Znd. Eng. C h e m . Res. 1991,30,939-946
Once the concentration profiles for component A are solved as functions of contact time for the reactive absorption and physical absorption cases, Q, and QphP can be calculated by integrating their respective instantaneous absorption rates over the contact time. The instantaneous absorption rate is determined by Fick's law for diffusion at the interface. For both cases the amount of gas absorbed was calculated by using the dimensionless masstransfer driving force at the liquid interface, (dA/dZ),,,.
The integrations were performed numerically by using Simpson's rule. Registry No. DGM,111-77-3; H2S,7783-06-4; SOz, 7446-09-5; S, 7704-34-9.
Literature Cited Astarita, G. Mass Transfer with Chemical Reaction; Elsevier: New York, 1967. Astarita, G.; Savage, D. W.; Bisio, A. Gas Treating with Chemical Soluents; Wiley: New York, 1983. Brian, P. L. T.; Hurley, J. F.; Hasseltine, E. H. Penetration Theory for Gas Absorption Accompanied by a Second Order Chemical Reaction. AZChE J. 1961,7, 226. Carslaw, H. S.; Jaeger, J. C. The Conduction of Heat in Solids; Oxford University Press: London, 1959; p 72. Chan, H.; Fair, J. R. Prediction of Point Efficiencies on Sieve Trays 1. Binary Systems. Znd. Eng. Chem. Process Des. Deu. 1984,23, 814. Crean, D. J. M.S. Thesis, University of California at Berkeley, 1987. Dankwerts, P. V. Gas-Liquid Reactions; McGraw-Hill: New York, 1970. Demyanovich, R. J.; Lynn, S. Vapor-Liquid Equilibria of Sulfur Dioxide in Polar Organic Solvents. Znd. Eng. Chem. Res. 1987, 26, 548. Glasscock, D. A,; Rochelle, G. T. Numerical Simulation of Theories for Gas Absorption with Chemical Reaction. AZChE J. 1989,35, 1271. Hix, R. M. Ph.D. Dissertation, University of California at Berkeley, 1989. Hikita, H.; Asai, S.; Ishikawa, H. Gas Absorption Accompanied By an Irreversible Second-Order Reaction with a Volatile Reactant.
939
Bull. Uniu. Osaka Prefect. 1979,A B , 57. Lynn, S.; Neumann, D. W.; Sciamanna, S. F.; Vorhis, F. H. A Comparison of the UCB Sulfur Recovery Process with Conventional Sulfur Recovery Technology for Treating Recycle Gas from a Crude Oil Residuum Hydrotreater. Enuiron. Prog. 1987,6,257. Neumann, D. W. Ph.D. Dissertation, University of California a t Berkeley, 1986. Neumann, D. W.; Lynn, S. Kinetics of the Reaction of H a and SO2 in Organic Solvents. Znd. Eng. Chem. Process Des. Deu. 1986,25, 248. Pangarkar, V. G. Gas absorption with reaction in a solution containing a volatile dissolved reactant. Chem. Eng. Sci. 1974,29, 877. Pohorecki, R.; Moniuk, W. Evaluation of Plate Efficiency for Absorption with First-Order Chemical Reaction. Znz. Chem. Procesowa 1983a,4,85. Pohorecki, R.; Moniuk, W. Evaluation of Plate Efficiency for Absorption with Chemical Reaction. Znz. Chem. Procesowa 198313, 4 , 353. Pohorecki, R.; Moniuk, W. Efficiency of Sieve Trays for Absorption with Chemical Reaction. Znz. Chem. Procesowa 1983c, 4, 545. Ramachandran, P. A.; Sharma, M. M. Simultaneous Absorption of Two Gases. Trans. Znst. Chem. Eng. 1971,49,253. Roper, G. H.; Hatch, T. F.; Pigford, R. L. Theory of Absorption and Reaction of Two Gases in a Liquid. Znd. Eng. Chem. Fundam. 1962,1 , 144. Sciamanna, S. F. Ph.D. Dissertation, University of California at Berkeley, 1986. Sciamanna, S. F.; Lynn, S. Solubility of H2S, SOz, COz,Propane, and n-Butane in Polyglycol Ether. Znd. Eng. Chem. Res. 1988a,27, 492. Sciamanna, S.F.; Lynn, S. An Integrated Process for Simultaneous Desulfurization, Dehydration and Recovery of Hydrocarbon Liquids from Natural Gas Streams. Znd. Eng. Chem. Res. 1988b, 27,500. Shaikh, A. A.; Varma, A. Gas absorption with chemical reaction: the case involving a volatile liquid reactant. Chem. Eng. Sci. 1984, 39, 1639. Sharma, M. M.; Gupta, R. K. Mass Transfer Characteristics of Plate Columns without Downcomers. Trans. Znst. Chem. Eng. 1967,45, T169. Stevens, C. A. Ph.D. Dissertation, University of California a t Berkeley, 1989. Received for review June 25, 1990 Revised manuscript received November 13, 1990 Accepted December 3, 1990
Enzyme-Catalyzed Interesterification of Triglycerides in Supercritical Carbon Dioxide Douglas A. Miller, Harvey
W.Blanch,* a n d John M . P r a u s n i t z
Department of Chemical Engineering, University of California, Berkeley, California 94720
The interesterification of trilaurin and myristic acid, catalyzed by a 1,3-specific lipase from Rhizopus arrhizus, has been investigated in supercritical carbon dioxide. Experimental data have been obtained from reactions conducted in a continuous-flow packed-bed reactor containing lipase covalently attached to glass beads. The reaction rate is not influenced by mass-transfer limitations over the range of flow rates studied, and lipase retains full activity at 1400 psi and 35 O C for up to 80 h. The carbon dioxide water content does not affect the intrinsic activity of the enzyme, but a higher water concentration causes a greater degree of unwanted hydrolysis. The selectivity of the reaction for interesterification over hydrolysis improves a t higher pressures as the extent of hydrolysis reaction is reduced. The activity and stability of lipase in supercritical carbon dioxide are similar to those in organic liquid solvents. Introduction The use of enzymes to catalyze reactions of commercial interest is now well established. The production of high
* To whom correspondence should be addressed. 0888-5885/91/ 2630-0939$02.50/0
fructose corn syrup catalyzed by glucose isomerase (Thompson et al., 1974) and the production of L-amino acids by aminoacylase (Chibata et al., 1972) provide two examples of industrial processes employing immobilizedenzyme reactors. However, many enzyme-catalyzed reactions of potential industrial interest involve nonpolar or 1991 American Chemical Society
940 Ind. Eng. Chem. Res., Vol. 30, No. 5, 1991
poorly water-soluble substrates, and most of these reactions cannot be conducted in aqueous solutions due to limited reaction rates and unfavorable reaction equilibria. As a result, research on the use of enzymes in nonaqueous solvent systems has grown, and it has now been shown that many enzymes can function in both water-miscible and water-immiscible solvents of varying polarity and water content (Klibanov, 1986; Zaks and Russell, 1988b). Supercritical fluids have proven useful for the procesaing of biological materials and may provide a promising alternative solvent for enzymatic catalysis. Supercritical fluids are used industrially in extraction processes, and several reviews on supercritical fluid extraction (McHugh and Krukonis, 1986; Randall, 1982; Williams, 1981) have been published. Examples of extraction processes that have been developed include extraction of caffeine from coffee (Roselius et al., 1974), extraction of vegetable oils (Friedrich, 1984), removal of cyclic oligomers from polyoxyalkylene (Copelin, 1981), and extraction of chemotherapeutic agents from plant materials (Krukonis et al., 1979). Supercritical fluids have several potential advantages over liquids for extraction processes; these also apply to reactive processes. First, the transport properties of supercritical fluids are generally intermediate between those of gases and liquids. The viscosities of supercritical fluids are about 1order of magnitude less than those of liquids; diffusion coefficients in supercritical fluids are about 1-2 orders of magnitude larger. These properties lead to greatly reduced mass-transport limitations for reactions conducted in supercritical fluids. Second, the solubilities of solutes in supercritical fluids are highly sensitive to pressure and/or temperature. In the critical region, solubility changes of orders of magnitude can result from relatively small changes in pressure (e.g. 10 bar). This high sensitivity of solubility to pressure may lead to a greatly simplified separation step with lowered energy costs and minimal solvent entrainment. However, supercritical fluid processing also has several limitations. Because the solubilities of solutes in supercritical fluids are relatively low, very high pressures (5000-10000 psi) may be needed to achieve solubilities similar to those in liquids. The lack of solubility and thermodynamic property data for supercritical fluids makes process design difficult; more data are needed for even the most widely studied supercritical fluid, carbon dioxide. Finally, the need for costly high-pressure equipment may make some technically feasible processes economically unattractive. Chemical reactions in supercritical fluids have been under investigation for many years; a review was published by Subramaniam and McHugh (1986). Enzymatic catalysis in supercritical fluids, first demonstrated in 1985 (Randolph et al., 1985), is now receiving widespread attention. In addition to reduced mass-transport limitations and simplified separations, supercritical fluids offer several other potential advantages for enzymatic reactions. Because most of the supercritical fluids of interest are nonpolar solvents that do not solubilize enzymes, separation and recovery of the catalyst after the reaction are facilitated. Nonpolar substrates such as lipids are more soluble in supercritical fluids than in aqueous solution, and the reduced water activity attainable in supercritical fluids may permit the reversal of many hydrolytic reactions. For example, cholesterol is about 50 times more soluble in carbon dioxide at 123 bar and 35 O C than in room-temperature water (Randolph et al., 1988). The critical temperature of carbon dioxide is 31.1 "C, low enough for
processing of many heat-labile biomaterials. Carbon dioxide is nontoxic, nonflammable, and inexpensive and posses no waste-disposal problems. There have been several reports of enzyme-catalyzed reactions in supercritical fluids, including the investigation of cholesterol oxidase in supercritical carbon dioxide and carbon dioxide/cosolvent mixtures (Randolph et al., 1988), and the study of polyphenol oxidase in supercritical carbon dioxide and fluoroform (Hammond et al, 1985). The use of lipases in supercritical carbon dioxide to catalyze interesterification reactions was studied by Chi et al. (1988) and Nakamura et al. (1986). The extent of interesterification at equilibrium and the amount of residual triglyceride were shown to depend on the water content in the reactor. The authors reported that the initial rates of hydrolysis and of interesterification increased with rising water content. The lipase-catalyzed transesterification of ethyl acetate and isoamyl alcohol was studied in supercritical carbon dioxide by van Eijs et al. (1988a). These authors determined the optimum water content in carbon dioxide to be 0.2 w t % for the production of isoamyl acetate at 100 bar and 60 "C. These studies show the importance of the water content, a parameter that can affect reaction rates, enzyme stabilities, and both reaction and phase equilibria. The effect of pressure on the lipase-catalyzed interesterification of trilaurin and palmitic acid in supercritical carbon dioxide was investigated by Erickson et al. (1990). The reaction rate decreased as the pressure was increased at constant reactant concentration. The authors reported that the reaction rate could be modeled by a rate law based on mole fractions of reactants, but they gave no physical explanation for this observation. The reaction investigated in this study is the lipasecatalyzed interesterification of triglycerides, a reaction that may be used to produce upgraded fats and oils. Interesterification, or more specifically acidolysis, occurs when a triglyceride reacts with a fatty acid, resulting in a rearrangement of the fatty acyl groups. The mixture of triglycerides becomes enriched with the new fatty acid, thereby changing its physicochemical properties. Some lipases catalyze the rearrangement of fatty acids only at the 1and 3 positions of triglycerides. This specificity can be exploited to produce well-defined mixtures of triglycerides, for example, cocoa butter substitutes derived from palm oil. Macrae provides a review of lipase-catalyzed interesterification of oils and fats (Macrae, 1983). Interesterification is well suited for study in supercritical carbon dioxide because the nonpolar reactants are soluble in carbon dioxide and no cofactor is required by the enzyme for activity. Further, a nonaqueous solvent is necessary to prevent the total hydrolysis of triglyceride to glycerol and fatty acids and to allow hydrolysis and esterification to occur simultaneously, resulting in interesterification. A discussion of the reaction mechanism is provided in a following section. We have studied the interesterification of trilaurin and myristic acid in supercritical carbon dioxide. Trilaurin and myristic acid were chosen as the substrates because their solubilities in supercritical carbon dioxide are larger than those of higher molecular weight triglycerides and fatty acids, facilitating determinations of reactant and product concentrations. Since lipases do not show a strong dependence on fatty acid chain length, our results should extend to other, similar interesterification systems. The primary goal of this investigation is to determine the suitability of supercritical carbon dioxide as a reaction medium for the enzymatic interesterification of tri-
Ind. Eng. Chem. Res., Vol. 30, No. 5 , 1991 941
reactor
sampling vaive substrate chamber
icezer
m a s flow meter
Figure 1. Continuous reaction apparatus, illustrating sampling loops and substrate addition.
glycerides. We have determined the enzyme's operational stability and the effects of pressure, temperature, and water content on the reaction. In addition, we have compared the rate of reaction in supercritical carbon dioxide with that in cyclohexane. Experimental Methods Materials. Lipase isolated from Rhizopus arrhizus (5 lo6 units/mL) was purchased from Sigma Chemical Co. A unit is defined as that which hydrolyzes 1.0 pequiv of fatty acid from a triglyceride in 1 h at pH 7.7 at 37 "C. Trilaurin and myristic acid with greater than 99% purity were also purchased from Sigma. Bone-dry carbon dioxide was purchased from Matheson Gas Products. Methylene chloride, cyclohexane, and methanol of spectrophotometric grade were obtained from Fischer Scientific. Enzyme Immobilization. A 1-g amount of porous, aminopropyl glass beads (75-A pore size, Sigma) was reacted with 10 mL of 2.5 vol % glutaraldehyde in 50 pM phosphate buffer at pH 7.5 for 30 min and then filtered and washed with buffer to remove unreacted glutaraldehyde. Aliquots of 300 pL of enzyme solution (used as received) and 10 mL of buffer were poured onto the glass beads and allowed to react for 4 h. Uncoupled enzyme was removed by washing with 50 pM phosphate buffer at pH 7.0. Apparatus and Procedure. Figure 1 presents the experimental apparatus used to conduct all experiments in supercritical carbon dioxide. The packed-bed reactor consisted of immobilized enzyme packed inside a in.-0.d. stainless steel tube, 10-cm length X 0.5-cm i.d. The glass beads were contained by glass wool plugs placed in each end of the reactor. The 175-mL substrate chamber was filled with 3 g of trilaurin and 5 g of myristic acid interspersed with 2-mm glass beads. The water chamber was filled with moistened cotton. Carbon dioxide passed through these two chambers, where it was saturated with water and substrate. The carbon dioxide stream then flowed through the reactor, where lipase catalyzed the reaction. The product stream exiting the reactor was sampled by using a 6-way value (Valco, C6U-HC) equipped with an isolatable 2.2-mL sample loop. Samples of the substrate stream were taken with no reactor in-line. The contents of the sample loop were bubbled through methylene chloride (CH2C12),and then the loop was flushed with several additional volumes of CH2C1,. The CH2C12was evaporated at room temperature, and the dry sample X
weighed. The sample was redissolved in cyclohexane for product analysis. Carbon dioxide was circulated with varying flow rate through the reaction system with a Milton Roy HPLC pump (Model No. 2396), and the mass flow rate was measured with a Micro Motion Model 6 mass flow meter. All valves and fittings were made of stainless steel and rated to 3000 psi. Safety features included a rupture disk rated to 2500 psi mounted on the substrate chamber and a polycarbonate box that served as a safety shield as well as a temperature-controlled air bath. The pressure was monitored with a pressure transducer (Omega, Model PX420-2KGI) and several pressure gauges; the temperature immediately upstream of the reactor was monitored with a copper-constantan thermocouple. Pressure was controlled by adjusting a micrometering valve downstream of the six-way valve. At this micrometering valve the pressure was reduced to about lo00 psi, causing complete precipitation of products. A three-way valve was used to recycle the carbon dioxide. Substrate and product concentrations were determined by supercritical-fluid chromatography (SFC) with a Lee Scientific Series 600 SFC. The mobile phase was carbon dioxide, and the column was a SB-Biphenyl-30 capillary SFC column. The conditions were isothermal at 100 "C with linear pressure programming from 125 to 300 atm at 15 atm/min after an initial isobaric period of 5 min. Triglyceride concentrations were alternatively determined by reversed-phase HPLC on a RP-18 column with a methanol mobile phase and UV detection at 210 nm. The rate of interesterification is defined as the moles of myristic acid incorporated into product triglyceride per liter reactor volume per second. The overall reaction rate is defined as the rate of depletion of trilaurin. Reaction-rate measurements were made at three different carbon dioxide water contents. A molecular sieve was used to dry the carbon dioxide completely (0.0 wt % water), and the water chamber described above was used to saturate the carbon dioxide to a known weight percent water (0.17 w t % at 1400 psi). Finally, two equal streams of dry and moist carbon dioxide were mixed to provide an intermediate water content (ca. 0.085 wt 5%). The reactor containing immobilized lipase was equilibrated at each water content by passing each stream over the reactor for approximately 1h prior to adding substrate to the carbon dioxide and taking samples. EPR Spectroscopy Studies. Electron paramagnetic resonance (EPR) spectroscopy was used to investigate possible conformational changes in the enzyme caused by variations in carbon dioxide pressure. EPR spectra were recorded on an IBM ER 200 D-SRC EPR spectrometer with a high-pressure quartz cell. A substrate analogue for lipase, 12-doxylstearicacid (Sigma, 95% purity), was used as a probe of enzyme structure. Enzyme and spin-labeled stearic acid were dried onto the walls of the EPR cell. The cell was then pressurized with carbon dioxide and allowed to equilibrate at 35 "C prior to recording EPR spectra. The spectra obtained were composite spectra consisting of spectra from separate populations of unbound and enzyme-bound spin-label. At each pressure the spectrum of bound spin-label was obtained by subtracting a spectrum of unbound spin-label (recorded with no enzyme present) from the composite spectrum. Kinetics of Interesterification The lipase-catalyzed interesterification of triglycerides is assumed to proceed via an acyl-enzyme mechanism. Evidence for this mechanism has been obtained with different lipases (Burdette and Quinn, 1986; Albro et al.,
942 Ind. Eng. Chem. Res., Vol. 30,No. 5, 1991
1976). The interesterification of trilaurin (TL) and myristic acid (MA) to product 1,2(2,3)-dilauroyl-3(l)-myristoyl-rac-glycerol (LLM), and 1,3-dimyristoyl-2-laurin (MLM) is believed to proceed via six steps:
-
+ TL * E-TL E-LA + DL E-LA + H20 E-LA E + LA E + MA w E-MA E-MA + H2O E-MA + DL w E-LLM E + LLM E + LLM w E-LLM E-LA + LM E-MA + LM * EeMLM E + MLM E
Q
Q
Q
-
1.0
"."
1
(1)
0
0 .o.
(2)
(3) 1.0
(5) 0.0
(6)
Results and Discussion Effect of Mass Transfer. To study the intrinsic kinetics of lipase-catalyzed interesterification, it was necessary ta determine the extent of mawtransfer limitations on the reaction rate. Because the enzyme is immobilized by covalent attachment to porous glass beads, external and/ or intraparticle mass-transfer limitations may exist. To ascertain the extent of external mass-transfer limitations, the interesterification rate was determined as a function of superficial velocity. Three reactors of different lengths were used to maintain a constant residence time of 42 s while the velocity was varied. Figure 2 shows that the observed interesterification rate did not change appreciably as the superficial velocity was varied, indicating
0
+
(4)
where E = lipase, TL = trilaurin, DL = dilaurin, LA = lauric acid, MA = myristic acid, E-LA, E-MA = acylenzyme intermediates, LM = 1(3)-myristoyl-2-lauroylrac-glycerol, LLM, MLM are defined above, and E-X = enzyme-bound species. Step 1 is a hydrolysis in which lipase binds trilaurin and catalyzes the release of dilaurin and the formation of the acyl-enzyme intermediate. Steps 2 and 3 are conversions between acyl-enzyme intermediates, free lipase, and fatty acids. Step 4 is an esterification between myristic acid and dilaurin to produce LLM, resulting from the nucleophilic attack of dilaurin on the acyl-enzyme intermediate containing myristic acid. Step 5 is the hydrolysis of LLM to produce LM and E-LA, and step 6 is the esterification of myristic acid and the diglyceride LM to produce MLM. All steps in the reaction pathway are reversible. The steps shown are only those leading to the desired products. Lipase can also bind the diglycerides LL and LM and hydrolyze them to produce monoglycerides, but a t the low water concentrations typical for interesterification this does not occur to an appreciable extent. Water plays a catalytic role in interesterification in that it is neither consumed nor produced as trilaurin is converted to LLM or MLM. However, since diglycerides are intermediates in interesterification and are always present at the completion of the reaction, some water is consumed overall. If excess water is present, the acyl-enzyme intermediates are depleted in steps 2 and 3 and the esterification steps that produce triglycerides are quenched. If too little water is available, E-LA and E-MA accumulate and less free enzyme is available for steps 1and 5, thereby slowing interesterification. The determination of the optimum water content for interesterification has thus been the subject of much research. The hydrolysis of triglycerides needs to be minimized while maximizing the rate of interesterification. The optimum water content, however, may be a strong function of the particular lipase used, the enzyme carrier or support, and the reactor configuration (Lilly, 1982; Wisdom et al., 1984).
'0
0
4
0.04
0.10
0.22
0.18
I !8
SUPERFICIAL mLOCITY (cm/s) Figure 2. Effect of superficial velocity on observed interesterification rate at 1400 psi and 35 OC. Carbon dioxide is watersaturated: residence time = 42 s. n
2.0
-?
3
1.8-
W
E w 2
8
1.2-
-0-
-
- &?.----
0
- - -0-
-
- - - %- -
OB-0.4--
N
8
0.0 T
that there are no external mass-transfer limitations. The possible effect of intraparticle mass-transfer limitations can be estimated by calculating the observable modulus (Bailey and Ollis, 1986) (7) where 9 = observable modulus, uo = observed maximum reaction rate (typically 7 X lo4 M s-l), D, = effective diffusion coefficient of trilaurin (5.4 X lo4 cm2s-l), So = bulk trilaurin concentration (1.7 X M), V = volume cm3),A = external surface area of of particle (6.5 X cm2). [$he diffusion coefficient of particle, (7.8 X trilaurin in cyclohexane at 35 "C is estimated to be 4.3 X lo4 cm2 s-l from the Stokes-Einstein equation. The diffusion coefficient of trilaurin in supercritical C02 (D%) is estimated to be approximately 10 times larger (Padaitis et al., 1983). The effective diffusion coefficient (D, = D, (porosity/tortuosity factor)) is calculated by using a glass-bead porosity of 0.5 (from supplier), and the tortuosity factor is assumed to be approximately equal to 4.1 The maximum value of 9 is of order of which is 3 orders of magnitude less than the critical value of 0.3, indicating that the effectiveness factor is approximately equal to unity. Therefore no internal mass-transfer limitations are present, and the reaction rate is solely controlled by enzyme kinetics. Operational Stability. To determine the resistance of lipase to deactivation under operational conditions in supercriticalcarbon dioxide, the interesterification rate was measured at intervals over a period of about 80 h of con-
Ind. Eng. Chem. Res., Vol. 30, No. 5, 1991 943
I
4.0
T
$ / ; ; ; ; I
t 0.0 -0.5
T
T
0.0
0.0
0.6
1.0
1.6
2.0
-0.6
Figure 4. Effect of water content on the interesterification rate at 1400 psi and 35 O C . Residence time = 55 s. Points are mean values of 10 samples.
tinuous reactor operation. Figure 3 shows that no loss of activity was observed. The stability of enzymes in supercritical carbon dioxide has also been investigated by other researchers. Randolph et al. (1988) showed that cholesterol oxidase from Gloecysticum chrysocreas was stable at 101.3 bar and 35 " C for at least 50 h. van Eijs et al. (1988b) showed that lipase remained active in supercritical carbon dioxide under operating conditions of 35 " C and 10 MPa for up to 12 days. However, Kasche et al. (1988) found that the enzymes a-chymotrypsin, trypsin, and penicillin amidase were partly denatured during depressurization from supercritical carbon dioxide at a rate of 100 bar in 5-10 min. The degree of denaturation was larger in humid C 0 2 than in dry C 0 2 . Loss of activity during depressurization is a disadvantage of a batch reactor for conducting enzymatic reactions in supercritical fluids. However, it may be possible to prevent denaturation upon depressurization if the pressure is reduced more gradually. It has been proposed that carbon dioxide could cause a decrease in the effective pH in the microenvironment of the enzyme, leading to a lower enzyme activity and possibly denaturation. Taniguchi et al. (1987) studied the effect of supercritical carbon dioxide on enzymatic activity of nine different enzymes and found no effect. The water content of the enzyme preparations was 5-7 w t % . However, the activity of lipase containing 50 w t % water was decreased to two-thirds of its original activity after contact with supercritical COP This decrease may be due to a pH shift caused by C 0 2 dissolving in the water surrounding an enzyme. With 5-7 wt 7% water, there may only be sufficient water present to hydrate the enzyme. Effect of Water Content. Figure 4 shows the effect of carbon dioxide water content on the rate of interesterification; the observed rate decreases as the water content rises. Our solubility measurements have shown that the solubility of trilaurin also decreases as C 0 2 water content rises, accounting for at least part of the rate decrease. The pseudo-first-order rate constant, calculated by dividing the reaction rate by the initial trilaurin concentration, remains constant as the water content changes, as shown in Figure 5. We conclude that the intrinsic activity of the enzyme is not significantly influenced by the water content in this concentration range. There appears to be sufficient water bound to the enzyme for interesterification to occur. Further, the retention of activity in dry (0 wt % water) carbon dioxide demonstrates that the water required by the enzyme for preservation of activity is very tightly bound and is not stripped away by the carbon dioxide stream.
0.0
0.6
1.0
1.6
2.0
WATER CONTENT (g water/kg CO,)
WATER CONTENT (g water/kg CO,)
Figure 5. Effect of water content on the pseudo-first-order rate constant for interesterification (rate constant = rate/ [TL],).
20
/
l106 l
- ,
-0.6
0.0
0.6
1.0
1.6
2.0
WATER CONTENT (g water/kg CO 2 ) Figure 6. Fraction of triglycerides hydrolyzed to partial glycerides as a function of water content.
Tight binding of water is an important feature of lipases. Zaks and Klibanov (1985) found that porcine pancreatic lipase was catalytically active in anhydrous pyridine, retaining its bound water even in the presence of this water-miscible solvent. This is a beneficial characteristic of lipases for catalyzing interesterification reactions because it allows the use of very low water concentrations which minimize the unwanted hydrolysis reaction. Figure 6 shows the effect of water content on the fraction of triglycerides converted to partial glycerides. As expected, the amount of hydrolysis increases as the water content rises. Steps 2 and 3 in the reaction pathway are pushed in the direction of free enzyme and fatty acid by higher water concentration. The concentration of acylenzyme intermediates is thus reduced and esterification is hindered. These observations are consistent with our kinetic studies of lipase-catalyzed interesterification in cyclohexane (Miller et al., 1991). The enzymatic turnover number, kat, was separately determined for hydrolysis and esterification,and it was found that hydrolysis was roughly three times slower than esterification under saturating conditions. Of the two steps comprising hydrolysis, step 1is slower than step 2. With COz as the solvent, a higher water content does not significantly affect the hydrolysis rate because water does not play a direct role in step 1, and there is sufficient water for the decomposition of E-LA (step 2) to occur at a high rate. As a result, the interesterification rate is not observed to change appreciably as the C 0 2 water content is varied. Effect of Pressure. To study the effect of pressure, reaction rates were measured at pressures from 1200 to 1600 psi in water-saturated carbon dioxide. The inter-
Ind. Eng. Chem. Res., Vol. 30, No. 5, 1991
0-0
A-A
INTRESTERIFICATION RATE OVEWFUACTIONRATE
-/'
1
800
k:;A7ATA
0 1160
1
1260
1360
1460
1560
1660
PRESSURE (psi) Figure 7. Effect of pressure on the overall reaction rate and the interesterification rate. Residence time = 28 s; carbon dioxide is water-saturated. 1
20.0,
~
w
2
I
4.04
l o -
O0 -n -n -
,
1160
1260
1360
1460
1560
1860
PRESSURE (psi) Figure 8. Effect of pressure on the pseudo-fiiborder rate constants for interesterification and the overall reaction.
esterification rate and the overall rate (based on total trilaurin conversion) increase as pressure rises, as shown in Figure 7. However, the interesterification rate increases much more rapidly than the overall rate, indicating that the selectivity of the reaction for interesterification over hydrolysis improves at higher pressures. Figure 8 shows that the pseudo-first-order rate constants for the overall reaction and for interesterification both decrease as pressure rises, but the overall reaction rate constant decreases by 64% while the rate constant for interesterification decreases by only 29%. The suppression of hydrolysis relative to interesterification could be the result of changes in enzymesolvent interactions that are dependent upon the density-dependent properties of the solvent. Enzyme-solvent interactions can have a marked effect on substrate binding and on the rate of reaction. The ratio k,/K,, an apparent second-order rate constant (see eq 4), for both subtilisin and a-chymotrypsin has been shown to correlate roughly with solvent hydrophobicity (Zaks and Klibanov, 1988a). If we assume that the reaction follows irreversible, one-substrate Michaelis-Menten kinetics, the rate expression is V = Vmax[Sl - Kcat[El~[Sl (8) K m + [SI K m + [SI The rate expression integrated for a plug-flow reactor is S[S], = V,=T = K , In (1- 6) (9) A plot of S[S], versus In (1 - 6) obtained by varying pressure (and therefore varying [SIo)should produce a straight line if K , and V, are independent of pressure.
I
I
3 27
I
I I
I
3 30
I
3 33
I
I
3 36
I
I
I
3 39
GAI.55 ( t h o u s a n d s )
Figure 9. EPR spectra of 12-doxylstearicacid bound to lipase from Rhizopus arrhizus in carbon dioxide at 35 "C and (a) atmospheric pressure and (b) 1600 psi.
However, our data are best fit by a curve, indicating that K, and/or V, do change as pressure and density change. Although the Michaelis-Menten model does not apply over the entire time course of interesterification, it may be applied here since trilaurin is the limiting substrate and the conversions are low at the residence times studied (less than 5% of trilaurin is converted to LLM and MLM). For Michaelis-Menten kinetics the rate can also be expressed
V = &[E][S] Km where the pseudo-first-order rate constant is equal to k,,[E]/K, and [E] is the free-enzyme concentration. If k,, and K, are pressure-dependent, either k, decreases or K, increases to account for the observed pressure dependence of the first-order rate constant. It is difficult to predict a priori which parameter may be more sensitive to changes in the density-dependent properties of the solvent. EPR spectroscopy was used to examine the effect of carbon dioxide pressure (or density) on the conformation of the enzyme. Conformational changes would provide a possible physical explanation for the observed pressure dependence of the first-order rate constant. EPR spectra of spin-labeled stearic acid bound to lipase at atmospheric pressure and at 1600 psi are shown in Figure 9. The spectra have very similar features, indicating that no large conformational changes occur near the enzyme-bound spin-label as a result of changing pressure. However, the possibility that subtle changes in conformation occur, below the detection limits of EPR, cannot be discounted. Effect of Temperature. The temperature dependence of the pseudo-first-order rate constant for interesterification was determined at 1400 psi from 30 to 40 "C. The rate constant attains an apparent maximum of 1.0 X s-l at 35 "C, increasing by 15% over its value at 30 "C. The rate constant decreases as the temperature rises to 40 "C, possibly due to a loss of enzyme activity caused by thermal denaturation. At the subcritical temperature of 30 "C, carbon dioxide exists as a liquid with a density of approximately 0.76 g/cm3. At this higher density, the solubilities of trilaurin and myristic acid are greater than at 35 "C; further, the likelihood of thermal denaturation is less at 30 "C than at 35 "C. For these reasons, liquid carbon dioxide may sometimes be preferable to super-
Ind. Eng. Chem. Res., Vol. 30, No. 5, 1991 945
RESIDENCE TIME (sec) Figure 10. Total conversion of trilaurin vs residence time at 1400 psi and 35 "C.
critical carbon dioxide for conducting enzymatic reactions. However, the transport properties of liquid carbon dioxide are not as favorable as those of supercritical carbon dioxide, and rate constants may be higher a t supercritical temperatures, as our results show. Comparison of Reaction Rates in C 0 2 and Cyclohexane. A determination of the enzymatic rate constants K , and kat in each solvent is necessary to make a meaningful comparison of the enzyme kinetics. Figure 10 shows the total conversion of trilaurin as a function of residence time in carbon dioxide at 1400 psi and 35 "C. To determine K , and V,, we attempted to fit the data to rate expressions integrated for a plug-flow reactor. The integrated rate expression for irreversible, one-substrate Michaelis-Menten kinetics (eq 3) did not give a reasonable fit as negative values were obtained for K,. A possible explanation is that the assumption of irreversibility breaks down at the high conversions attained. However, the integrated rate expression for reversible Michaelis-Menten kinetics also was not fit by the data. Apparently, the reaction cannot be considered simply as a reversible conversion of trilaurin to products (LLM, MLM, DL, LM). Lipase binds all of the triglycerides, diglycerides, and fatty acids present and catalyzes many interconversions that do not involve trilaurin. Thus, the one-substrate assumption does not apply except for low conversions (i.e. 0-5%). The maximum rate of reaction, V, can be obtained from the initial rate of reaction, provided that the initial trilaurin concentration is substantially greater than K,. Since K , is unknown for this reaction, only apparent turnover numbers may be compared. The enzymatic turnover number calculated from out data at 1400 psi and M was 35 "C with a trilaurin concentration of 2.9 X 0.1 s-l. Reaction-rate measurements performed in cyclohexane with the same enzyme preparation and a trilaurin M yielded a turnover number concentration of 7.8 X of 0.3 s-l. Considering that these turnover numbers were possibly determined at nonsaturating conditions, it appears that the rates of reaction in COz and cyclohexane are roughly similar. Conclusions This paper discusses the suitability of supercritical carbon dioxide as a reaction medium for an enzyme-catalyzed reaction of commercial interest: the lipasecatalyzed interesterification of triglycerides. Lipase is active and stable in supercritical C02at 35 "C and pressures ranging from 1200 to 1600 psi. The water content of the carbon dioxide has little effect on intrinsic enzyme activity, and the use of dry carbon dioxide decreaes substrate hydrolysis. Higher pressures increase the selectivity of the reaction
for interesterification over hydrolysis, thereby maximizing the conversion of triglycdrides to the desired interesterification products. The kinetics and stability of lipase in supercritical carbon dioxide appear to be similar to those in organic liquid solvents such as cyclohexane. The economic feasibility of an industrial-scale lipase-catalyzed interesterification on C02may depend upon possible cost advantages realized from simplified separations and lower solvent costs versus higher capital costs for high-pressure equipment. However, the most important advantage of supercritical carbon dioxide as a reaction medium may follow from its nontoxicity, as required for meeting increasingly stringent health and environmental regulations. Acknowledgment This work was supported by a National Science Foundation Graduate Fellowship (D.A.M.) and a grant from NSF Engineering Division, CBT-8715908. We thank Mr. Christian Lockemann for assistance in conducting experiments and Prof. Douglas Clark for assistance in interpreting EPR spectra. Nomenclature E = lipase TL = Trilaurin DL = dilaurin LA = lauric acid MA = myristic acid E-LA, E-MA = acyl-enzyme intermediates LM = 1(3)-myristoyl-2-lauroyl-rac-glycerol LLM = 1,2(2,3)-dilauroyl-3(l)-myristoyl-rac-glycerol MLM = 1,3-dimyristoyl-2-laurin E-X = enzyme-bound species 9 = observable modulus, dimensionless uo = observed maximum reaction rate, M s-l D, = effective diffusion coefficient of trilaurin, cm2s-l D, = diffusion coefficient of trilaurin, cm2 s-l So = bulk trilaurin concentration, M V , = volume of particle, cm3 A = external surface area of particle, cm2 v'= reaction rate, M s-1 V,, = maximum reaction rate, M s-l [SI = substrate (trilaurin) concentration, M K, = substrate concentration where V = V-12, M k,, = enzymatic turnover number, s-l [E], = total enzyme concentration, M 6 = conversion of trilaurin [SI,= initial trilaurin concentration, M T = residence time, s Registry No. COz, 124-38-9; trilaurin, 538-24-9; myristic acid, 544-63-8; lipase, 9001-62-1.
Literature Cited Albro, P. W.; Corbett, B. J.; Hass,J. R. The Mechanism for Nonspecific Lipase from Rat Pancreas. Biochim. Biophys. Acta 1976, 431,493-506. Bailey, J . E.; Ollis, D. F. Biochemical Engineering Fundamentab, 2nd Ed.; McGraw-Hill: New York, 1986. Burdette, R. A.; Quinn, D. M. Interfacial Reaction Dynamics and Acyl-Enzyme Mechanism for Lipoprotein Lipase-Catalyzed Hydrolvsis of Liuid D-Nitrouhenvl - - Esters. J. Biol. Chem. 1986.261 (26);12016-i202i. Chi. Y. M.: Nakamura. K.: Yano. T. Enzvmatic Interesterification in Supercritical Carbon Dioxide. Agric: Biol. Chen. 1988,52 (6), 1541-1550. Chibata, I.; Tam, T.; Sato, T.;Mori, T. In Fermentation Technology Today; Terui, G., Ed.; Society of Fermentation Technology: Osaka, Japan, 1972;pp 383-389. Copelin, H. B.Method for Reducing Oligometric Cyclic Ether Content of a Polymerizate. US. Patent 4,306,058,1981.
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Erickson, J. C.; Schyns, P.; Cooney, C. L. Effect of Pressure on an Enzymatic Reaction in a Supercritical Fluid. AIChE J. 1990,36 (2),299-301. Friedrich, J. P. Supercritical COz Extraction of Lipids from LipidContaining Materials. US. Patent 4,466,923,1984. Hammond, D. A.; Karel, M.; Klibanov, A. M.; Krukonis, V. J. Enzymatic Reactions in Supercritical Gases. Appl. Biochem. Biotechnol. 1985,11, 393-400. Kasche, V.; Schlothauer, R.; Brunner, G. Enzyme Denaturation in Supercritical COz: Stabilizing Effect of S-S Bonds During the Depressurization Step. Biotech. Lett. 1988,10(8),569-574. Klibanov, A. M. Enzymes That Work in Organic Solvents. CHEMTECH (June) 1986,354-359. Krukonis, V. J.; Branfman, A. R.; Broome, M. G. Supercritical Fluid Extraction of Plant Materials Containing Chemotherapeutic Drugs. Presented at the AIChE Meeting, Boston, MA, Aug 1979. Lilly, M. D. Two-Liquid-Phase Biocatalytic Reactions. J. Chem. Technol. Biotechnol. 1982,32,162-169. Macrae, A. R. Lipase-Catalyzed Interesterification of Oils and Fats. J . Am. Oil Chem. SOC.1983,60 (2),291-294. McHugh, M. A,; Krukonis, V. J. Supercritical Fluid Extraction: Principles and Practice; Butterworth Boston, MA, 1986. Miller, D. A,; Prausnitz, J. M.; Blanch, H. W. Kinetics of LipaseCatalyzed Interesterification of Triglycerides in Cyclohexane. Enzyme Microb. Technol. 1991,13,98-103. Nakamura, K.;Chi, Y. M.; Yamada, Y.;Yano, T. Lipase Activity and Stability in Supercritical Carbon Dioxide. Chem. Eng. Commun. 1986,45, 207-212. Paulaitis, M. E.; Krukonis, V. J.; Kurnik, R. T.; Reid, R. C. Supercritical Fluid Extraction. Rev. Chem. Eng. 1983,I, 179-250. Randall, L. G. The Present Status of Dense (Supercritical) Gas Extraction and Dense Gas Chromatography: Impetus for DGC/MS Development. Sep. Sci. Technol. 1982,17 (l),1-118. Randolph, T. W.; Blanch, H. W.; Prausnitz, J. M.; Wilke, C. R. Enzvmatic Catalvsis in a Suuercritical Fluid. Biotechnol. Lett. 1985,7 (5),325-328. Randolph, T. W.; Clark, D. S.; Blanch, H. W.; Prausnitz, J. M. Enzvmatic Oxidation of Cholesterol Aggregates in Suuercritical Carbon Dioxide. Science 1988,239,387-390.
Roselius, W.; Vitzthum, 0.; Hubert, P. Method for the Production of Caffeine-Free Coffee Extract. U S . Patent 3,843,824,1974. Subramaniam, B.;McHugh, M. A. Reactions in Supercritical Fluids-A Review. Ind. Eng. Chem. Process Des. Deu. 1986,25, 1-12. Taniguchi, M.; Kamihira, M.; Kobayashi, T. Effect of Treatment with Supercritical Carbon Dioxide on Enzymatic Activity. Agric. Biol. Chem. 1987,51 (2),593-594. Thompson, K.N.; Johnson, R. A.; Lloyd, N. E. US.Patent 3788945, 1974. van Eijs, A. M. M.; deJong, J. P. J.; Doddema, H. J.; Lindeboom, D. R. Enzymatic Transesterification in Supercritical Carbon Dioxide. In Proceedings of the International Symposium on Supercritical Fluids; Perrut, M., Ed.; Societe Francaise de Chimie: Paris, 1988a. van Eijs, A. M. M.; deJong, J. P. J.; Oostrom, H. H. M.; Doddema, H. J.; Visser, M. A.; Stoop, R. Enzymatic Synthesis of Nonylacetate and Isoamylacetate in Supercritical Carbon Dioxide and Organic Solvents. In Proceedings of the 2nd Netherlands Biotechnology Congress; Breteler, H., van Lelyveld, P. H., Luyben, K. Ch. A. M., Eds.; Netherlands Biotechnological Society: Zeist, The Netherlands, 198813. Williams, D. F. Extraction with Supercritical Gases. Chem. Eng. Sci. 1981,36(ll),1769-1788. Wisdom, R. A.; Dunnill, P.; Lilly, M. D.; Macrae, A. Enzymic Interesterification of Fats: Factors Influencing the Choice of Support for Immobilized Lipase. Enzyme Microb. Technol. 1984,6, 443-446. Zaks, A.; Klibanov, A. M. Enzyme-Catalyzed Processes in Organic Solvents. Proc. Natl. Acad. Sci. USA 1985,82,3192-3196. Zaks, A.; Klibanov, A. M. Enzymatic Catalysis in Nonaqueous Solvents. J . Biol. Chem. 1988a,263 (7),3194-3201. Zaks, A.; Russell, A. J. Enzymes in Organic Solvents: Properties and Applications. J. Biotechnol. 198813,8,259-270. Received for review August 17, 1990 Revised manuscript received December 4, 1990 Accepted December 6, 1990
A Laboratory Fixed-Bed Reactor for (Just About) All Occasions Alan S.Foss Department of Chemical Engineering, University of California, Berkeley, California 94720
A packed-bed catalytic reactor is synthesized with a coordinated combination of physical equipment and a computer simulation of the rate of chemical reaction. Several electrical heaters immersed in the granular packing supply the heat of reaction, which is calculated by the on-line computer using current temperature measurements made thoughout the bed and (just about) any concentration function declared by the user. Air is used as the reactant stream and granules of pumice as the packing. The thermal response time of the reactor is a few minutes. The ease of operating such an apparatus makes it suitable for a number of control system investigations in our instructional laboratory. The reactor can be operated adiabatically or with cooling a t the wall. Autothermal operation is also possible through use of a feed-effluent heat exchanger that is attached to one end of the reactor. A “quench” stream of reactant or diluent may be injected into the bed at the halfway point and may be used in accomplishing certain control tasks. Chemical reactors intended for use in instructional laboratories need to be easy to operate but not so simple in their operating characteristics that they are unrepresentative of reacting systems. Those two criteria are next to impossible to satisfy when one wants to offer students experience in operating and controlling a fixed-bed exothermic reactor. Reactant preparation and supply, catalyst activation, concentration measurement, and assurance of safe conditions can overwhelm everyone involved, student and instructor, and can swamp one’s efforts in attaining the primary objective of the laboratory experience. The reactor system described here is free of all such nuisances. Nuisance-free is but one of its merits. The reactor can be
operated either adiabatically or with wall cooling (countercurrent or cocurrent), autothermally (with a feed-effluent heat exchanger), or with quench stream injection at the halfway point; can be started up and lined out in a matter of minutes; and can be run with just about any reaction one would want to present to the students-just about any reaction. All this is accomplished with a hybrid of physical equipment and a computer simulation. Reactor Concept The elimination of chemical reactants is the key to breaking free of the numerous hindrances just mentioned. However, chemical reaction phenomena and effects must
oaaa-5aa5pi /2630-0946$02.50/0 0 1991 American Chemical Society
