Kinetic Modeling of Immobilized Lipase Catalysis in Synthesis of n

Apr 10, 2008 - Department of Chemical Engineering, University Institute of Chemical Technology (UICT), University of Mumbai, Matunga, Mumbai−400 019...
0 downloads 0 Views 110KB Size
3358

Ind. Eng. Chem. Res. 2008, 47, 3358-3363

Kinetic Modeling of Immobilized Lipase Catalysis in Synthesis of n-Butyl Levulinate† Ganapati D. Yadav* and Indrakant V. Borkar Department of Chemical Engineering, UniVersity Institute of Chemical Technology (UICT), UniVersity of Mumbai, Matunga, Mumbai-400 019, India

n-Butyl levulinate is used as an important intermediate having diverse applications. The present work focuses on the synthesis of n-butyl levulinate by esterification of levulinic acid with n-butanol by using immobilized lipases. Novozym 435 (Candida antarctica lipase) was found to be the most efficient catalyst, and tetrabutyl methyl ether was the best solvent. Effects of various parameters were studied to analyze the kinetics and mechanism of the lipase action. Ping-pong bi-bi mechanism with n-butanol substrate inhibition was found to describe the kinetics of the reaction. The kinetic parameters evaluated from initial rate data were used to simulate the experimental results. There was a very good agreement between theory and experiment. 1. Introduction Lipases are the most widely used enzymes for the synthesis of organic chemicals, mainly in aqueous media and in some cases nonaqueous media, because they are inexpensive, stable, and easy to recycle.1,2 Lipases possess wide substrate specificity, have an ability to recognize chirality, and do not require labile cofactors.3-6 Of late, lipases have been used to catalyze a number of reactions in nonaqueous media such as esterification, transesterification, amidation, hydrolysis, hydrazinolysis, and epoxidation.5,7-19 The versatility of lipase catalysis in the synthesis of other groups of chemicals needs to be explored. Massive efforts are directed worldwide to identify attractive chemical transformations to convert biomass into organic bulk chemicals on a commercial scale. An attractive option is the conversion of lignocellulosic biomass into levulinic acid (4oxypentanoic acid) by acid treatment at relatively mild conditions. Levulinic acid contains a ketone group and a carboxylic acid group. These two functional groups make levulinic acid an essentially very versatile building block for the synthesis of various bulk chemicals. Leuvlinic acid has been identified as a promising green, biomass-derived platform chemical. n-Butyl levulinate is an important commercially available intermediate used in organic process industries for different purposes such as plasticizing agents, solvents, and odorous substances.20 There is a dearth of literature on enzyme-catalyzed synthesis of esters in nonaqueous media. Besides, quantitative information on kinetics and modeling of esterification of levulinic acid is totally lacking. Esterification of levulinic acid in the presence of a suitable acid catalyst, such as sulfuric acid, yields n-butyl levulinate. Esterification of the carboxyl group is relatively slow and needs activation either by high temperature or by a catalyst to achieve equilibrium conversion in a reasonable amount of time. Biochemical-catalyzed routes have unique advantages over chemical catalysis. Hence, it was thought worthwhile to study the † This paper is dedicated to Professor Bruce Nauman on the occasion of his 70th birthday. G.D.Y. has known Bruce’s impressive contributions to RTD theory ever since he was at the Loughborough University UK in 1981. Bruce was then spending time at Loughborough on writing his book on ‘Mixing in Continuous System’ with Bryan Buffham. * To whom correspondence should be addressed. Tel.: 91-22-24102120. Fax: 91-22-2414-5614. E-mail: [email protected].

synthesis of n-butyl levulinate from levulinic acid and n-butanol, by using a variety of immobilized lipases, including reaction mechanism and kinetics. 2. Experimental Section 2.1. Enzymes. Some enzymes were procured as gift samples from reputed firm Novo Nordisk (Denmark): Novozym 435, Lipozyme RM IM, and Lipozyme TL IM. Novozym 435 is Candida antarctica lipase immobilized on a macroporous polyacrylic resin. Lipozyme RM IM is Rhizomucor miehei immobilized on an anionic resin. Lipozyme TL IM is Thermomyces lanuginosus immobilized on silica. Thermomyces lanuginosus is produced by genetically modified Aspergillus oryzae. 2.2. Chemicals. All chemicals were procured from firms of repute: levulinic acid and n-butanol (E. Merck (India), Mumbai) and tetra-butyl methyl ether, n-nonane, and other analytical reagents (s. d. Fine Chemicals Pvt. Ltd., Mumbai, India). 2.3. Experimental Setup. The experimental setup consisted of a 3 cm i.d. fully baffled mechanically agitated glass reactor of 50 mL capacity, equipped with four baffles and a six-bladed turbine impeller. The entire reactor assembly was immersed in a thermostatic water bath, which was maintained at a desired temperature with an accuracy of (1 °C. A typical reaction mixture consisted of 0.01 mol of levulinic acid and 0.02 mol of n-butanol diluted to 15 mL with tetrabutyl methyl ether as a solvent. The reaction mass was agitated at 30 °C for 15 min at a speed of 500 rpm, and then 35 mg of immobilized enzyme (Novozym 435) was added to initiate the reaction. Liquid samples were withdrawn periodically from the reaction mixture and filtered to remove traces particles, if any. The filtrate was analyzed by GC. 2.4. Analysis. The concentrations of the reactants and products were determined on Chemito Gas chromatograph (model 8610) equipped with a flame ionization detector. A 4 m × 3.8 mm stainless steel column packed with SE-30 was used for analysis. Synthetic mixtures were prepared of pure samples, and calibration was done to quantify the collected data for conversions and rates of reactions. n-Butyl levulinate was confirmed by GC-MS. 2.5. Determination of Initial Rates. To determine the initial rates of enzymatic reaction, the concentrations of levulinic acid were varied from 0.66 to 2.64 M for known concentrations of n-butanol (between 0.66 and 2.64 M). The total volume was

10.1021/ie800193f CCC: $40.75 © 2008 American Chemical Society Published on Web 04/10/2008

Ind. Eng. Chem. Res., Vol. 47, No. 10, 2008 3359 Scheme 1. Esterification of Levulinic Acid with n-Butanol

made to 15 mL with tetra-butyl methyl ether, after which 35 mg of Novozym 435 was added to initiate the reaction and the reaction was continued until 30% conversion at 50 °C. Concentration-versus-time plots were made for different substrate concentrations, and the curves were fitted by using a polynomial. The derivative of the polynomial was equated to zero, and initial rates were calculated by solving that equation at zero time. 3. Results and Discussions The reaction is represented by Scheme 1. 3.1. Screening of Different Lipases. Activities of Novozym 435, Lipozyme TL IM, and Lipozyme RM IM were evaluated under otherwise similar conditions (Figure 1). Among all the lipases, Novozym 435 was found to be the best with maximum initial rates and 85% conversion in 2 h. Novozym 435 is a versatile enzyme and has been found to be very effective in nonaqueous media. Dimensional analysis of Candida antarctica lipase B (Novozym 435) based on the crystal structure clearly shows that amino acid residue A281 is a part of an R-helix (R10) located at the top of the substrate-binding pocket in a highly hydrophobic environment.21 This microenvironment around the active site pocket of Novozym 435 favors proper interaction of substrate in nonaqueous media. The flapping lid of Rhizomucor miehei (Lipozyme RM IM) and Thermomyces lanuginosus (Lipozyme TL IM) projects into the binding pocket there by creating steric hindrance in binding of levulinic acid at the active site.22 Since Novozym 435 is highly active in anhydrous media and does not contain a flapping lid, there is less steric hindrance as compared to other lipases and it shows more activity.23 Therefore, it was used in all further experiments. 3.2. Mass Transfer Analysis. This is a solid-liquid system having levulinic acid and n-butanol in the liquid phase and the solid polymer-supported enzyme (Novozym 435). The pore space of the solid is filled with aqueous phase, and a thin film of aqueous phase surrounding the matrix should be retained for the enzyme to be active. The effect of external mass transfer resistance to the transfer of the reactants toward the outer surface of the solid particle was studied in the range of speed of agitation of 250-1000 rpm. The initial rate of reaction increased with speed of agitation from 250 to 500 rpm and remained constant at higher speeds (Figure 2). Indeed, the conversion after 2 h was increased correspondingly from 55 to 85% in the same range and remained practically constant beyond 500 rpm. Thus, there was no resistance to external mass transfer. So further experiments were performed by using a speed of 500 rpm. 3.3. Effect of Catalysis Loading. The effect of catalyst loading was studied from 10 to 50 mg under otherwise similar Scheme 2. Ping-pong bi-bi Lipase Action Mechanism

Figure 1. Efficacy of different lipases. Reaction conditions: levulinic acid ) 0.01 mol, n-butanol ) 0.02 mol, solvent tetra-butyl methyl ether up to 15 mL, speed of agitation ) 500 rpm, temperature ) 50 °C, and lipase ) 16.33 units/mL.

Figure 2. Effect of speed of agitation. Reaction conditions: levulinic acid ) 0.01 mol, n-butanol ) 0.02 mol, tetra-butyl methyl ether up to 15 mL, temperature ) 50 °C, and catalyst ) 35 mg.

conditions (Figure 3). The rate of reaction increased with increasing catalyst loading, and the overall conversion also increased correspondingly from 50 to 89%. The initial rate of reaction was found to be linearly dependent on catalyst loading in the liquid phase (Figure 4). 3.4. Effect of Temperature. The effect of temperature activity of Novozym 435 was monitored from 30 to 60 °C (Figure 5). The maximum initial rate of reaction was obtained at 60 °C, and the conversion after 2 h was 90%. The Arrhenius plot was made on the basis of ln(initial rates) vs reciprocal of temperature. An apparent activation energy was calculated from the observed initial rates at different temperature under otherwise

3360

Ind. Eng. Chem. Res., Vol. 47, No. 10, 2008

Figure 6. Arrhenius plot. Figure 3. Effect of catalyst loading. Reaction conditions: levulinic acid ) 0.01 mol, n-butanol ) 0.02 mol, tetra-butyl methyl ether up to 15 mL, speed of agitation ) 500 rpm, and temperature ) 50 °C.

Figure 4. Initial rates versus enzyme loading. Reaction conditions: levulinic acid ) 0.01 mol, n-butanol ) 0.02 mol, tetra-butyl methyl ether up to 15 mL, speed of agitation ) 500 rpm, and temperature ) 50 °C.

Figure 5. Effect of temperature. Reaction conditions: levulinic acid ) 0.01 mol, n-butanol ) 0.02 mol, tetra-butyl methyl ether up to 15 mL, speed of agitation ) 500 rpm, and catalyst ) 35 mg.

similar conditions. Figure 6 shows the Arrhenius plot. The activation energy was found to be 1.653 kcal/mol. Several authors have reported activation energy for enzymatic reaction from 0.9 to 9 kcal/mol.21 In general, the activation energy values

Figure 7. Effect of mole ratio of n-butanol. Reaction conditions: levulinic acid ) 0.01 mol, tetra-butyl methyl ether up to 15 mL, temperature ) 50 °C, speed of agitation ) 500 rpm, and catalyst ) 35 mg.

for enzyme-catalyzed reactions are much lower than those for chemical catalysis. The activation energy of 1.2 kcal/mol is lower than those reported for most lipase-catalyzed reactions.23 Kim and Chung24 have reported a value of 7.0 kcal/mol for the lipase-catalyzed hydrolysis of palm kernel oil in reversed micelle systems. Desnulle25 has reported a value of 5.3 kcal/mol in an aqueous emulsion system. In the current work, the apparent activation energy is lower but still comparable to those reported in the literature. 3.5. Effect of Mole Ratio of n-Butanol. The mole ratio of n-butanol to levulinic acid was varied, keeping the moles of levulinic acid constant and with a constant liquid volume using tetra-butyl methyl ether. Figure 7 shows that the maximum conversion (88%) and rate of reaction were obtained with a 1:2 mole ratio. By increasing the mole ratio of n-butanol from 1 to 3, the conversion and intial rates increased (Table 1.). With a further increase in n-butanol mole ratio to 4, there was a decrease in the conversion of the reaction. The decrease in the conversion and rate of reaction with increasing concentration of n-butanol could be attributed to the inhibitory effect of n-butanol on the Novozym 435. n-Butanol contains a hydrophobic tail and a polar head. Since the enzyme is hydrophobic, there may be hydrophobic-hydrophobic inter-

Ind. Eng. Chem. Res., Vol. 47, No. 10, 2008 3361 Table 1. Initial Rates for Different Mole Ratiosa mole ratio (n-butanol/levulinic acid)

initial rates (mol/L‚min)

1:1 2:1 3:1 4:1

0.238 0.295 0.402 0.4222

a Reaction conditions: levulinic acid ) 0.01 mol, tetra-butyl methyl ether up to 15 mL, temperature ) 30 °C, and catalyst ) 35 mg.

Figure 9. Linewear-Burk plot for different n-butanol concentrations.

Figure 8. Reusability of lipase. Reaction conditions: levulinic acid ) 0.01 mol, n-butanol ) 0.02 mol, tetra-butyl methyl ether up to 15 mL, speed of agitation ) 500 rpm and catalyst ) 35 and temperature ) 50 °C.

action between the enzyme and n-butanol, which will increase the residence time of n-butanol. Because of a close contact with the neighboring hydrophobic residues, the enzyme-n-butanol complex would have to be partially dehydrated, which may destabilize the native conformation of the enzyme; this leads to “molecular lubrication”.14 The enzyme is immobilized within macroporous polyacrylic resin, which is hydrophilic; it may trap water within the support as well as it may form a film around the resin.26 Because the active-site pocket of Novozym 435 is hydrophilic and the environment around that pocket is hydrophobic, the concentration of water and its residence time at the active site is greater than that of levulinic acid. Therefore, there may also be deactivation of the enzyme due to water. External mass transfer limitation was eliminated by increasing the speeds of agitation. Because the speed of agitation is very low, the force of interaction between support and water is greater than the shear created at the surface and there is no deactivation of the enzyme at the outer surface.12 3.6. Reusability of Catalyst. Novozym 435 was reused thrice in this study. After each run, the enzyme was washed with tetrabutyl methyl ether and solvent was evaporated prior to reuse. The activity was reduced after each reuse, which indicates that it strongly inhibited substrates (Figure 8). 4. Kinetic Model To investigate the influence of substrate concentration on the rate of esterification, the concentration of n-butanol were varied from 0.66 to 2.65 mol/L at different levels of levulinic acid concentration (0.66 to 2.65 mol/L). Samples were taken from the reaction mixture periodically and analyzed by GC. The Lineweaver-Burk plots of reciprocal rate versus reciprocal concentration of levulinic acid are illustrated in Figure 9. The family of lines in Figure 9 has no common intersection, and therefore, a sequential mechanism can be ruled out. At low

concentrations of n-butanol and levulinic acid, the slope of the lines is not influenced by the concentration of the fixed substrate. This is indicative of a mechanism that requires the dissociation of one product before the association of the second substrate to the enzyme-substrate complex. The shape of the curve at higher concentrations of n-butanol can be used as an indication for substrate inhibition. This inhibition is stronger at low concentrations of levulinic acid and can be neglected at saturation concentrations. A mechanism in which a product is released between the additions of two substrates is called ping-pong bi-bi mechanism. This mechanism is common in group transfer or “substituted enzyme” reactions. This mechanism involves two different stable forms and two sets of central complexes. Since each substrate addition is followed by a product release, the mechanism could be called “ping-pong bi-bi” mechanism, as shown in Scheme 2. From these observations, a ping-pong bibi mechanism with alcohol inhibition is postulated. These assumptions are used to design a reaction mechanism that is depicted in Cleland’s notation, as shown below.

By analogy to the classical mechanism of esterification by lipases, it is assumed that levulinic acid (A) binds first to the free enzyme (E) and forms a noncovalent enzyme-acid complex (EA), which releases the first product, water (P) and E’ modified enzyme. The second substrate, n-butanol (B), reacts with E’ to give the complex E’B and gives the product n-butyl levulinate (Q) and free enzyme (E). Along with this, B also forms the dead-end complex [EiB] by binding to free enzyme [E]. The rate equation is as follows,27

V)

Vm[A][B]

(

)

[B] KmB[A] + KmA[B] 1 + + [A][B] Ki

(1)

where KmA is the Michaelis constant for levulinic acid, KmB is the Michaelis constant for n-butanol, and Ki is the inhibition constant due to n-butanol.

3362

Ind. Eng. Chem. Res., Vol. 47, No. 10, 2008

Figure 10. Secondary plot. Table 2. Kinetic Parameters for Esterification of Levulinic Acid with n-Butanol

Figure 11. Parity plot for experimental and simulated initial rates.

kinetic constants

initial guess from primary and secondary plot

refined value by Polymath 5.1

Vm (mol/L‚min) KmA (mol/L) KmB (mol/L) Ki

2.27 3.12 0.8 3.55

2.340 2.837 1.263 4.084

By taking the reciprocal of eq 1 and arranging terms, we get

(

)

Vm KmB KmA [B] 1+ +1 + ) V Ki [B] [A]

(2)

Proper manipulation of eq 2 leads to

(

)

5. Conclusions

(

[B] KmA 1 1 1 KmB + +1 ) 1+ V Ki Vm [A] Vm [B]

)

(3)

This is an equation of a straight line, which is called the primary plot.

Slope1 )

KmA KmA [B] + VmKi Vm

Intercept1 )

KmB 1 1 + Vm [B] Vm

(4)

(5)

Intercept1 can be plotted against 1/[B] to get the following:

Intercept2 ) Slope2 )

1 Vm

KmB Vm

mental initial rates with respect to initial concentrations, all the rate constants were calculated through nonlinear regression to the rate equation using Polymath 5.1. The best-fit values obtained are presented in Table 2. These values were then used to generate simulated initial rate values. Equation 1 was integrated numerically using Euler’s method with a step size of integration of 0.01. All data collected in this study are used to make the parity plot. A plot of simulated versus experimental values is made at various concentrations of A, which demonstrates that the simulated and experimental data fit very well (Figure 11).

(6)

(7)

Because the intercept of this primary plot depends on the concentration of [B], for different [B] there is a change in intercepts of the lines for the ping-pong bi-bi mechanism model. Preliminary values of kinetic parameters for the initial guess were calculated from the primary and secondary plots (Figures 9 and 10), as explained above. These results agree with the ping-pong bi-bi mechanism with n-butanol inhibition. These parameters were utilized in the final equation to evaluate the remaining parameters. The algorithm makes it possible to better estimate the constants. Kinetic parameters were refined by using the Polymath 5.1 software package. By knowing the experi-

Esterification of levulinic acid is very important in a number of industries. This is typically produced by using chemical catalysis at higher temperatures. Synthesis of n-butyl levulinate was accomplished at room temperature by esterification of levulinic acid with n-butanol by using different lipases, among which Novozym 435 was found to be the most active catalyst. The effects of various parameters on conversion and rates of reaction were studied systematically with Novozym 435 as the catalyst and tetra-butyl methyl ether as the solvent. The kinetics was found to obey the ping-pong bi-bi mechanism with n-butanol substrate inhibition. Acknowledgment The authors thank Novo Nordisk, Denmark, for the gifts of enzymes. I.V.B. acknowledges DBT-SRF award from Government of India. G.D.Y. acknowledges support from the Darbari Seth Professor Endowment. G.D.Y. has enjoyed the immense hospitality of Purdue University as a Distinguished Visiting Scholar under their President’s Asian Initiative during the writing of this manuscript. Nomenclature V ) rate of reaction (mol/L‚min) Vm ) maximum rate of reaction (mol/L‚min) [A0] ) initial concentration of levulinic acid (mol/L) [B0] ) initial concentration of n-butanol (mol/L) KmA ) Michaelis constant for levulinic acid (mol/L) KmB ) Michaelis constant of n-butanol (mol/L) Ki ) inhibition constant of n-butanol

Ind. Eng. Chem. Res., Vol. 47, No. 10, 2008 3363

Literature Cited (1) Lilly, M. D.; Eighth, P. V. Danckwerts Memorial lecture. Presented at Glaziers’ Hall, London, U.K., May 13, 1993; Advances in biotransformation processes. Chem. Eng. Sci. 1994, 49, 151. (2) Turner, M. Biocatalysis in organic chemistry. Part II. Present and future. Trends Biotechnol. 1995, 13, 253. (3) Bommarius, A. S.; Schwarm, M.; Drauz, K. Biocatalysis to amino acid-based chiral pharmaceuticalssExamples and perspectives. J. Mol. Catal., B 1998, 5, 1. (4) Gotor, V.; Brieva, R.; Gonzalez, C.; Rebolledo F. Enzymatic aminolysis and transamidation reactions. Tetrahedron 1991, 47, 9207. (5) Weber, N.; Klein E.; Vosmann, K.; Muherjee, K. D. Preparation of long-chain acyl thioesters-thiowax esters by the use of lipase. Biotechnol. Lett. 1998, 20, 687. (6) Yadav, G. D.; Sivakumar, P. Enzyme-catalysed optical resolution of mandelic acid via RS(()-methyl mandelate in non-aqueous media. Biochem. Eng. J. 2004, 19, 101. (7) Reslow, M.; Adlercreutz, P.; Mattiasson, B. Organic solvents for bioorganic synthesis. 1. Optimization of parameters for a chymotrypsin catalysed process. Appl Microbiol. Biotechnol. 1987, 26, 1. (8) Rizzi, M.; Stylos, P.; Riek A. A. A kinetic study of immobilized lipase catalysing the synthesis of isoamyl acetate by transesterification in n-hexane. Enzyme Microbiol. Technol. 1992, 14, 709. (9) Santaniello, E.; Ferraboschi, P.; Grisenti, P. Lipase-catalyzed transesterification in organic solvents: Applications to the preparation of enantiomerically pure compounds. Enzyme Microbiol. Technol. 1993, 15, 367. (10) Tramper, L. C. J.; Lilly, M. D. Biocatalysis in Organic SolVents; Elsevier: Amsterdam, The Netherlands, 1987; p 147. (11) Weber, N.; Klein, E.; Mukherjee, K. D. Long chain acyl thioesters by solvent free thioesterification and transthioesterification catalyzed by lipase. Appl. Microbiol. Biotechnol. 1999, 51, 401. (12) Yadav, G. D.; Borkar, I. V. Kinetic modeling of microwave-assisted chemoenzymatic epoxidation of styrene. AIChE J. 2006, 52, 1235. (13) Yadav, G. D.; Lathi, P. S. Synthesis of citronellol laurate in organic media catalyzed by immobilized lipases: Kinetic studies. J. Mol. Catal., B 2004, 27, 113. (14) Yadav, G. D.; Lathi, P. S. Synergism between microwave and enzyme catalysis in intensification of reactions and selectivities: Transesterification of methyl acetoacetate with alcohols. J. Mol. Catal., A 2004, 223, 51.

(15) Yadav, G. D.; Manjula Devi, K. A kinetic model for the enzymecatalyzed self-epoxidation of oleic acid. J. Am. Oil Chem. Soc. 2001, 78, 347. (16) Yadav G. D.; Manjula Devi, K. Enzymatic synthesis of perlauric acid using Novozym 435. Biochem. Eng. J. 2002, 10, 93. (17) Yadav, G. D.; Manjula Devi, K. Kinetics of hydrolysis of tetrahydrofurfuryl butyrate in a three phase system containing immobilized lipase from Candida antarctica. Biochem. Eng. J. 2003, 17, 57. (18) Yadav, G. D.; Manjula Devi, K. Immobilized lipase-catalysed esterification and transesterification reactions in non-aqueous media for the synthesis of tetrahydrofurfuryl butyrate: Comparison and kinetic modeling. Chem. Eng. Sci. 2004, 59, 373. (19) Yadav, G. D.; Trivedi, A. H. Kinetic modeling of immobilizedlipase catalyzed transesterification of n-octanol with vinyl acetate in nonaqueous media. Enzyme Microbiol. Technol. 2003, 32, 783. (20) Bart, H. J.; Reidetschlager, J.; Schatka, K.; Lehmann, A. Kinetics of esterification of levulinic acid with n-butanol by homogeneous catalysis. Ind. Eng. Chem. Res. 1994, 33, 21. (21) Uppenberg, J.; Patkar, S.; Bergfors, T.; Jones, T. A. Crystallization and preliminary X-ray studies of lipase B from Candida antarctica. J. Mol. Biol. 1994, 235, 790. (22) Jaeger, K. E.; Thorsten, E. Lipases for biotechnology. Curr. Opin. Biotechnol. 2002, 13, 390. (23) Baily, J. E.; Ollis, D. F. Biochemical engineering fundamentals, second ed.; McGraw Hill Book Company: New York, 1986. (24) Kim, T.; Chung, K. Some characteristics of palm oil kernel olein hydrolysis by Rhizopus arrhizus lipase in reversed micelle of AOT in iso-octane and additive effects. Enzyme Microbiol. Technol. 1989, 11, 528. (25) Desnulle, D. Pancreatic lipase. AdV. Enzymol. 1961, 23, 129. (26) Braden, M. The absorption of water by acrylic resins and other material. J. Prosthet. Dent. 1964, 14, 307. (27) Segel, I. H. Enzyme Kinetics; Wiley-Interscience Publication: John Wiley and Sons, Inc., New York, 1975; p 273.

ReceiVed for reView February 1, 2008 ReVised manuscript receiVed March 4, 2008 Accepted March 5, 2008 IE800193F