치토산을 분산제로 사용하여 졸-겔법으로 만든 유기/무기 혼성막에 고정화한 효소의 특성연구

장 종관／, 조 규진*／, 문 일식／, 이 재석／／

／순천 대학교 공과대학 화학공학과, 전남 순천시 매곡동 315번지

／／광주 과학기술원 신소재공학과, 광주광역시 쌍암동 572번지

Preparation and Characterization of □-Amylase Immobilized Inorganic/Organic Hybrid Membrane Using Chitosan as a Dispersant in the Sol-Gel Processing.

Jongkwan Jang／, Gyoujin Cho*／, Il-Shik Moon／, and Jae-Suk Lee／／

／Department of Chemical Engineering, Suncheon National University, Suncheon, Chunnam, 540-742 Korea

／／Department of Material Science and Engineering, Kwangju Institute of Science and Technology (K-JIST), Kwangju, 506-303 Korea

Introduction

Inorganic glasses composed of oxides of boron, aluminum, silicon, titanium, zirconium, vanadium, etc. can offer rigid, amorphous, three-dimensional structures often with excellent optical clarity [Brinker and Sherrer, 1990]. Therefore, they have been considered as host materials for the incorporation of various organic, and inorganic materials. However, the processing temperatures of the most performed inorganic glasses often exceed the decomposition temperatures of common organic materials and the denature temperatures of living organisms.

Fortunately, clear inorganic and modified inorganic/organic glasses can often be prepared in situ by low temperature polymerization of appropriate monomers in a sol-gel process [Klein, 1988]. The sol-gel method opened a new way in preparing host matrices to encapsulate unique guest molecules. We reported the sol-gel method to offer the thermal stability of nonlinear optical organic chromophores in silica matrice [Lee et al, 1996]. As an obvious entension of the sol-gel method, it may be very plausible to investigate whether living organisms can be incorporated in the glass matrice without losing any viability and activity using the sol-gel method. The enzyme incorporated glass membrane may have potential to use in the field of membrane bioreactor [Srivastava et al, 1996].

As an adaption of interpenetrating polymer network (IPN) method, the microscopically homogeneous morphology of organic/inorganic IPNs synthesized with an organic polymer phase made by copolymerization of both methylmethacrylate (MMA) and 3-(trimethoxysilyl) propylmethacrylate (TSM) and an inorganic SiO2 phase made by a sol-gel hydrolysis of both tetra ethyl orthosilicate (TEOS) and TSM with a dispersant (chitosan) are used as a membrane matrice in this study. Since the chitosan has been well known as a biocompatible host to provide cell viability and activity [Groboillot et al, 1993], the chitosan may act not only dispersing the organic phase in the matrice but also providing the friendly environment to the guest enzyme (□-amylase).

In this paper, we wish to report the optimum condition in preparing the □-amylase immobilized inorganic/organic hybrid membrane and the results from characterization of the membranes by FT-IR, TGA, scanning electron microscopy (SEM), water stability, and enzyme activity.

Experimental

The copolymerization of TSM, and MMA occurs under acidic condition at 50℃ where the chitosan, and tetraethoxy orthosililte (TEOS) mixed, initally TSM, chitosan, and TEOS standing at room temperature until observing the gelation. The gelation was determined by the point at which the solution quit flowing as a liquide. Once polymerization had ocuured, the rasulting gel was allowed to cure at 50℃ for an additional 24h. During this time, no shrinkage and separation from the walls of the teflom dish. After curing, the clear and flexible membrane can be easily pulling away from the dish for further characterization. The immobilization of enzyme was carried out using same procedure in preparing the membrane besides adding termamyl into chitosan solution. □-amylase (Termamyl, NOVO. Co).

Results and discussion.

We observed no macro and micro phase separation during the polymerization. However, when the sol-gel processing started at 50℃ without the initial standing at room temperature, we did observe the phase separation and there results were consistant. Based on this result, we specqulate that when the free radical copolymerization of TSM and MMA is fast with respect to the sol-gel reaction rate, the visible phase separation occured during the polymerization.

The FT-IR spectrum of the membrane is charactrized by three intence band, two in the carbonyl stretching regions at 1717 □-1 and 1690 □-1 respectly for PMMA and chitosan, and the other in the Si-O stretching at 980∼1100 □-1. The carbonyl stretching of PMMA was not shown from chitosan/silica membrane. In situ copolymerization of TSM and MMA at 50℃ in the gel can be followed by visual inspection of carbonly stretching regions;  without heating, the carbonyl peak of appeared at the higher energy region (1717 □-1) but the carbonyl peak of the same membrane preparead with heating showed at 1690 □-1. This observation indicates that only at 50℃ the TSM and MMA copolymerization can be initiated in the gel without phase separation.

Since our interest is developing a host matrix to encapsulate enzymes, we tested the stability of the resulting membrane in aqueous solution and showed that the membrane was swollen little bit. After drying the membrane, the weight loss was only less than 5% for all samples tested. However, the membrane prepared from chitosan/silica was completely destroy in the aqueous phase. This excellent water stability of the TSM/MMA/chitosan/SiO2 membrane may be due to the hydrophobicity of homogeneonsly distributed the copolymer in the membrane. The thermal stabilities of the hybrid membrane and the chitosan silicate membrane were determined by the TGA analysis. The TGA curves are shown that the thermal stability of the hybrid membrane was improved by the in situ copolymerization of TSM comparing the chitosan/silicate membrane.

The morphology of the enzyme immobilized membrane was strongly affected by the amount of enzyme loading. The optimum condition was obtained when the enzyme was loaded 0.1□ per prepared sol-gel solution (20□). Enzyme activity was estimated by measuring the amount of oligosaccharides produced enzymatically, using the starch as a substrate. The amount of oligosaccharides formed was determined by UV-vis. In the reaction of native enzyme, the amount of enzyme used was calculated based on the same amount of immobilized enzyme in the prepared membrane. The reaction of both native enzyme  and immobilized enzyme were carried out in the mixed solution of 1□ of starch solution for 3 min. When the immobilized enzyme was used, a piece of the membrane (size : 1 □) was immersed in the reaction solution. All the reactions were carried out without agitation. Storage stability for the immobilized enzyme on the function of time were showed in the Figure 1. Immobilized one was calculated based on by

Activity (%)=[eqn: { B} over {A }]×100

where A is the amount of the oligosaccaride produced by native enzyme (added to the same amounts to the immobilized one); B is the amount of the oligosaccaride produced by the immobilized enzyme.

As shown in Figure 1, the acitivity of immobilized □-amylase fell rapidly in an early stage of storage. This acitivity dropping may be due to releasing of some enzyme from the membrane since the membrane stored in distilled water at room temperature.

The membrane morphology, as shown in Figure 2 and 3 respectively for the surface and cross-section of the membrane, is proven to be a typical porous symmetric membrane by SEM.

Conclusions.

We have prepared water stable chitosan incorporated organic/inorganic hybrid membrane by using the in situ copolymerization of TSM and MMA in the gel phase. In this process, the chitosan acts as a dispersant to homogeneously disperse TSM and MMA in the sol-gel solution. Without the chitosan, we could not prepare the microscopically homogeneous water stable membrane. In addition, we have shown that □-amylase immobilized in the resulting membrane remained viable and showed stable activity for a couple of days in water storage at room temperature. The morphology of the membrane showed a typical porous symmetry from the SEM invistigation.

References.

Brinker, C. J. and Sherrer, G. W., Sol-Gel Science, Academic Press, NY         (1989).

Groboillot, A. F., Champagne, C. P., Darling, G. D. and Poncelet, D., Neufeld,    R. J., “Membrane Formation by Interfacial Cross-linking of Chitosan for      Microencapsaalation of Lactococcus Lactis.” Biotechnol. Bioeng. 42. 1157.     (1993).

Klein, L.,“Sol-gel Technology for Thin Films, Fibers, Preforms, Electronics,     and Speciality Shapes” Noyes Publications: Park Ridge, Nj. (1988).

Lee, J. S. Cho, Y. S. Cho, G. Jang, K. T. Shul, Y. G. and Choi, M. G., “UV    Study on the Thermal Stability of Disperse Red 1 Films In Coporated with    Reactive Silica” Mol, Cryst. Liq. Cryst.. 280, 53 (1996).

Srivastava, S., Nighojkar, A. and Kwmar, A., “Immobilization of Cuscuta       Reflexa Starch Hosphorylase: Production of Glucose-1-Phosphate Using        Bioreactors” J. Ferment. Bioeng. 81, 354 (1996).

Fig 1. Storage stability of immobilized enzyme

Fig 2. Surface morphology of enzyme    Fig 3. Cross sectional morphology

immobilized membrane by SEM.       of enzyme immobilized membrane

by SEM.