Ind. Eng. Chern. Res. 1994,33, 2168-2173
2168
Tyrosinase Reaction/Chitosan Adsorption: Potential for Removing Polymerization Storage Inhibitors Anami
R. Patel, Wei-Qiang Sun, and Gregory F. Payne'
Department of Chemical and Biochemical Engineering and Center for Agricultural Biotechnology, University of Maryland Baltimore County, Baltimore, Maryland 21228
Polymerization inhibitors are added to monomer solutions to prevent the inadvertent initiation of free radical polymerization during monomer storage and transportation. Residual levels of these inhibitors can be a source of irreproducibility in polymerization kinetics and can, therefore, disrupt efforts to control polymerization reactions for process optimization. We examined the feasibility of a non-capital-intensive separations approach for simply and efficiently removing the tertbutylcatechol storage inhibitor. The separations approach employs the enzyme tyrosinase to selectively oxidize the phenolic inhibitor, and the oxidation products are then strongly bound to a chitosan sorbent. For practicality we covalently immobilized the tyrosinase within chitosan gels to yield beads which served as both catalyst and sorbent for the phenolic inhibitor. These tyrosinasecontaining chitosan gels were shown to be capable of removing low levels of tert-butylcatechol from both aqueous and organic phases and thus have the potential to be exploited for removing phenolic inhibitors in a range of polymerization operations. The use of tyrosinase-containing chitosan gels for waste minimization is discussed. Introduction Due to increased environmental concerns, chemical manufacturers are reevaluating production processes to reduce or eliminate the generation of wastes. One approach in waste minimization is to more efficiently convert raw materialsinto the desiredproduct with a corresponding reduction in the waste associatedwith undesired byproduct formation. In practice, improved conversion efficiencies generallyrequire that reactions be conducted under tightly controlled conditions. For polymers, the product is not a single compound but rather a series of compounds which vary according to the number of monomer units incorporated into the polymer. Since the properties of polymer products strongly depend on the molecular weight, it is desirable to synthesize polymers with an optimal average molecular weight and a narrow molecular weight distribution. To synthesize such polymers, it is necessary to precisely understand polymerization kinetics (Baillagou and Soong, 1985a,b; Levy, 1992; Soroush and Kravaris, 1993;Kalfas and Ray, 1993a,b; Karaman et al., 1993)and control these reactions (Soroush and Kravaris, 1992). In the absence of synthetic capabilitiesto limit polydispersity, it is necessary to separate polymers of incorrect molecular weights from a polymer product. Obviously, this latter option is undesirable because of the difficulties in separating the various polymers and because the polymers of incorrect molecular weight are likely to be disposed as a waste. One problem in the control of free radical polymerization reactions is the presence of polymerization inhibitors in monomer solutions. Inhibitors are purposefully added to monomer solutions to prevent the inadvertent initiation of the free radical reactions during storage and transport of the monomers. When polymerization reactions are to be conducted, residual storage inhibitors can disrupt free radical initiation reactions and are generally believed to be responsible for variabilities in free radical polymerization kinetics. To obtain reproducible kinetics, some manufacturers remove these inhibitors from the monomer solutions (often by distillation) prior to polymerization. However, due to the hazard associated with uninhibited monomer solutions, it is often desirable to remove the inhibitor just prior to, or even after, adding the monomer 0888-5885/94/2633-2168$04.50/0
to the polymerization reactor. Further, for small-volume producers of specialty polymers, it would be desirable to be able to remove the storage inhibitor without incurring the capital expense of traditional separations equipment. In the study reported here, we investigatedthe potential of using a new, non-capital-intensiveseparations approach for removing phenolic-type storage inhibitors. The inhibitor used in this study was tert-butylcatechol, and the separations approach involves two steps. In the first step, the enzyme tyrosinase (alsoknown as polyphenol oxidase) is used to oxidize the inhibitor-presumably to its quinone (Duckworth and Coleman, 1970). Tyrosinase enzymes oxidize various phenolic compounds, and since they use molecular oxygen as the oxidant, complex biological coreactants are not required. The quinone products of the tyrosinase-catalyzed reaction are unstable and, if allowed to sit, will react with themselves to produce oligoor polyphenols (Mason and Wright, 1949;Dec and Bollag, 1990; Bollag, 1992). In the second step, an adsorbent of appropriate surface chemistry is used to chemisorb the unstable products of the tyrosinase-catalyzedreaction. For our work, we have used chitosan as our adsorbent because it can be derived fromchitin,a biopolymer readily available from the seafood industry (e.g., from waste crabshells). Also, chitosan has primary amine groups which appear to be able to bind with quinones in reactions analogous to those studied elsewhere (Par&, 1980;Simmonset al., 1989; Nithianandam and Erhan, 1991). The unique features of this two-step separations approach are that the substrate specificity of the tyrosinase enzyme confers selectivity to the adsorption (Payne et al., 1992) and the strength of quinone-chitosanbinding confers high affinity to adsorption (Sunet al., 1992). Thus, the combination of tyrosinase and chitosan can be considered as a separating agent which can achieve very high separations factors, which reduces or eliminates the need for multiple-equilibrium-staged operation. Practically, by eliminating the need for multiple-equilibrium stages, the capital investment required to achieve separations is greatly reduced compared to traditional separations approaches (e.g., distillation) which do not exploit separating agents of such high separation factors. The objectives of this study were to (1)demonstrate the 0 1994 American Chemical Society
Ind. Eng. Chem. Res., Vol. 33, No. 9, 1994 2169 Chitosan Solution (PH = 4)
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technical feasibilityof tyrosinase and chitosan for removing the tert-butylcatechol inhibitor, (2) investigate a configuration of tyrosinase and chitosan which would be practical in operation, (3) test the tyrosinase/chitosan configuration under conditions likely to be encountered in practice. Throughout this study, we used two different tyrosinase enzymes, one derived from mushroom and the other originally derived from the bacteria Streptomyces (Lerch and Ettlinger, 1972;Yoshimoto et aZ.,1985;Philipp et al., 1991).
Materials and Methods Materials. Mushroom tyrosinase was obtained from Worthington Biochemical Corporation (Freehold,NJ) and was reported to have a specific activity of 754 units/mg as determined by the assay of L-tyrosine. The bacterial tyrosinase was donated by Biosource Genetics (Vacaville, CA) and was reported to have specificactivity of 17.4units/ mg solid based on the reaction of L-dihydroxyphenylalanine. Due to differences in assay methods we standardized the amount of enzyme used in our study by determining the amounts of each enzyme required to yield comparable rates of tert-butylcatechol oxidation. We report our activities in the figure legends in terms of the suppliers' units. Chitosan from crabshells was purchased from Sigma Chemicals (St. Louis, MO), and tert-butylcatechol was purchased from Lancaster (Windham, NH). The concentration of tert-butylcatechol used in this study was 0.5 mM since this level is industrially relevant and easily measured experimentally. Immobilizationof Tyrosinasewithin Chitosan Gel Beads. Figure 1 outlines the procedure for producing tyrosinase-containing chitosan gels. Chitosan flakes (4 g/lOO mL) were added to 0.3 M acetate buffer (pH = 4) and dissolved overnight. After dissolving, 15 mL of the viscous chitosan solution was mixed with 2 mL of an enzyme solution containing either 1600 units/mL of mushroom tyrosinase or 100 units/mL of the bacterial tyrosinase. To this enzyme-chitosan mixture 1mL of a 0.05 5% glutaraldehyde solution was added. This enzyme immobilization reaction was allowed to proceed for 15min at 4 "C. After binding the enzyme to chitosan, the liquid was added dropwise into a 0.05 M NaOH solution. The beads were removed from the caustic after 1-2 min and then contacted with 100mL of a 100mM phosphate buffer (pH = 6.8) for 15 min. These beads were subsequently washed twice for 15 min with 100 mL of distilled water. In experiments, all beads from the 15-mLchitosan solution were added to 25 mL of solutions containing tertbutylcatechol. Enzyme activities reported in these ex-
Figure 2. Oxidation of tert-butylcatechol by soluble tyrosinase. Aqueous solutions containing 0.5 mM tert- butylcatechol in 50 mM phosphate (pH = 6.8) were incubatedat room temperature. Solutions contained either noenzyme (control),mushroom tyrosinase (20units/ mL), or bacterial tyrosinase (0.095 unita/mL). Differences in enzyme activities are discussed in the Materials and Methods section.
periments are the total initial enzyme added to the chitosan divided by the volume of the tert-butylcatechol solution. Experimental Methods. The experimental methods have been described previously (Payne et al., 1992; Sun et aZ., 1992). To monitor the progress of the enzymatic reaction, loss of the oxygen coreactant (Mayer et al., 1966) was followed using a dissolved oxygen (DO) probe (Microelectrodes, Londonderry, NH). To follow losses in tertbutylcatechol and its oxidation products, we used ultraviolet-visible (UV-visible) spectrophotometry.
Results Technical Feasibility: Soluble Tyrosinase and Chitosan Flakes. To determine whether the tyrosinase enzymes are capable of reacting with the phenolic polymerization inhibitor, we dissolved the enzyme in aqueous solutions containing 0.5 mM tert- butylcatechol and monitored the consumption of the oxygen coreactant. As shown in Figure 2, tyrosinases are capable of catalyzing the oxidation of tert-butylcatechol. For the two-step operation to be successful, it is necessary for the tyrosinase-catalyzed oxidation products to be adsorbed onto chitosan. To test for adsorption, we conducted an experiment involving soluble enzyme and chitosan flakes. The curves labeled "A" in Figure 3 are the UV absorbances of aqueous solutions containing 0.5 mM tert- butylcatechol. When mushroom tyrosinase (Figure 3a) or the bacterial tyrosinase (Figure 3b) were incubated with the tert-butylcatechol solutions, curves B show a broadening of the absorbance with a peak appearing at 400 nm. This peak is consistent with the formation of a quinone product (Duckworth and Coleman, 1970)from the tyrosinase-catalyzed reaction. Curves C in Figure 3 show the absorbance of solutions after chitosan, but not tyrosinase, was incubated with the tert-butylcatechol. The similarities of the absorbances in curves A and C indicate that tert-butylcatechol does not adsorb onto the chitosan flakes. This observation is consistent with previous studies which have shown chitosan to have limited capabilities for adsorbing organics from aqueous solutions (Sun et al., 1992; Payne et al., 1992). Curves D in Figure 3 show the absorbance when both tyrosinase and chitosan were incubated with tert-butylcatechol. As shown, both the tert-butylcatechol and the products of the tyrosinasecatalyzed reaction are removed from solution by the combination of tyrosinase and chitosan.
2170 Ind. Eng. Chem. Res., Vol. 33, No. 9, 1994 1
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Figure 3. Tyrosinase reaction/chitosanadsorption of tert-butylcatechol by mushroom (a) and bacterial (b) tyrosinases. Curves A control containing 0.5 mM tert-butylcatecholin phosphate buffered (50 mM and pH = 6.8) aqueous solutions. Curves B tertbutylcatechol plus soluble tyrosinases.C w e s C: tert - butylcatechol plus chitosan flakes (5 w/v % ). Curves D: tert-butylcatechol plus soluble tyrosinases and chitoean. When added,mushroomtyrosinase was added at 20 unita/mL (a) and the bacterial tyrosinase was added at 0.096 unita/mL (b). Absorbances were measured after 1 h of incubation.
Immobilization of Tyrosinase within Chitosan Gel. Although Figures 2 and 3 demonstrate the technical feasibility for removing the tert - butylcatechol storage inhibitor, it is desirable to develop more practical configurationsfor using tyrosinase and chitosan. Specifically, to avoid contamination of the process stream with the enzyme, it is desirable to immobilize tyrosinase on or in a solid. Further, to improve the accessibility of the amine groups of chitosan for chemisorption, it is desirable to utilize chitosan in a gel (and not a flake) form. To address these issues, we chose to immobilize tyrosinase within the chitosan gel to yield a bead which is capable of both catalyzing the oxidation of tert -butylcatechol and chemisorbing the oxidation products. Chitosan gel beads are readily formed by first dissolving chitosan in an acidic solution and then adding the viscous chitosan solution dropwise into a basic solution (Mitani et al., 1991;Rorrer et al., 1993). Also, enzymes have been immobilizedin or on chitosan using either physical (Stanley et al., 1976;Synowiecki et al., 1981;Nozawa et al., 1982) or covalent (Synowiecki et ai.,1982;Hirano and Miura, 1979; Iyengar and Rao, 1979; Yamaguchi et al., 1982; Carrara and Rubiolo, 1994;Itoyama et al., 1994)bonding. The method we used for covalently immobilizing the tyrosinases within the chitosan gels is illustrated in Figure 1,which showsthat enzyme andglutaraldehyde were added
Figure 4. Oxidation of tert- butylcatecholby tyrwinaee immobilized within chitosangels.Aqueous solutionsof 0.5 mM tert-butylcatechol in 100mM phosphate (pH = 6.8)were incubatedat room temperature with chitosan gels containing no enzyme (control), mushroom tyrosinase (128 units/mL), or bacterial tyrosinase (8 unita/mL). Enzyme activities are reported as the initial activity used for immobilization per milliliter of tert-butylcatechol reaction liquid.
to acidic chitosan solutions prior to forming the gel beads. To reduce inactivation of the enzymea during immobilization, we minimized the extremes in pH and the time the enzymes were exposed to such pH extremes. Further details of the immobilization procedure are provided in the Materials and Methods section. To determine whether the tyrosinase enzymes immobilized within the chitosan gel retained activity, we incubated these gels with aqueous solutions containing 0.5 mM tert-butylcatechol and monitored the loss of the oxygen coreactant. Figure 4 shows that both the immobilized mushroom and bacterial tyrosinases were capable of oxidizing tert-butylcatechol. As a control, a chitosan gel was prepared in an identical manner except that tyrosinase was not added prior to forming the gel beads. As shown in Figure 4, oxygen consumption was not observed when these control beads were incubated with tert- butylcatechol. Thus, Figure 4 demonstrates that, after immobilization within chitosan gel, sufficient activity of tyrosinase can be retained for tert-butylcatechol oxidation. We did not attempt to optimize the immobilization procedure to minimize the loss of enzyme activity. Testing of Tyrosinase-Containing Chitosan Gels. One potential application of tyrosinase-containingchitosan gels is to remove storage inhibitors in suspension or emulsion polymerizationsafter the aqueous phase has been added. In this application the tyrosinase-containing chitosan gel would need toadsorb tert-butylcatechol from an aqueous continuous phase. To simulate such conditions, we conducted the experiment in Figure 5. Curves A of Figure 5 show the UV absorbance of the control containing 0.5 mM tert- butylcatechol in an aqueous solution. When chitosan beads were added which did not contain tyrosinase, curves B show there was a small reduction in the UV absorbance compared to the control (curves A). This small reduction in absorbance is presumably due to the dilution of tert-butylcatechol by the aqueous chitosan gel. When tyrosinase-containing chitosan beads were added to aqueous solutions containing tert-butylcatechol, curves C show a large reduction in UV absorbance indicating that the tert - butylcatechol was removed from solution and the oxidation products were effectively bound to chitosan. Supporting this conclusion were observations that the clear to white colored chitosan gels turned light brown when both tyrosinase and tert-
Ind. Eng. Chem. Res., Vol. 33, No. 9, 1994 2171
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