cid,f ollowedb y neutralizatioann d

2367
precipitation with anhydrous ammonia: G. M. Whitesides, M. Siegel, and P. Garrett, J. Ag Ctem., 40, 2516 (1975). The material used was 80-85% pure, with ammonium acetate and acetamide as the principal impurities. The acetyl phosphate solution was maintained at 0 "C before addition to minimize hydrolysis. (8) Preliminary experiments indicated that, under simulated reactor conditions, the rate ol G-6-P formation was faster at pH 6.7 than at higher pH where the soluble enzymes would be expected to be more active (A. Sols, G. delaFuente, C. D. Villar-Palasi, and C. Ascensio, Biochem. Biophys. Acta, 30, 92 (1958); l. A. Rose, M. Grunberg-Manago, S. T. Korey, and S. Ochoa, J. Biol. Chem., 2'11,737 (1954)). (9) H. U. Bergmeyer, Ed., "Methods of Enzymatic Analysis", Verlag Chemie Weinheim, Academic Press, New York and London, 1974, p 1238. ('10) Hexokinase, A. Sols et al., Biochim. Biophys. Acta, 30,92 (1958); acetate kinase, C. A. Janson and W. W. Cleland, J. Biol. Chem., 249,2567 (19741, R. S. Langer, C. R. Gardner, B. K. Hamilton, and C. K. Colton, AIChE J., in press. (11) H. A. Lardy and H. O. L. Fischer, Biochem. Prep.,2,39 (1952). (12) W. A. Wood and B. L. Horecker, Biochem. Prep.,3,71 (1953). (13) M. Dixon and E. C. Webb, "Enzymes", 2nd ed, Academic Press, New York, N.Y., 1964, p 216, and references cited therein. (14) G. M. Whitesides, in ref 3, part 2, Chapter Vll.

Alfred Pollak, Richard L. Baughn, George M. Whitesides* Deport ment of C he mi st r y M as s ac hus et t s I nst it ut e of Tec h nol og1: Cambridge,M assachusetts0 2 I 39 Receiued December 7. I976