Electrically Immobilized EnzymeReactors: Bioconversion of aCharged Substrate. Hydrolysis ofPenicillin G by Penicillin G Acylase

Alessandra Bossi, Stefania Guerrera, Pier Giorgio Righetti

University of Verona, Department of Industrial and AgriculturalBiotechnologies, Strada Le Grazie, Ca’ Vignal, 37134 Verona, Italy;telephone/fax: +39 045 809 8901; e-mail: righetti@imiucca.csi.unimi.it

Received 21 August 1998; accepted 29 December 1998

Abstract: The possibility of using the multicompartmentimmobilized enzyme reactor (MIER) in presence of acharged substrate is here explored. Penicillin G acylase isused to convert penicillin G (a free acid, with a pK of 2.6)into two charged products: phenyl acetic acid (PAA, witha pK of 4.2) and 6-aminopenicillanic acid (6-APA, a zwit-terion with a pI of 3.6). The enzyme is trapped by anisoelectric mechanism in a chamber of the electrolyzerdelimited by a pI 5.0 and a pI 9.0 amphoteric, isoelectricmembranes. Under normal operating conditions (con-tinuous substrate feeding in the presence of an electricfield), only a low substrate conversion can be achieved,due to rapid electrophoretic transport of unreacted peni-cillin G out of the reaction chamber towards the anode.Excellent conversion rates (>96%) are obtained under a“doubly-discontinuous” operation mode: a time-lapsesubstrate feeding, accompanied by short times (4–8 min)of electric field interruption. The product of interest (6-APA, a precursor of semisynthetic penicillins), by virtueof its amphoteric nature, is trapped in a chamber delim-ited by a pI 3.5 membrane and a pI 5.5 membrane, adja-cent to the reaction chamber on its anodic side. The othercontaminant product (PAA) first accumulates in the samechamber and then progressively vacates it to collect inthe anodic reservoir, leaving behind a pure 6-APA solu-tion. In this operation mode, vanishing amounts of un-reacted substrate (penicillin G) leave the reaction cham-ber to contaminate the adjacent, anodic chambers. Anovel class of zwitterionic buffers is additionally re-ported, able to cover very thoroughly any pH value alongthe pH 3–10 interval: polymeric, zwitterionic buffers, syn-thesized with the principle of the Immobiline (acrylamidoweak acids and bases) chemicals. Enhanced enzyme re-activity is found in this macromolecular buffers as com-pared to conventional ones. © 1999 John Wiley & Sons, Inc.Biotechnol Bioeng 64: 383–391, 1999.Keywords: isoelectric membranes; immobilized reactors;penicillin G acylase

INTRODUCTION

Biocatalysis and bioseparation evolved as separated fieldsfor the study of biotransformation processes. Usually suchprocesses involve the enzyme conversion of substrates into

products of chemical or pharmaceutical interest. While bio-catalysis explores enzyme immobilization techniques forenhancing the catalytic properties and the resistance againstdenaturation of the macromolecular catalysts, bioseparationconcerns the recovery of products. A growing number ofattempts have recently been made in coupling enzyme im-mobilization–stabilization and product recovery, and an in-creasing interest has been devoted to the application of elec-tric fields for product recovery, such as electrodialysis(Nidetzky et al., 1996; Ishimura and Suga, 1992). Amongthe developments of integrated bioprocesses, a membraneimmobilized enzyme reactor (MIER) was first described byus in 1996 (Chiari et al., 1996), and a mathematical modelfor the related transport equations was developed by Nembriet al. (1997).

The MIER is peculiar because of the trapping of theenzyme between two isoelectric membranes in the presenceof an electric field, which acts as a driving force in remov-ing the products of the biotransformation. Immobilization isbased on the principle of isoelectric focusing, thus the en-zyme does not have to be subjected to any structuralchanges: it is soluble in the reaction chamber aqueous so-lution and is confined between two charged membraneshaving isoelectric points (pI) encompassing the pI value ofthe macromolecular catalyst (Righetti and Bossi, 1998). Thepossibility of preparing membranes with the most appropri-ate pI values and the great flexibility of the reactor configu-ration, in terms of number of chambers and recirculatedvolumes, make the MIER an interesting alternative to con-ventional reactors and suggests that it might be helpful inthe catalysis of multistep reactions, where several enzymesare involved.

When exploring a variety of reactions (Chiari et al., 1996;Nembri et al.; 1997, Righetti et al.; 1997; Bossi et al., 1998)in order to define the fields of advantageous applications ofthe MIER reactor, a few general guidelines were derived:(1) the MIER is adequate for enzymes inhibited by prod-ucts; (2) the MIER could be exploited for separation andrecovery of charged products; (3) the presence of an electricfield imposes limitations in buffer choice: only zwitterionic,Correspondence to:P. G. Righetti should be addressed.

© 1999 John Wiley & Sons, Inc. CCC 0006-3592/99/040383-09

isoelectric compounds are allowed as buffering species. Inthe present paper we study the hydrolysis of penicillin G bypenicillin G acylase in the MIER reactor. This well-knownreaction, which leads to the formation of 6-aminopenicil-lanic acid (6-APA), an intermediate for the synthesis ofsemisynthetic antibiotics, and phenylacetic acid (PAA),which severely inhibits the enzyme, allows us to work outseveral aspects of the influence of the electric field. Becausethe substrate contains a carboxyl group, its migration to-wards the anode has to be taken into consideration. Sec-ondly, the chemistry of Penicillium is dominated by reac-tions, usually nucleophilic attacks, at the highly labileb-lac-tam ring carbonyl at 7-position (Arnott and Weatherley,1995); therefore, substrate degradation effects, caused eitherby the field strength or by sudden decrements in the pHvalue during the reaction, have to be evaluated. The stabilityof the enzyme in the reactor is considered too, and a novelbuffering system is proposed: a polymeric, zwitterionicbuffer. This macromolecular buffer is synthesized, and itschemico-physical properties, as well as its stabilizing effectonto the enzyme catalyst, are evaluated. Finally we consid-ered the efficiency of the reactor, in terms of yield of reac-tion at the end of the process, arising from different ways ofoperating the MIER: A comparison between continuoussubstrate infusion mode, time-lapse substrate feeding modeand discontinuous electric field operation mode was made.

MATERIALS AND METHODS

Equipment and Reagents

Penicillin G acylase (E.C. 3.5.1.11) fromEscherichia coli(22 U/mg),D-histidine, MOPS, and penicillin G potassiumsalt were from Sigma (St. Louis, MO); Immobiline chemi-cals for the preparation of membranes were from PharmaciaBiotech (Uppsala, Sweden); Immobilines of pK 7.0 and 8.5,for the polymerization of macromolecular buffers, were pur-chased as powders from Fluka (Buchs, Switzerland).NaOH, acetic acid, formic acid, 2-propanol, glycerol, Bi-cine, borate, acetonitrile, and trifluoroethanol were fromMerck (Darmstadt, Germany). Acrylamide,N,N8-methylenebisacrylamide (Bis), ammonium persulphate, andN,N,N8,N8-tetramethylethylene diamine (TEMED) werefrom Bio-Rad (Hercules, CA, USA). The MIER reactor wasfrom Talent S.r.l. (Trieste, Italy). The capillary electropho-resis Quanta 4000 apparatus was from Waters (Bedford,MA, USA) and the capillaries (50-mm I.D., 375-mm O.D.)were from Polymicro Technologies (Phoenix, AZ). Dialysismembranes (cutoff 6,000–8,000 Da) were from Spectra/Por(Laguna Hills, CA) and the ultrafitration system, Microcon30,000, was purchased from Waters (Bedford, MA).

Polymerization of the Acrylamido-Buffer

The recipes for the zwitterionic polymeric buffers were cal-culated with the computer program of Giaffreda et al.

(1993) and are shown in Table I. Immobilines were added ata concentration of 10 or 20 mM and admixed with 9% Tacrylamide monomers (in the absence of crosslinker) and2-propanol to a final concentration of 3%. The solutionswere degassed for 1 h, polymerization was then carried outat 70°C for 2 h, and finally the polymeric buffer was ex-tensively dialyzed against distilled water.

Batch Reactions

Batch reactions were performed at a temperature of 25°C inthe following buffers: 50 mM Bicine–NaOH, pH 8.0; 50mM MOPS–acetate, pH 7.0; 50 mM D-histidine, pH 7.6;zwitterionic polymeric buffers, pH 7.5 and 8.2 (either assynthesized or at a 1:1 dilution). 0.99 U/mL of enzyme and10 mM of substrate were utilized. At different time periodsaliquots were withdrawn and reactions were blocked bycentrifugation at 10,000 g for 5 min with Microcon 30,000microconcentrators and subsequently analyzed by capillaryzone electrophoresis (CZE).

Stability of Penicillin G in Acidic Solutions

Penicillin G (10 mM) was dissolved in a solution of 5 mMformic acid at pH 3 and the degradation was monitored over3 h by CZE analysis of the sampled aliquots. Another ex-periment of degradation was carried out in the same condi-tions, but by thermostating the solution at 40°C.

Capillary Electrophoresis Analysis

Capillary electrophoresis was used as an analytical tool formonitoring the enzymatic hydrolysis in batch and in theMIER reactor. Samples were added with 2 mM acrylamideinternal standard and 20% acetonitrile, then injected by hy-drostatic pressure for 4 s in a 32-cm long, 50-mm I.D.,uncoated capillary. Runs were performed in 25 mM borateNaOH buffer, pH 10.0, at a potential of 10 kV with a cor-responding current of about 18mA. The detection wave-length was 214 nm. Peak quantitation was obtained by in-tegration of the normalized peak areas. The Maximaprogram, supplied with the Quanta 4000 capillary electro-phoresis unit, was used for data treatment. The reaction

Table I. Chemical composition of zwitterionic, polymeric buffers.

Chemical species

Zwitterionic polymeric buffer

pH 7.5 pH 8.2

Immobiline pK 7.0 20 mM —Immobiline pK 8.5 — 20 mMImmobiline pK 3.6 5.94 mM 9.79 mMAcrylamide 9% 9%2-Propanol 3% 3%Calculatedb-power 20 mequ/(litre?pH)a 20 mequ/(litre?pH)Experimentalb-power 19.1 mequ/(litre?pH) 18.2 mequ/(litre?pH)

amequ4 milliequivalents.

384 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 64, NO. 4, AUGUST 20, 1999

progress was followed via determination of substrate con-centration decrements and product formation as a functionof time. The quantitation of substrate and products (6-APAand PAA) was obtained by calibration curves. The maindegradation product, penicillenic acid (Arnott, 1995) wasidentified by its migration time, whereas additional degra-dation products could not be recognized but were presentonly in minute amounts in the MIER.

Membrane Preparation and Setup of theMIER Instrument

The isoelectric membranes were prepared as described byRighetti et al. (1989, 1990): Glass fiber filters WhatmannGF/D were used as support for the polymerization of solu-tions made of 7% T, 4% C neutral monomers (acrylamideand N,N8-methylene bisacrylamide) and the appropriateconcentration of Immobiline chemicals. Three membraneswith pI 3.5, 5.5, and 9.0 were cast with the following reci-pes: 20 mM Immobiline pK 3.6 titrated with 9.48 mM Im-mobiline pK 9.3 for the pI 3.5; 15 mM Immobiline pK 4.6and 15 mM of Immobiline pK 6.2 for the pI 5.5; and 20 mMImmobiline pK 9.3 titrated with 16.62 mM Immobiline pK3.6 for the pI 3.6 membranes. The polymerization condi-tions were 1 h at 50°C, then each polymerized filter wasextensively washed in distilled water, so as to remove un-reacted monomers and catalysts. The MIER instrument wasset up as shown in Fig. 2. The electrolyzer was assembledwith four chambers: anodic, chamber 1, reaction chamber,and cathodic chamber, each having a volume of 7.5 mL.Each chamber was connected to a reservoir, thus the finalvolume of each compartment was 35 mL and recirculationwas provided by a membrane pump with a flow rate of 5mL/min. The anodic solution was 100 mM acetic acid, pH2.85 (conductivity 39mS/cm), and the cathodic solution was1.5 mM NaOH, pH 11.17, with a conductivity of 43mS/cm.Chamber 1 was filled with distilled water whereas the re-action chamber contained the zwitterionic polymeric buffereither at pH 8.2 or at pH 7.5. The enzyme, penicillin Gacylase fromE. coli,was isoelectrically trapped between thepI 5.5 and pI 9.0 membranes at a concentration of 0.64UE/mL. Runs were performed at different voltage condi-tions: 50, 100, and 200 V. The temperature of operation was25°C, and it was kept constant by the presence of Peltierunits. One MIER experiment was carried out with the ad-dition of 10% trifluoroethanol to all the chambers of thereactor.

Mode of Operation

The following definitions apply.Time-lapse substrate feeding:in this operation mode the

electric field is continuously applied, whereas the substrateis added discontinuously to the reaction chamber at well-defined time intervals (at 0, 47, 94 and 145 min).

Doubly discontinuous mode:both the substrate and theelectric field are applied discontinuously. The electric field

is stopped for a defined time period (8 min) after eachaddition (for 2 min) of substrate at the following time in-tervals: 0, 48, 92 and 143 min.

Continuous infusion mode:the electric field is continu-ously applied and the substrate is continuously infused at arate of 1 mM/min.

RESULTS

Choice of the Appropriate Buffering System

Preliminary batch reactions were carried out in differentbuffer conditions: MOPS–NaOH, pH 7.0; Bicine–NaOH,pH 8.0; or the zwitterionic, isoelectric D-histidine buffer,pH 7.6. In MOPS and Bicine buffers, 10 mM penicillin Gwas converted at rates of 1.8 and 3.7mmol/min ? UE re-spectively, indicating that increasing the pH from a value of7.0 to 8.0 will improve significantly the reaction rate (Fig.1). The data of chemical catalysis were collected by moni-toring the development of products with batches prepared inthe same buffering conditions but in the absence of catalyst:The contribution of chemical catalysis was negligible ascompared with the rates indicated above. The reactions inbatch were monitored over a time period of 60 min, and theyield did not exceed 50%.

Another set of experiments was performed in D-histidinebuffer, whose zwitterionic properties and whose (pI − pK)value, which is≅1.5, as listed by Svensson in his table ofpossible carrier ampholytes (Svensson, 1962; Righetti,1983), make it suitable for use in the MIER. The substrateconcentration and the units of enzyme of batches run inD-

Figure 1. Reaction rate of penicillin G acylase in presence of differentbuffers. Crosses, 50 mM MOPS acetate, pH 7.0; squares, 50 mM BICINE–NaOH, pH 8.0; diamonds, zwitterionic, polymeric buffer, pH 7.5. Initialsubstrate concentration: 10 mM.

BOSSI, GUERRERA, AND RIGHETTI: ISOELECTRICALLY TRAPPED ELECTROENZYMIC BIOREACTOR 385

histidine were the same as in the previous experiments withBicine and MOPS buffers. However, when capillary elec-trophoresis separations were performed using the aliquotswithdrawn at different time periods for quantitative analy-sis, the absorption of the imidazole ring of the histidinebuffer at the detection wavelength of 214 nm produced amarked peak, which interfered with the quantitation proce-dure. Therefore we opted for a UV-transparent zwitterionicbuffering system and thus we selected and testeda,b-diaminopropionic acid [pI 4 8.20 and (pI − pK) 4 1.4].The results were not encouraging, however, because thechemical structure of the buffering compound has the fun-damental geometrical requirements for fitting within the ac-tive site of the enzyme. As a consequence, several undesir-able byproducts, arising from acylation reactions, were re-covered at the end of the process.

Design and Synthesis of a PolymericZwitterionic Buffer

In order to resolve the buffering problems in the MIERreactor, as described above, a new generation of macromo-lecular buffers was designed, based on the Immobiline tech-nology. Immobiline chemicals are acrylamido-based buff-ers, synthesized by Chiari et al. (1989a,b) and successfullyadopted for the preparation of immobilized pH gradients(IPG) for both analytical and preparative purposes (Righetti,1990). Immobilines were built up to carry a weak acidic ora weak basic group as substituent of the nitrogen involved inthe amido bond. By cross-polymerizing stochiometric quan-

tities of acidic and basic Immobilines, admixed with theneutral monomer acrylamide, an amphoteric isoelectricpolymer with the desired pI value and buffering powerproperties will be generated.

With the aid of the computer program of Giaffreda et al.(1993), recipes for polymeric buffers were calculated andare shown in Table I: buffers were prepared with pI 7.5 and8.2 and with a calculated buffering power (b) of 10 and 20mequiv/L. Polymerization was conducted at high tempera-tures and in the presence of a terminating agent, so as tocontrol the chain length of the polymer, which is directlyrelated to its viscosity (Gelfi et al., 1995). Experimentaltitration, by sequential additions of 2 mM of a standardsolution of 1N HCl, was used for evaluating the expectedbpower. The experimentalb power, as shown in Table I, isdirectly related to the amount of Immobilines incorporatedduring the polymerization reaction, and the data indicatethat the protocol of synthesis allows a very high incorpora-tion efficiency (>90%). Batch reactions in presence of thepH 7.5 polymeric buffer (20 mM Immobiline, 9% T neutralmonomers) are shown in Fig. 1: The enzyme displays areaction rate of 1.94mmol/min ? UE, and the yield of con-version is 88%, which is significantly higher as compared toresults obtained with MOPS and Bicine buffers.

Preliminary Experiments with the MIER: The Rateof Substrate Feeding and SubstrateDegradation Problems

A first set of experiments was conducted in the MIER re-actor equipped as described in the scheme of Fig. 2. Be-cause of its chemical structure, penicillin G is negativelycharged over a wide range of pH values, due to the freecarboxylic group of theb-lactam ring (pK 2.6; see Fig. 3).When the substrate is added to the reaction chamber therewill be a competition between its hydrolytic conversion bythe enzyme and its escape induced by the electric field.

The rate of escape of penicillin G through the electrolyzerwas experimentally determined by introducing 10 mM (75mmol) of substrate in the reaction chamber and by quanti-tating the amount of penicillin G migrated anodically as afunction of time (Fig. 4). The anodic migration rate was 3.5mmol/min at 50 V and 8.7mmol/min at 100 V, which sug-gests that the leakage due to electric transport is quite ap-preciable and is proportional to the applied electric field.The substrate conversion rate calculated from batch experi-ments in the polymeric zwitterionic buffer of pI 7.5 is 1.9mmol/(min ? EU); since the MIER contains a total of 22.4EU, this means that total conversion of 43mmol/min ofsubstrate should occur in the reaction chamber. By simplycombining anodic migration rates and substrate conversionrates obtained from batch experiments, we established theupper limit of the optimal substrate feeding rate for theMIER experiments, in presence of 22.4 total enzyme units(EU) and 100 V applied voltage. The enzyme convertedtotally 43.4 mmol/min of penicillin G; the aliquot of sub-strate converted and subjected to the electric field is pro-

Figure 2. Assemblage of the MIER. Symbols: A, anodic reservoir; 1,chamber 1; R, reaction chamber; C, cathodic reservoir. P1–P4: four peri-staltic pumps. The three isoelectric membranes are marked as pI 3.5, pI 5.5,and pI 9.0 septa. The arrowheads indicate liquid recirculation from thevarious reservoirs in and out of the electric field.

386 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 64, NO. 4, AUGUST 20, 1999

portional to the liquid volume contained in between the twoisoelectric membranes delimiting the reaction chamber andis 8 mmol/min, whereas the substrated leakage due to elec-tric transport would be 8.7mmol/min; therefore the optimalsubstrate infusion should be 43.4mmol/min.

The stability of penicillin G in the reactor was then in-vestigated. It is well known that penicillin G undergoesacidic degradation with first-order kinetics (Benedict et al.,1945); the complete degradation scheme is given by Blaha

et al. (1976). The following mathematical expression givesthe rate of formation of 6-aminopenicillanic acid:

Rate=k@H+# ? @penicillin#

Ka + @H+#, (1)

wherek is the specific first order rate constant andKa is theacid dissociation constant. The MIER reaction chamberbuffered at pH 7.5 and at pH 8.2 was fed with 10 mMpenicillin G, the anodic migration took place at 50 V, andthe pH dependent degradation of the substrate was moni-tored by sampling aliquots from all the chambers of theelectrolyzer at different time periods. Penicillin G and itsdegradation forms were separated by capillary zone electro-phoresis (CZE); Fig. 5 shows a typical electropherogram ofdegraded penicillin G.

In order to assess if degradation was increased by thepresence of the electric field a control experiment was setup: 10 mM penicillin G was dissolved in 5 mM formic acid,pH 3, and aliquots were analyzed by CZE. No significantdifferences were detected between the two sets of data,therefore under our experimental conditions there does notseem to be additional field-induced degradation. Our resultsare in accordance with the work of Arnott and Weatherley(1995). Another control experiment was run at constanttemperature (40°C) under the conditions described above:The degradation process seemed to be three times quicker athigh temperatures, therefore the MIER reactions were con-ducted at 25°C.

Eq. (1) is the mathematical expression of the rate of for-mation of 6-aminopenicillanic acid in a solution with agiven proton concentration. In the MIER system the H+

concentration is not constant: A gradient of protons will be

Figure 4. Rate of electric transport of the substrate (penicillin G) out ofthe enzyme reaction chamber in the MIER apparatus in the presence of avoltage gradient of 50 V.

Figure 3. Reaction scheme of penicillin G acylase. The substrate (penicillin G, a free acid with a pK of 2.6) is converted into phenylacetic acid (PAA,pK 4.2) and 6-aminopenicillanic acid (6-APA, an amphoteric compound with pI 3.6, pK 2.6 for the free carboxyl, and pK 4.6 for the free amino group).

BOSSI, GUERRERA, AND RIGHETTI: ISOELECTRICALLY TRAPPED ELECTROENZYMIC BIOREACTOR 387

established immediately after voltage application, and theH+ concentration will thus be different in the various cham-bers. Moreover when the enzymatic reaction takes placethere is a continuous development of acidic products, whichwill migrate along the reactor compartments and collect inchamber 1 or at the anode according to their respective pKor pI values (see the reaction scheme of Fig. 3). A timedependence of the H+ concentration could be considered.We could write Eq. (2) as the rate of formation of 6-ami-nopenicillanic acid specific for each reactor chamber:

RateChi =k@H+#Chi @penicillin#Chi

Ka + @H+#Chi

, (2)

and include the time dependence in Eq. (3)

­Rate

­t=

k ?­@H+#Chi

­t? @penicillin#Chi

Ka ?­@H+#Chi

­t

, (3)

where­@H+#Chi

­t

is related to field strength, permeation through the mem-branes and migration coefficient.

Enzymatic Hydrolysis of Penicillin G in theMIER Reactor

The MIER reactor was set up as described in Fig. 2, thereaction chamber was buffered with the zwitterionic poly-meric buffer, pH 8.2, and the enzyme was added to a finalconcentration of 0.64 U/mL. Different ways of substrateaddition were adopted in order to reduce the anodic leakageof penicillin G and to study which operation method wouldincrease the efficiency of conversion of the reactor.

The first operation mode was called time-lapse substratefeeding: a final concentration of 3 mM penicillin G wasadded to the reaction chamber at 0, 47, 94, and 145 min,such times corresponding to minima in the plot of currentversus time of Fig. 6. This plot is characterized by a cycliccourse; a sudden increase of current to a maximum is theconsequence of the substrate addition, since, when hydro-lysis takes place, one mole of penicillin G is converted into2 mol of charged products and there is a double concentra-tion of ionic species in the MIER, generating the currentpeak. When all the substrate is either enzymatically con-verted or migrated to the anode, the current returns to theinitial value. The partial yield of the reaction in the time-lapse substrate feeding operation mode was about 95% at 45min; however, as time passed, the amount of penicillin Gwhich migrated through the anode increased, thus at the endof the process (time4 160 min) the yield was only 80%.

In order to ameliorate the total yield of the process westudied the effect of a double discontinuity, of substratefeeding and of application of the electric field in the MIER.The reaction chamber was fed with substrate at a final con-centration of 2 mM at 0, 48, 92, and 143 min. As the sub-strate was added in the MIER reaction chamber the voltageapplication was stopped for 8 min, thus a complete mixingof all the components was allowed and the enzymatic ac-tivity towards penicillin G began as a semi-batch process.The monitoring of this doubly-discontinuous process isshown in Fig. 7A: in the enzyme reaction chamber (R in

Figure 5. Check for degradation of penicillin G, incubated in the MIERreaction chamber at pH 8.2 and subjected to electrophoretic transport at 50V. CZE runs performed in 25 mM borate–NaOH buffer, pH 10, at 10 kVtotal voltage (18mA) in a 32-cm long, 50-mM I.D. fused silica capillary,detection at 214 nm. Peaks: 1, internal standard (2 mM acrylamide); 2,undegraded penicillin G; 3–6, various degradation products of penicillin G,of which peak 6 was identified as penicillenic acid.

Figure 6. Monitoring the progress of enzyme reaction in the time-lapsesubstrate feeding. A 3 mM penicillin G solution was added to the reactionchamber of the MIER at the time intervals 0, 47, 94, and 145 min, undera constant electric field of 100 V. The sudden increments of current are dueto the production of PAA and 6-APA in equimolar amounts.

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Fig. 2) there is a rapid decay of the substrate, to whichcorresponds a rapid accumulation of the two charged prod-ucts, 6-APA and PAA. As the current is switched on 8 minafter substrate addition, both charged products leave thereaction chamber, reaching vanishing concentrations after40 min of applied voltage. At this point, a new substrateinjection is made, as indicated by the time points and ver-tical arrows and a new cycle began. What happens in theadjacent chamber (Ch 1, see Fig. 2) is shown in Fig. 7B:6-APA (which is an amphoteric ion, having a pI of 3.6)accumulates in Ch 1, since it is isoelectrically trapped be-tween the pI 5.5 and 3.5 membranes. On the contrary PAA,which is a free weak acid (pK 4.2), after an initial phase ofaccumulation, keeps leaching out of Ch 1 and is collected inthe anodic chamber (as monitored by CZE). Evacuation ofPAA from the 6-APA collection chamber is probably drivenby two mechanisms: electric transport and slow diffusiontowards the anode of the protonated form in equilibriumwith dissociated PAA. Nevertheless the total combinedtransport is slow, due to extensive protonation of the disso-ciated form as it crosses the pI 3.5 membrane, since on theother side of this membrane a pH 2.85 acetic acid solutionis present as anolyte. Subsequent experiments with the ad-

dition of a new chamber in between Ch 1 and the anodicreservoir, delimited by a pI 3.2 membrane, greatly acceler-ated the evacuation of PAA from the 6-APA collectionchamber (not shown). The yield of the process was dramati-cally improved as it was 96.6% at 175 min, which indicatesthat short-time phases in the field-stopped mode could bevery useful for avoiding anodic leakage of the charged sub-strate molecules. It is also seen from Fig. 7B that this modeof operation leads to negligible losses of unreacted substrate(penicillin G) out of the enzyme chamber: the level of PenG in chamber 1 (solid circles), as monitored by CZE, doesin fact oscillate around zero.

As a final test, yields were calculated from MIER experi-ments carried out with a continuous infusion of penicillin G:the substrate feeding rate was calculated as described above,infusion was continued for 1 h, with constant 50 V appliedvoltage, but at the end the yield was only 30%. It can thusbe concluded that continuous substrate infusion, in presenceof a continuous electric field, is vastly inferior as comparedwith the doubly-discontinuous operation mode.

As a final remark, it is noted that, in all the operationmodes, the yields of of the MIER reactor are far better thanthose obtained in batch operations, indicating that in theMIER reaction chamber the electric depletion of the enzymereaction products (notably PAA) was efficient enough tominimise the product inhibition effect of phenylacetic acid,known to dramatically lower the enzymatic activity. Finally,we considered the problem of the low solubility of 6-APAin water solutions. In order to avoid precipitation phenom-ena in the reaction chamber, which would be deleterious toseparation and recovery of products, an attempt was made atameliorating the solubility of 6-APA, by adding 10% tri-fluoroethanol to the MIER apparatus. The reactor experi-ment was conducted as described in the previous session, byapplying a voltage of 50 V and infusing the substrate at 0,20, 40, 60, and 80 min. At the end of the process the sub-strate conversion was 60%. The trifluoroethanol waschoosen because its well documented structuring effect onproteins and peptides (Castagnola et al., 1996), but no datawere available on its effect on the mechanism of catalysis.Since the enzyme catalyzes both the hydrolytic and the syn-thetic reaction, we could hypothesize that the change inwater activity, which is a consequence of the addition oforganic solvent, favoured the synthetic direction, or that thepresence of the trifuoroethanol solvent changed the rate ofconversion.

DISCUSSION

Although we believe we have proven quite convincinglythat the MIER reactor we have invented offers a uniqueperformance in enzyme catalysis (for a review, see Righettiand Bossi, 1998), we were still dissatisfied with its perfor-mance due to two major limitations: (a) the inability of

Figure 7. Monitoring substrate conversion and product accumulation inthe “doubly-discontinuous” operation mode. (A) 2 mM penicillin G ali-quots (Pen G, solid circles) are added at times 0, 48, 92, and 143 min. Theproduction and subsequent electrophoretic transport of 6-aminopenicillanicacid (6-APA, solid triangles) and phenylacetic acid (PAA, crosses) arequantified by CZE. (B) Accumulation of the same three compounds inchamber 1.

BOSSI, GUERRERA, AND RIGHETTI: ISOELECTRICALLY TRAPPED ELECTROENZYMIC BIOREACTOR 389

working properly with charged substrates, since they couldnot be confined into the enzyme reaction chamber in pres-ence of an electric field; (b) the lack of suitable amphotericbuffers covering efficiently the pH 3–10 interval. We be-lieve that the present report offers a valid solution to bothproblems, as discussed below.

Operation with Charged Substrates

As demonstrated in the present investigation, charged sub-strates are not easily amenable to operation in a MIER re-actor. As shown in Fig. 4, in the case of penicillin G (a fairlystrong acidic compound, with pK 4 2.6), even relativelylow electric fields cause a rapid transport of the substrateoutside the reaction chamber. This electrophoretic transportcompetes with the lytic activity of the enzyme on its sub-strate, so that only low reaction yields can be expected.Different modes of operating the MIER (use of rather lowvoltage gradients, intermitted substrate feeding) broughtonly a minor amelioration of enzyme performance. How-ever, excellent yields could be obtained with an operationmode here dubbed as “doubly discontinuous”: When a time-lapse substrate feeding mode was coupled with an intermit-ted electric field mode, almost quantitative reaction yields(96% conversion) could be obtained. Typically, after aninitial substrate injection of 2–4 mM (depending on theamount of enzyme used and on the solubility of penicillin Gin water solutions) the electric field is interrupted for vari-able periods of time (here 4–8 min) so as to allow almostquantitative conversion of the substrate. At this point rela-tively high voltage gradients can be applied (up to 200 V) soas to remove the reaction products from the enzyme cham-ber. This process is efficiently monitored in real time byCZE, which produces electropherograms of the variouscompounds in a matter of minutes. When evacuation ofproducts from the reaction chamber is deemed satisfactory(e.g., PAA severely inhibits penicillin G acylase, so that lowlevels of this compound in the enzyme chamber are highlydesirable) a second cyclic process of this type can be re-peated and so on, as shown in Fig. 7A. Figure 7B demon-strates that this mode of operation offers another advantage:continuous collection, during enzyme catalysis, of the de-sired product (6-APA) and its separation from an unwantedcontaminant, phenylacetic acid. This is made possible bythe different chemical nature of these two compounds:Whereas 6-APA is an amphoteric compound, with a pI of3.6, which can be efficiently retained in Ch 1 by an isoelec-tric trapping mechanism, PAA is a free acid which contin-ues to migrate and to accumulate into the anodic chamber.In the particular example of Fig. 7B, this separation was notbrought to completion because the experiment was termi-nated prematurely. In addition, it was later found that in-sertion of an additional chamber (delimited by an extramembrane with pI 3.2) in between chamber 1 and the anodicreservoir, considerably accelerated the process, since thefree PAA acid was kept more dissociated by the higher pHencountered in this extra chamber. We believe that this

mode of operation can be successfully adopted in all casesin which the substrate is a charged product, thus broadeningthe field of application of the MIER reactor.

Use of Polymeric, Amphoteric Buffers

The other major drawback for the use of the MIER is thelack of suitable amphoteric buffers to uniformly cover thepH 3–10 interval. This problem plagued in the past conven-tional isoelectric focusing and forced Vesterberg (1969) todevise the synthesis of carrier ampholytes. As shown bySvensson (1962) (see also Table 1.2 in Righetti, 1983), thereis a dramatic paucity of commercially available ampholytesexhibiting the hallmark of a good carrier ampholyte, i.e., (pI− pK) < 1.5, which is to say good conductivity and goodbuffering capacity in the isoprotic state. Moreover, in thelist of these 42 compounds compiled by Svensson (1962)there is a huge gap with practically no species acting ascarrier ampholytes in the pH 5–7 interval. In previous work,we adopted the few compounds available (mostly freeamino acids and dipeptides), but even this approach wasbesieged by problems, such as interference of these buffer-ing ions with enzymatic activity assays. The adoption, aspresented here, of polymeric, amphoteric buffers, solvescompletely this problem. First of all, such buffers are madewith the Immobiline chemicals (Righetti, 1990) which areacrylamido weak acids and bases with pK values well dis-tributed in the pH 3–10 interval. Secondly, all the basicImmobilines are produced so as to contain a tertiary aminogroup as buffering ion. Such group is thus unreactive to-wards all typical reagents for primary amino groups, thus itsinterference with a host of detection reactions is a mostunlikely event. Thirdly, due to the precise know-how builtover more than 15 years of operation with the Immobilinetechnology and to the sophisticated computer algorithmsdeveloped for modelling and calculating not only any de-sired pH gradient (linear, concave, convex, exponential) butalso any fixed pH value along the pH scale (Giaffreda et al.,1993), it is today an easy matter to devise and synthesizeany such polymeric, amphoteric buffer along the pH scale,with a precision down to 1/1000 of a pH unit and with anydesired buffering power.

CONCLUSIONS

We feel we have proven that the field of action of the MIERreactor has been considerably expanded by the introductionof the “doubly discontinuous” mode of operation and by thesimultaneous adoption of macromolecular buffers. Muchworks still remains to be done in order to assess the physico-chemical characteristics of such macromolecular buffers,such as average chain length, kinematic viscosity measure-ments and optimization of synthetic parameters. Neverthe-less, it appears that these polymeric buffers allow a properoperation of the MIER reactor and offer distinct advantagesover conventional buffers, such as unreactivity towards allreagents detecting primary amino groups. We are also in-

390 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 64, NO. 4, AUGUST 20, 1999

vestigating the possibility that such macromolecular bufferscould further stabilize the enzyme in the reactor upon pro-longed operation, perhaps by providing a milieu with a highcharge density on its surface.

Supported by grants from Agenzia Spaziale Italiana (ASI, Roma,Grant No. ARS-98-179) and by Progetto Finalizzato Biotecnolo-gie e Biostrumentazione (CNR, Roma, Grant No. 97.01199.PF9).

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