Solvent-free 시스템에서의 효소 반응을 위한

컴퓨터를 이용한 수분 활성도 제어

원 기훈, 이 선복

포항공과대학교 화학공학과

Computer-Aided Control of Water Activity

for Enzyme Reactions in Solvent-Free Systems

Keehoon Won, Sun Bok Lee

Dept. of Chemical Engineering, Pohang University of Science and Technology

Introduction

Enzymatic catalysis in non-aqueous solvents is such a rapidly developing research area that it has attracted interest from chemists, biochemists, and chemical engineers [1]. Under these conditions, the industrial utility of enzymes is improved because of enhanced thermostability, ease of product and enzyme recovery, the ability to catalyze reactions that are kinetically or thermodynamically impossible in aqueous solution and so on. However, if feasible, it would be technologically more attractive to perform the enzymatic reaction in a mixture of the substrates themselves without any bulk solvents. This approach can combine the advantage of non-aqueous enzymology with the high levels of productivity. In addition recent environmental legislation is severely restricting the use of many traditional organic solvents such as chlorinated hydrocarbons.

For the successful implementation of a solvent-free system, there should be a driving force for the reaction to proceed in the desired direction until completion. In the case of ester synthesis this is usually achieved by removal of water produced during the reaction.

When implementing any such synthesis, one has to remember that excessive dehydration of the reaction mixture can lead to a dramatic deterioration of enzyme performance. This is because water plays a major role in enzymatic catalysis, and more specifically, in the maintenance of the unique catalytically competent conformation of the enzyme. It has been amply demonstrated that enzyme activity and stability depend critically on the level of biocatalyst hydration. In practical terms, the introduction of thermodynamic water activity (aw) as a generic parameter has significantly simplified the optimization and control of biotransformations performed in non-aqueous media [2]. Too low water activity can not provide flexibility enough for enzymes to function in non-aqueous systems. However, the high level of enzyme hydration does not always accompany the increase of its activity. In many cases, the reaction rate decreases sharply at high water activity. Recently enzyme agglomeration was shown to make the largest contribution to this serious loss in catalytic efficiency [3]. Consequently, it would be more desirable to control water activity in the system at an optimum rather than to exclusively remove water produced by the reaction.

We previously constructed a water removal system by continuously sparging dried air through the organic solvent as the reaction medium [4]. In this work, the computer-aided  water activity control system by air bubbling has been developed for enzymatic esterifications in solvent-free systems.

Experimental

Lipase OF from Candida rugosa was purchased from Meito Sangyo (Tokyo, Japan) and used as received. n-Capric acid and n-decyl alcohol were products of Sigma (St. Louis, Mo, USA). Silica blue was from Shinyo (Osaka, Japan). All the chemicals were used without further purification.

Esterification reactions were carried out in the enzyme reactor. Enzyme powders were suspended in 100 ml of n-decyl alcohol for some 30 min. Enzymatic esterifications  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 500 rpm.

A schematic diagram of the water activity control system is shown in Figure 1. Compressed air passed through an air filter (0.2 □m). The air flow rate was controlled by the mass flow controller. The inlet air stream was split by an Y connector into two flows, which had different humidity: one obtained water by bubbling through distilled water in two bottles in series; the other lost water by flowing through silica blue in other two bottles in series. But only one of these two flows was allowed to enter the enzyme reactor through the sintered glass disc at the bottom of the reactor. Sparged air either provided water for the reaction medium or removed water from it.

Figure 1. A schematic diagram of the computer-aided water activity control system.

Consequently water activity can be controlled by manipulating the flow rate and type of air supplied into the enzyme reactor. Both of them were determined by the output value from a feedback digital controller. The controlled variable, say water activity, was measured, and then sent in the form of digital signals to the digital computer where the measured output value was compared to the desired, or set point, value. Based on the error, the deviation between desired and measured value, control action was determined. The sign and magnitude of the output determine the type and flow rate of inlet air gas, respectively.

The substrates and product were determined by GC analysis. At predetermined intervals, about 0.5 ml of suspension was taken from the reaction medium and centrifuged to settle enzyme particles. 0.4 □l of sample diluted with cyclohexane was analyzed by GC with a flame ionization detector and a HP-INNOWax capillary column (inside diameter: 0.32 mm and length: 30 m). The column temperature was programmed to be increased from 160 ℃ to 240 ℃ at 20 ℃/min and maintained at 240 ℃ for 3.0 min. Injector and detector temperature were 250 ℃ and 260 ℃, respectively. Helium as a carrier gas was supplied into the column at 3.8 ml/min.

Results and Discussion

Several methods of controlling water activity have been reported in the literature which include pervaporation [5] and the use of saturated salt solutions [6] or salt hydrate pairs [7]. In this work we used both humidified air and dried one first for water activity control, which was achieved by controlling the flow rate and type of air sparged into the enzyme reactor. A digital computer as a feedback controller was implemented for this purpose. The performance of the digital PID controller during the enzymatic esterification is shown in Figure 2. Although water was produced continuously by the esterification, the relative humidity in the enzyme reactor was observed to be maintained constant at the desired point. In addition, water activity responded quickly to the set point change. The air flow rate is also shown to be changed by the PID control algorithm. The positive and negative sign of it represent humidified air and dried air, respectively.

Figure . Performance of the digital PID controller during the enzymatic esterification (Kc = 3000 sccm/%, □I = 6000 min, □D = 1 min, and time interval = 2 min).

The results of a batch reaction without and with water activity control at a relative humidity of 90% are shown in Figure 3. The reaction rate at controlled water activity was marginally higher than that without control. However, the final conversion was in marked contrast with the corresponding value attained without water activity control. Water activity control improved the reaction yield. This occurs since the continuous increase in the relative humidity by the esterification shifts the thermodynamic equilibrium in favor of the reverse, hydrolysis reaction.

For the first time, we have developed the computer-aided water activity control system using both humidified air and dried air for enzymatic esterifications in solvent-free media. The simplicity of this system renders it suitable for use over a wide range of water activities, and facilitates easy scale-up.

Figure 3. Progress curves of lipase-catalyzed esterifications with PID control or without control of relative humidity of 90%. Used enzyme: 1.0g; PID control: Kc = 3000 sccm/%, □I = 6000 min, □D = 1 min, and time interval = 2 min.

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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6. Wehtje, E., Kaur, J., Adlercreutz, P., Chand, S., and Mattiasson, B.: Enzyme Microb. Technol., 21, 502 (1997).

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