Methods 21 Pervaporation

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Pervaporation is defined as a separation technique in which a liquid feed mixture is separated by partial vaporization through a nonporous permselective (selectively permeable) membrane (11). Transport phenomena in pervaporation are different when compared to any other membrane processes such as dialysis, reverse osmosis, and ultrafiltration because of multiple interactions

From: Methods in Biotechnology, Vol. 15: Enzymes in Nonaqueous Solvents: Methods and Protocols Edited by: E. N. Vulfson, P. J. Halling, and H. L. Holland © Humana Press Inc., Totowa, NJ

between the feed components and the membrane polymer. The driving force for the mass transfer of permeants from the feed to the permeate is a gradient in chemical potential, which is established by the difference in the partial pressure of the permeants across the membrane. This difference in partial pressure can be created principally by using a condenser and vacuum pump system, or by sweeping an inert gas on the permeate side of the membrane. Pervaporation by using a condenser and a vacuum pump is the more dominant mode of the operation. Recently, pervaporation has been applied as a way to mediate esterification and interesterification reactions that are catalyzed by lipase (3,12,13). The rate, conversion, and the selectivity of lipase are greatly affected by the water concentration in the reaction mixture. Pervaporation can be easily used to control the water concentration of the reaction mixture. As shown in Fig. 1, the conversion yield was 65% at 120 min. After equilibrium, the water was removed by the pervaporation process. The conversion yield increased to about 85% after 240 min. Consequently, the thermodynamic equilibrium in the synthesis of «-butyl oleate was shifted toward synthesis by water removal.

2.1.1. Removal of Water Produced from Lipase-Catalyzed Esterification

2.1.1.1. Membrane Preparation

The membrane was prepared according to the method of Mulder and Smolders (14) as follows:

1. Dissolve cellulose acetate in acetone.

2. Cast the cellulose acetate solution upon a glass plate.

3. The acetone was allowed to evaporate. The membrane was completely transparent and the thickness of the membrane was 20 ^m.

2.1.1.2. Pervaporation

1. Set up a pervaporation reactor (3). The membrane was supported by a filter paper placed on a sintered stainless-steel disk.

2. Add equimolar substrates (40 mmol each of oleic acid and «-butanol), Lipozyme® (Novo, 0.4 g), and isooctane (30 mL) into the reactor.

4. Apply the vacuum at the permeate side (2 mbar). The water separated from the reaction mixture was condensed in the condensor.

2.1.2. On-Off Dewatering Control by Tubular-Type Pervaporation System

When lipase was used as a biocatalyst in organic synthesis, aw control was essential. Unfortunately, there is no aw sensor available that can monitor aw continuously in the solvent system in a large-scale production system. Therefore, it is impossible to control aw directly in our pervaporation system.

Fig. 1. Equilibrium shift by water removal using pervaporation. The reaction mixture contained 40 mmol of oleic acid, 40 mmol of n-butanol, and 0.4 g of Lipozyme® in 30 mL of isooctane at 25°C.

As an alternative, we used a sorption isotherm so that the dissolved water concentration corresponding to the aw was controlled by an on-off control mode (13); that is, the water formed was removed by pervaporation with the control based on manual analysis of water in the organic phase at the optimal water solubility corresponding to the optimal aw. A computer-controlled pervaporation system made it possible to control the aw indirectly in lipase-catalyzed esterification. In this system, a tubular-type membrane was used instead of the sheet-type membrane (3,15). A tubular-type system has several advantages in durability at relatively high temperature and high flow rate, and in the ability to withstand exposure to harsh chemicals (16). Also, this tubular-type pervaporation system was able to increase the membrane area exposed to the reaction mixture by inserting several tubular-type membrane modules when compared to the sheet type system having the membrane area localized on the bottom of the reactor. As shown in Fig. 2, the synthesis rate of the controlled reaction was about twice faster than that of the uncontrolled reaction, and the experimental data fitted the calculated ester curve quite well (13). In addition, the water-solubility curve calculated from the dewatering control algorithm fitted the experimental data well (Fig. 3). Therefore, the water formed from the enzymatic esterification could be removed while maintaining the optimal aw range of the reaction mix-

Fig. 2. Time-course for the lipase-catalyzed esterification of «-butyl oleate with the on-off dewatering control (•) and without the control (A). Solid line: Ester concentrations (Eq. 3) integrated from the adaptive step-size Runge-Kutta method. The reaction mixtures contained 50 mg Candida rugusa lipase, 400 mM butanol, and 400 mM oleate in 25 mL n-hexane.

Fig. 2. Time-course for the lipase-catalyzed esterification of «-butyl oleate with the on-off dewatering control (•) and without the control (A). Solid line: Ester concentrations (Eq. 3) integrated from the adaptive step-size Runge-Kutta method. The reaction mixtures contained 50 mg Candida rugusa lipase, 400 mM butanol, and 400 mM oleate in 25 mL n-hexane.

ture, and the reaction was able to proceed toward synthesis by the irreversible reaction mode. We hope that this method can be used to control the aw in various enzymatic syntheses of various esters, peptides, and glycosides in the presence of organic solvents where the direct aw control is impossible.

2.1.2.1. Tubular-Type Membrane Module Preparation

The tubular-type membrane module was prepared according to the method of Song and Hong (16) as follows:

1. Prepare the tubular-type porous ceramic support (the average pore diameter was about 0.1 ^m.)

2. Coat the porous support with 20 wt% cellulose acetate solution in acetone using dip-coating and rotation-drying techniques. The thickness of the active layer of cellulose acetate was about 30 ^m.

0 100 200 300 400 500

Time (min)

0 100 200 300 400 500

Time (min)

Fig. 3. Time-course for the water solubility in the reaction mixture. Solid line: The change in water calculated from the balance between production in reaction (Eq. 3) and removal (Eq. 1); •: experimental value.

2.1.2.2. Determination of Optimal Water Activity

1. Pre-equilibrate the Candida rugosa lipase (50 mg, Lipase OF, Meito) with the salt hydrates in a desiccator. [The salt hydtrates used in the following: Na2HPO4-2/0 H2O (aw = 0.18), NaAc-3/0 H2O (aw = 0.3), Na4P2O7-10/0 H2O (aw = 0.52), Na2HPO4-7/2 H2O (aw = 0.85) (5).]

2. Pre-equilibrate 400 mM substrate solution (1:1 molar ratio of oleic acid to butanol in n-hexane) with the same salt hydrates with direct contact for 3 d in a cylindrical reactor with screw caps and Teflon-lined septa.

3. Initiate the reaction by adding the 50 mg of lipase into the cylindrical reactor in which the pre-equilibrated substrate solution and 2 g of the salt hydrates were included.

4. Mix vigorously on a shaker (175 strokes/min) at 30°C.

5. Samples taken from the reaction were analyzed by a Hewlett-Packard Model 5890 series II GC with a flame ionization detector (13).

6. The optimal water activity was shown to be in the range 0.52-0.65 because the synthetic activity was similar over this range.

2.1.2.3. Determination of Sorption Isotherm Curve

Denature the lipase by adding 1N HCl, and dry the denatured lipase. Pre-equilibrate the reaction mixtures including the denatured lipase with 2 g of different salt hydrates at 30°C.

3. Analyze the water contents in the organic phase of the reaction mixtures with a Karl Fischer titrater.

4. The water solubility range corresponding to the optimal aw range (0.52-0.65) was 0.015-0.026 mmol/mL.

2.1.2.4. Determination of Dewatering Rate of Reaction Mixture by Pervaporation

1. Pre-equilibrate the reaction mixture including the denatured lipase with water (aw = 1).

2. Add the water saturated reaction mixture into the pervaporation system.

3. Set up the tubular-type pervaporation system to remove the water (13).

4. Remove the water in the reaction mixture by pervaporation at 30°C.

5. Take the samples at the predetermined time intervals and analyze the water content of the samples using the Karl Fischer titrater.

6. The dewatering rate was as follows:

log[water (mmol/mL)] = -1.367 - (0.037) Time (min) (1)

2.1.2.5. Strategies for Controlling aw in Lipase-Catalyzed Esterification

1. If the water is removed at the same rate that the ester is formed at the optimal water concentration, the original reversible reaction would be shifted predominantly to the following irreversible reaction mode;

The reaction rate is d [Ester] (3) -dF- = k [FA] [OH]

where [FA] is the R1COOH concentration (mmol/mL), [OH] is the R2OH concentration (mmol/mL), and [Ester] is the RjCOOR2 concentration (mmol/mL).

2. The reaction rate constants (k) at different water activities were obtained from the experiment by controlling the aw using salt hydrates (Table 1). The k value at the optimal aw was 0.01075 ± 0.00038 mL/(mmol/min) at 30°C.

3. The ester concentration (Eq. 3) was integrated from the fourth-order adaptive step-size Runge-Kutta method. The change in water present was calculated from the balance between production in reaction (Eq. 3) and dewatering rate (Eq. 1).

4. The water was removed while maintaining the optimal water concentration range corresponding to the aw range.

5. We put the initial water concentration in solution (S0) and the values at the upper (SU) and lower (SL) limits into the algorithm. The SU and SL were 0.026 and 0.015 mmol/mL, respectively, which corresponded to the optimal aw range (0.520.65). If the water concentration increased above SU, a switch was allowed to be turned on; the switch was turned off if the water was decreased below SL. The detailed algorithm was explained by Kwon and Rhee (13).

Table 1

Reaction Rate Constant (k) of C. rugosa Lipase-Catalyzed Esterification of n-Butyl Oleate in n-Hexane at Different Water Activities

Table 1

Reaction Rate Constant (k) of C. rugosa Lipase-Catalyzed Esterification of n-Butyl Oleate in n-Hexane at Different Water Activities

aw buffer used

a a uw

Rate constant (k) (mL/mmol/min)

CH3COONa-3/0 H2O

0.30

0.00375 ± 0.00050

Na4P2O7-10/0 H2O

0.52

0.01070 ± 0.00045

Na2HPO4-7/2 H2O

0.65

0.01075 ± 0.00038

Na2HPO4-12/7 H2O

0.80

0.00742 ± 0.00048

11 The aw value was from ref. 5.

2.1.2.6. The Lipase-Catalyzed Esterification with Continuous aw Control

1. Pre-equilibrate the lipase (50 mg) with Na2HPO4-7/2 H2O(aw = 0.65) in the vapor phase for 3 d.

2. Pre-equilibrate the substrate solution (400 mM) with Na2HPO4-7/2 H2O (aw = 0.65) by direct contact for 3 d.

3. Initiate the reaction by adding 25 mL of the pre-equilibrated substrate solution into the cylindrical reactor in which the pre-equilibrated lipase was included.

4. Mix vigorously on a shaker (175 strokes/min) at 30°C for 480 min. During the reaction, no salt hydrates was included in the reactor.

5. Control the water concentration using the computer-controlled tubular-type per-vaporation system.

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