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원 기훈, 이 선복

포항공과대학교 환경연구소, 포항공과대학교 화학공학과

On-Line Monitoring of Enzymatic Ester Synthesis Reaction in Solvent-Free Media

with Computer-Aided Water Activity Control

Keehoon Won and Sun Bok Lee

Institute of Environmental and Energy Technology, POSTECH,

Department of Chemical Engineering, POSTECH

Introduction

Enzymatic catalysis in nonaqueous media has gained considerable interest in recent years as an efficient approach to the synthesis of natural products, pharmaceuticals, fine chemicals and food ingredient [1,2]. Particularly, if it is feasible to perform the enzymatic reaction in a mixture of the substrates themselves without any bulk solvents, this approach can combine the advantages of nonaqueous enzymology with the high levels of productivity. In addition recent environmental legislation is severely restricting the use of many traditional organic solvents. The major advantages of these solvent-free systems are that no solvent is used, minimizing the environmental impact and the reaction volume, lowering the size of the equipment required [3].

When implementing such solvent-free synthesis, we have to remember the critical role of water in catalytic activity of the enzyme and reaction equilibrium. It has been amply demonstrated that enzyme activity and selectivity depend critically on the level of biocatalyst hydration. Product yield is also affected by the hydration level in the reaction system, which is often expressed as thermodynamic water activity (aw) [4]. Therefore, a number of methods of controlling water activity for water-producing reactions such as esterifications have been reported. The conventional methods, most of which use saturated salt solutions or solid salt hydrate pairs [5,6], are not adequate for solvent-free systems where a great amount of water is produced during the reaction because of a low water transfer rate. We previously developed a computer-aided water activity control system using air bubbling for lipase-catalyzed esterification reactions in solvent-free media. Water activity was successfully kept very close to the set point despite massive production of water during the ester synthesis [7]. In the present study, on-line monitoring of the esterification conversions with computer-aided aw control has been described.

Experimental

Lipozyme IM, Rhizomucor miehei lipase immobilized on anion-exchange resin, was a generous gift from Novo Nordisk (Bagsvaerd, Denmark) and used as received. n-Capric acid and n-decyl alcohol were purchased from Sigma (St. Louis, MO, USA). Silica gel (particle size: 3-6 mm) was from Shinyo (Osaka, Japan). Other chemicals were of analytical grade. Enzyme powders were stored in a refrigerator before use.

Esterification reactions were carried out in a water-jacketed, stirred-tank type enzyme reactor. Enzyme powders (1.0 g) were suspended in 100 ml of n-decyl alcohol for 30 min. The reactions were started by the addition of the substrate mixture (22 ml of n-decyl alcohol and 62 ml of n-capric acid). All the reactions were performed at 30 ℃ and the reaction medium was stirred vigorously at 500 rpm for a rapid equilibration between the enzyme and the liquid medium.

A schematic diagram of the water activity control system is shown in Figure 1. The flow rate of compressed air passing through an air filter (0.2 □m) was controlled by a mass flow controller. The inlet air stream was split by a Y-connector into two flows: one obtained water by flowing it through distilled water bottles in series, whereas the other lost water by flowing it through silica blue bottles in series. Only one of these two flows was allowed to pass through the solenoid valve and then entered the enzyme reactor through the sintered glass disc at the bottom of the reactor.

Water activity was controlled by manipulating the flow rate and type of air supplied into the enzyme reactor as described previously [7]. The computer as a digital PI controller determined the flow rate and type of inlet air by comparing the set point to the actual aw in the reactor measured with a relative humidity sensor. In this work, humidified air was programmed not to be supplied after the relative humidity reached the set point. Previous study showed that the performance of the PI controller with constant parameters was different depending on the reaction time due to time-varying and nonlinear features of the system. Hence, in the present study the PI controller parameter was changed with the time.

Figure 1. Schematic diagram of the computer-aided water activity control system: (1) air pump; (2) air filter; (3) mass flow controller; (4) silica gel; (5) distilled water; (6) digital to analog converter; (7) solid state relay; (8) solenoid valve; (9) stirrer; (10) relative humidity sensor; (11) enzyme reactor; (12) analog to digital converter; (13) computer.

The reaction mixtures composed of substrates and product with five compositions and the enzyme powder were equilibrated separately with a variety of saturated salt solutions at 30 ℃ for 18 days. Equilibration was achieved through the vapor phase in sealed vials. After the equilibration, the water contents in the liquid medium were measured with a Karl-Fischer titrator. The amounts of water bound to the enzyme powders were determined from the weight difference. The concentration of substrates and product were determined by GC analysis. The analytical conditions were the same as described previously [7].

Results and Discussion

The conversion of esterification reactions can be determined from the amount of water produced by the reaction, because water is formed as a byproduct in the same molar ratio to the product, ester. We have estimated the reaction conversion without any analysis of the reaction medium by on-line monitoring the amount of produced water through a mass balance equation.

Changes in the water content in the reaction system with the time depend on both water transfer rate by air bubbling and water production rate by the esterification reaction. Therefore, a material balance on the water in the reaction system can be written as

[eqn: {E DELTA {C}_{w,e}+V DELTA {C}_{w,m}} over { DELTA t}={M}_{w} LEFT ( {q} over {RT} RIGHT ) LEFT({P}_{w,i n} - {P}_{w,out } RIGHT ) + {r}_{w}]

[eqn: = {M}_{w} LEFT ( {q} over {RT} RIGHT ) LEFT ( { {P}_{vap} } over {100} RIGHT ) LEFT({RH }_{i n } - {RH}_{out } RIGHT ) + {r}_{w}]	(1)

[eqn: X= { SUM LEFT ( r/mw RIGHT )DELTA t } over { {S}_{0} } TIMES 100]	(2)

where Cw,e = the water concentration bound to the enzyme particles (g/g); Cw,m = the water concentration in the reaction medium (g/l); Pw = the partial pressure of water; rw = the rate of water production by the reaction (g/min); q = air flow rate (sccm); Mw = molecular weight of water; R = the gas constant; V = the reaction volume (l); E = the enzyme amount (g); Pvap = the vapor pressure of water; RH = the relative humidity (%); X = the reaction conversion (%); and S0 = initial amount of the substrate (mol).

The previous study revealed that the water activity in the reaction medium was equal to that in the reactor headspace. Therefore, Cw,e and Cw,m can be determined by measuring the relative humidity in the gas phase from water adsorption isotherm and water solubility curves, respectively. Each correlation between water activity and water content was expressed in the third order equations through nonlinear regression analysis of experimental data. On the other hand, the dependence of Cw,m on the reaction conversion was found to be nearly linear. The relative humidity of the inlet and outlet air gas was respectively measured with two RH sensors.

PI control of water activity was carried out for lipase-catalyzed esterification reaction in solvent-free media. As shown in Figure 2a, once the relative humidity reached the setpoint (60%), the relative humidity was successfully kept constant in spite of massive production of water by manipulating the rate of dried air bubbling. The water activity calculated from the measured water content and the water solubility curves (closed circles) confirmed that the water activity in the reaction medium was equal to that in the reactor headspace (dotted line). Figure 2b shows the flow rate of air gas supplied into the enzyme reactor for aw control. The reaction conversion calculated from the equations (1) and (2) agreed with that measured by GC analysis as shown in Figure 2c. Thus on-line monitoring of the ester synthesis conversion has been achieved in solvent-free media.

Figure 2. Computer-aided control of water activity for solvent-free esterification reaction. PI control: Kc = 1,000 (X＠70%), 500 (70≤X＠90), 250 (90≤X) sccm/%, □I = 2,000 min, and time interval = 1 min. (a) the relative humidity of the inlet and outlet air (temperature compensated), (b) air flow rate, (c) on-line monitoring of the reaction conversion.

Acknowledgement

The authors wish to acknowledge the financial support of the Korea Research Foundation made in the program year of 1998.

References

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2.	Kim, M. G. and Lee, S. B.: J. Ferment. Bioeng., 81, 269 (1996).

3.	Pepin, P. and Lortie, R.: Biotechnol. Bioeng., 63, 502 (1999).

4.	Halling, P. J.: Enzyme Microb. Technol., 16, 178 (1994).

5.	Ujang, Z., Al-Sharbati, N., and Vaidya, A. M.: Biotechnol. Prog., 13, 39 (1997).

6.	Zacharis, E., Omar, I. C., Partridge, J., Robb, D. A., and Halling, P. J.: Biotechnol. Bioeng., 55, 367 (1997).

7.	Won, K. and Lee, S. B.: Theories and Applications of Chemical Engineering, 5, 1021 (1999).