Bioconjugate Chem. 1993, 4, 509-514
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Preparation and Properties of Thermoreversible, Phase-Separating Enzyme-Oligo(N4sopropylacrylamide) Conjugates Guohua Chen and Allan S. Hoffman' Center for Bioengineering, FL-20, University of Washington, Seattle, Washington 98195. Received June 10, 1993'
A thermoreversible N-isopropylacrylamide (NIPAAm) oligomer with an N-hydroxysuccinimide (NHS) ester functional end group has been prepared for coupling to an enzyme, 0-D-glucosidase, to form a thermoreversible, phase-separating polymer-enzyme conjugate. This conjugate can be used for separation, recovery, and recycle of an enzyme simply by applying small temperature changes to the reaction medium. In contrast to the random polymer-enzyme conjugates previously reported by us and others in the literature, in this study the conjugate is formed by a single, end attachment of each oligomer chain to the enzyme. Preliminary studies show that the conjugated enzyme exhibits very high retention of activity, even higher than native enzyme, and shows improved thermal stability compared to native enzyme.
INTRODUCTION Immobilized enzymes have been widely used in chromatographic columns (1). In addition, because of the ease of recovery and recycle as well as enhanced stability, immobilized enzymes on particulates have also been used in fluidized or packed-bed bioreactors (2, 3). However, diffusion limitations of substrate into and/or product out of the solid carrier is a fundamental problem with insoluble enzyme conjugates, particularly when a macromolecular substrate is utilized. Furthermore, it is impractical for solid substrates to be used with immobilized enzyme catalysts. To overcome these problems, soluble polymer-enzyme conjugates have received more attention since they exhibit many advantages over enzymes immobilized on or within porous solid carriers, especially when used for macromolecular or solid substrates (4). In particular, when enzymes are conjugated to reversible, phase-separating polymers, the enzymatic reaction can occur in solution, after which the enzyme can be recovered in an insoluble state by small changes of pH (5-8) or temperature (9-12). Our aim in this study is to prepare thermoreversible, phase-separating, polymer-enzyme conjugates and to apply them to reactions with macromolecular or solid substrates. One case would be the use of polymer-conjugated cellulase for conversion of cellulose into glucose. It is well-known that poly(N-isopropylacrylamide) (PNIPAAm) is a thermoreversible water-soluble polymer and that aqueous solutions of this polymer exhibit a lower critical solution temperature (LCST) ca. 33 "C (13). The reversible phase-separation behavior of PNIPAAm has been utilized to reversibly precipitate and then redissolve a PNIPAAm-antibody conjugate (9) and other protein conjugates (14,151. However, in all of those studies, the polymers used have been random copolymers of NIPAAm with comonomers containing reactive groups for conjugation of the proteins. Since the copolymers may have more than one reactive group in each polymer chain, it is hard to know (a) if the enzyme is coupled to a polymer chain by a single or multiple attachment, (b) whether more than one enzyme is conjugated to one polymer chain, and also (c) at what point along the chain the conjugation
* Correspondence should be addressed to Allan S. Hoffman. * Abstract published in Advance ACS Abstracts, October 1, 1993. 1043-1802/93/2904-0509$04.00/0
occurs, all of which might significantly influence the activity and properties of the conjugated enzymes. In some cases, the conjugates were not completely water-soluble because a cross-linked network of enzyme and polymer may have formed (11). In order both to simplify and to define more precisely the structure of the conjugated enzyme,we have previously synthesized a new type of NIPAAm oligomer having an end-capped carboxyl group and used this group for conjugation to 8-D-glucosidase,one of the three enzymes comprising cellulase (16). In that preliminary study, the coupling reaction was run by mixing the oligomer and enzyme together with the activation reagent, a watersoluble carbodiimide, l-ethyl-3-[3-(dimethylamino)propyllcarbodiimide (EDC). Therefore, it is possible that besides the polymer-enzyme conjugate, enzyme-enzyme conjugates, which could be either water-soluble or waterinsoluble, might be formed during the coupling reaction. Although no insoluble aggregateswere noted after reaction, soluble enzyme-enzyme conjugates could coexist in the end product since both carboxyl and amino groups are present in the enzyme and they might also be conjugated to each other along with the polymer, to form polymerenzyme-enzyme conjugates. This could also yield poorly defined structures of the conjugates. In order to overcome this problem, in this study the carboxylated NIPAAm oligomer waa first separately activated by esterification with N-hydroxysuccinimide in methylene chloride in the presence of N,N-dicyclohexylcarbodiimide (DCC) (17)to obtain the NIPAAmoligomer with a functionally reactive NHS ester group at one end. It is well-known that the NHS ester is very reactive toward amino groups and has widely been used as a functional group for coupling reactions with proteins, ligands, etc. (18). This NHS-activated end group on the oligomer was coupled with P-D-glucosidaseto form a thermoreversible, phase-separating PNIPAAm-P-D-glucosidase conjugate with the polymer chain uniquely attached to the enzyme by a single end attachment, and without formation of enzyme-enzyme conjugates. Properties of this unique polymer-conjugated enzyme are reported here. EXPERIMENTAL PROCEDURES Materials. P-D-Glucosidase (from almonds), p-nitrophenyl 0-D-glucopyranoside (pNPG), dicyclohexylcarbodiimide (DCC), and N-hydroxysuccinimide (NHS) were 0 1993 American Chemical Society
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purchased from Sigma, St. Louis, MO. PNIPAAm oligomer terminated with a carboxyl group at one end with a molecular weight of 4000 was prepared as described previously (16). All other reagents used were of analytical grade. Preparation of N-Hydroxysuccinimide Esterified PNIPAAm Oligomer. A 4-g (1 mmol) sample of the carboxylated PNIPAAm oligomer was dissolved in 40 mL of anhydrous methylene chloride and cooled to 0 "C. NHS (2mmol) and DCC (2 mmol) were added under stirring. The stirring was continued for 4 h, while the temperature was raised to 20 "C. After reaction, the insoluble dicyclohexylurea was removed from the reaction mixture by filtration and the polymer was isolated by precipitation into anhydrous diethyl ether. The degree of NHS esterification was evaluated spectrophotometrically by the release of N-hydroxysuccinimide in alkaline solution (17). Conjugation of 8-D-Glucosidase with PNIPAAm. The NHS-esterified PNIPAAm oligomer (40 mg) was dissolved in a solution of P-D-glucosidase in 0.05 M phosphate buffer (the enzyme concentration and the volume of the enzyme solution used are summarized in Table I) at 4 "C and resultant solution was gently shaken at 4 "C for 16 h. After reaction, 0.5 mL of a saturated ammonium sulfate solution (which reduces the precipitation temperature of PNIPAAm (12,141)was added and the mixture was diluted to ca. 8 mL with the same buffer and warmed up to 30 "C. The resulting precipitate was recovered by centrifugation (12000 rpm for 10 min at 30 "C). The precipitate was redissolved at 4 "C in 8 mL of 0.05 M citric acid + phosphate buffer (CP buffer having a p H of 5.0, which is the optimal pH for the native enzyme). To this solution 0.5 mL of saturated ammonium sulfate was added and the mixture was warmed up to 30 "C. The resulting precipitate was recovered again by centrifugation. This procedure was repeated another two times. The final precipitate was dissolved in 10 mL of buffer (pH 5.0) and stored at 4 "C for further study. The enzymatic activity of the solution was tested (see below). The amount of protein conjugated by PNIPAAm was determined by the BCA protein assay method (19). Enzyme Assay. A 0.01-mg portion of the native or conjugated P-D-glucosidase was incubated in 3.0 mL of 0.05 M CP buffer and pNPG was added as a model substrate (0.45 mM, pH 5.0, 25 "C, unless otherwise indicated) for 5 min. The reaction was stopped by adding 1.0 mL of 0.25M sodium carbonate solution to adjust the pH to around 9. The enzyme activity was determined by measuring the released p-nitrophenol spectrophotometrically at 405 nm. One unit of the enzyme activity was expressed as the production of 1 pM p-nitrophenollmin under the assay conditions (20). Recycling Process of Solution Reaction-Phase Separation Recovery of the PNIPAAm-p-D-glucosidase Conjugate. In 6 mL of 0.45 mM substrate (pNPG)buffer solution (pH 5.0),0.02 mg of conjugated enzyme plus 3.0 mg of NIPAAm oligomer was added (the latter was added to enhance the precipitation and to aid complete recovery ofthe conjugate) (14). The mixture was incubated at 25 "C for 10 min. The reaction was stopped by addition of 2.0 mL of 0.25 M sodium carbonate solution to adjust the pH to ca. 9. Then 1.0 mL of saturated ammonium sulfate solution was added and the mixture was warmed up to 30 "C to precipitate the conjugate along with the added oligomer. The resulting precipitate was recovered by centrifugation (12000 rpm for 10 min at 30 "C). The enzyme activity was assayed by spectrophotometrically measuring the concentration of p-nitrophenol in the
Chen and Hoffman
supernatant produced during the enzymatic reaction. The collected precipitate was redissolved in the same buffer as above at 25 "C for the next cyclic enzymatic reaction process, which was carried out under all the same conditions as before. Each cycle the enzyme activity was assayed by measuring the supernatant solution. A control was run using a physical mixture of P-D-glucosidasewith free NIPAAm oligomer in the same buffer solution at similar enzyme and polymer concentrations, and following the same solution-precipitation cycles, i.e., dissolving at 25 "C and warming to 35 "C to precipitate the polymer (except in this case the enzyme activity was assayed by taking samples from the solution after each cycle, to run the enzymatic reaction at 25 "C). Optimal pH. The activities of native enzyme, PNIPAAm-conjugated enzyme, and a physical mixture of native enzyme with free PNIPAAm were measured at different pHs (ranging from 3.5 to 8.5) at 25 "C with a pNPG concentration of 0.45 mM in 0.05 M buffer. Kinetic Constants. The kinetic parameters for free, native P-D-glucosidase and PNIPAAm conjugated P-Dglucosidase were determined using pNPG as substrate in the concentration range of 0.11-0.68 mM at 25 "C,pH 5.0 and 7.0, respectively. K, values were calculated from Lineweaver-Burke (double, double reciprocal) plots. Optimal Temperature and Thermal Inactivation. The optimal temperature of hydrolysis of the substrate by native and polymer-conjugated P-D-glucosidase was tested over a range of temperatures. The enzyme was incubated with pNPG at a particular temperature for 15 min with gentle stirring, and the reaction was stopped by the addition of 0.25M sodium carbonate solution to adjust the pH to ca. 9, at which the enzyme is inactivated. After cooling to room temperature, the p-nitrophenol concentration was measured (405 nm). To examine thermal stability over time, native and PNIPAAm-conjugated P-D-glucosidase were incubated either at 60 "C for 45 min with sampling at 5 min intervals or at a variety of temperatures for 15 min each with gentle stirring. After incubation, the samples were cooled to room temperature and kept there for at least 2 h, assuring that all the samples were at the same temperature before the activity assays. The activities were then measured at 25 "C, pH 5.0. For comparison, controls were based on physical mixtures of native enzyme and free NIPAAm oligomer with the same amount of each as for the native or conjugated enzymes. Storage Stability. Solutions with concentrations of ca. 0.1 mg/mL of native and polymer-conjugated P-Dglucosidase were stored at 4 "C in 0.05M CP buffer at pH 5.0. The activities were measured at 25 "C, pH 5.0,as a function of storage time. RESULTS AND DISCUSSION
Preparation of NHS End-Capped NIPAAm Oligomers and PNIPAAm-0-D-Glucosidase Conjugates. N-Isopropylacrylamide (NIPAAm) oligomer with a carboxyl group at one end of the chain was synthesized by radical polymerization using 2,2'-azoisobutyronitrile (AIBN) and 0-mercaptopropionic acid (MPA) as initiator and chain-transfer reagent, respectively, as described previously (16) and also shown in Figure 1, step I. The polymer used in this work was prepared by solution polymerization using tert-butanyl alcohol as solvent, at 60 "C for 20 h, with the weight ratio of monomer to initiator to chain transfer reagent of 100:1:4. The polymer has a
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Enzyme-Oligo(N1PAAm) Conjugates
AH I CH H3C' 'CH,
AH AH H3C/ 'CHI
Table I. Effect of Polymer/Enzyme Concentration on the Conjugation and Resultant Enzyme Activity reaction medium product [P + E]a P/E mg E/g polymer retention of (coniueated) activitvb ( % ) exD (ma/mL) DH (wt/wt) 1 6.3 8.0 4:l 34.0 f 1.1 83 f 2 2 12.5 8.0 4:l 38.0 f 1.4 85 f 4 3 25.0 8.0 41 41.0 f 1.1 89 f 5 4 50.0 8.0 41 48.7 f 4.8 96 f 14 5c 50.0 8.0 41 negl negl 6d 50.0 8.0 41 negl negl 7 50.0 8.0 42 39.6 f 5.6 107 f 4 8.0 4:3 41.4 f 5.6 105 f 5 8 50.0 9 50.0 8.0 44 43.5 f 1.7 106 f 3 10 50.0 6.0 41 22.3 f 2.5 104 f 6 11 50.0 7.0 4:l 45.1 f 5.0 101 f 10 12 50.0 9.0 41 77.9 f 10.9 117 f 20 P = PNIPAAm oligomer, E = fl-D-glucosidase. The retention of activitywas calculated by dividingthe specificactivityof conjugated fl-D-glucosidaseby the specific activity of the same amount of native enzyme (the specific activity of native enzyme is taken as 100%). This control was run with the mixture of fl-D-glucosidasewith the unactivated carboxyl-terminated NIPAAm oligomer (negl for negligible). This control was run with the mixture of @-D-glucosidase with free PNIPAAm which had no functional group. e Conjugation was run in 0.05 M phosphate buffer at 4 OC for 16 h. ~~
Step I
45 "C) than either the native enzyme or the physical mixture of native enzyme with free PNIPAAm. Figure 6 shows the time course for the thermal inactivation of the enzymes at 60 "C. (The samples were incubated a t 60 "C for various times, then cooled to 25 "C where activities were measured.) Free PNIPAAm does not protect the enzyme from the thermal inactivation. The native enzyme retains only ca. 20 % of ita initial activity by a heat treatment at 60 "C for 45 min, whereas the conjugated 8-D-glucosidasestill retains 40%of its original activity. It is clear that the conjugate is more thermally stable, indicating that the conjugation of j3-D-glucosidase with NIPAAm oligomer provides protection against thermal inactivation of the enzyme at temperatures well above the LCST of the polymer. This is different from the wellknown stabilization of proteins conjugated with PEO (24) or polysaccharides (25, 261, since the PEO or polysaccharide chains remain soluble. The protection of PNIPA-
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Time (min)
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Figure 5. Effect of incubation at various temperatures at pH
20
Figure 6. Effect of incubation time at 60 "C (pH 5.0) on the retained activity of native and conjugated 8-D-glucosidase. The activity was measured for each sample at 25 "C using pNPG as substrate in 0.05 M CP buffer, pH 5.0,120 min after incubation. (The retained activity of the original enzymes without incubation at 60 "C is taken as loo%.) Am-conjugated enzyme against thermal inactivation may be due to the reduction in mobility for the precipitated conjugate above 34 "C. Many examples have been reported in the literature of the protection against thermal inactivation for immobilized enzymes on solid carriers, and this protection has been explained as due to the reduction of mobility of the enzyme after immobilization on the solid carriers (27-29). Nevertheless, it is interesting to note in our case (with a small substrate and product) that the kinetics up to the temperature at the maximum rate are the same for the conjugated or free enzyme (Figure 4). Solutions of both native and conjugated 8-D-glucosidase in 0.05 M CP buffer (ca. 0.1 mg/mL, pH 5.0) were stored at 4 "C, and the activities of these solutions were measured as function of storage time. After 2 months storage the PNIPAAm-8-D-glucosidase conjugate retains 85 9% of its initial activity, while the native enzyme only retains 70 9% of its original activity. The relatively high stability of the conjugates at 4 "C as compared to native j3-D-glucosidase may be due to the expanded and hydrated PNIPAAm chain (24,30)to form a "protective colloid" surrounding the enzyme (26,31),which could protect the enzyme from slow deactivation in solution or on the vessel walls. This is similar to the proposed action of PEO-enzyme conjugates (24). CONCLUSIONS
Thermally-reversible soluble-insoluble PNIPAAm-BD-glucosidase conjugates have been successfully prepared via the reaction of a terminal active ester group on the NIPAAm oligomer with amino groups on the enzyme. The conjugated j3-D-glucosidaseretains a high percent of the free enzyme activity and can repeatedly undergo solutionprecipitation cycles without significant loss of specific activity. Improved stability against activity loss at high temperature (60 "C) or after long storage a t 4 "C was observed for the conjugated 8-D-glucosidase compared to the native enzyme. More extensive study of ita use with macromolecular or solid substrates in a recycle process is underway. ACKNOWLEDGMENT
The National Science Foundation (NSF) (Grant No. BCS-9101716) and the Washington Technology Center (WTC) are gratefully acknowledged for their financial support of this project.
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