β-Fructosidase (inulase) from $\underline{Kluyveromyces fragilis}$ was purified 106 folds using Sepharose 6B column (5.0 × 95 cm). The purified enzyme was immobilized on Tygon tube and aminoethyl cellulose for the purpose of studies on production of fructose from Jerusalem artichoke tuber extract and mass transfer limitation.
Silanization in chloroform at 65℃ and 10% glutaraldehyde treatment were found to be critical points for the immobilization on Tygon tube, while 2% glutaraldehyde was effective for the immobilization of the enzyme on aminoethyl cellulose. The characteristics of the immobilized enzymes were examined. Optimal pH's of invertase and inulase activities were 4.0 and 5.5, respectively for the enzyme immobilized on aminoethyl cellulose, while 3.5 and 4.5, respectively for the immobilized enzyme on Tygon tube. Optimal temperatures of invertase and inulase activities were shown to be 60℃ and 45℃, respectively for the enzyme immobilized on aminoethyl cellulose. Compared with soluble enzyme both activities were unstable in this temperature.
The activation energies were 5.9 kcal/mole for the enzyme immobilized on Tygon tube and 7.0 kcal/mole for the immobilized enzyme on aminoethyl cellulose with sucrose as substrate. With inulin as substrate these were 3.4 kcal/mole for the former and 5.6 kcal/mole for the latter. Km for sucrose hydrolysis was 10 mM for the immobilized enzyme on Tygon tube, and 7.7 mM for the enzyme immobilized on aminoethyl cellulose. When 2% inulin was used instead of 5% inulin, the activity ratios (invertase/inulase) were 6.8 in soluble enzyme, 7.5 in the immobilized enzyme on aminoethyl cellulose, and 10 in the case of the enzyme immobilized on Tygon tube. Considering the higher Km value, decrease in activation energy and the activity ratio, there existed a significant limitation of internal diffusion in the case of the enzyme immobilized on Tygon tube using the inulin as substrate.
Among two immobilized enzymes, the enzyme immobilized on aminoethyl cellulose was selected for the operation of the reactor because of its higher activity and yield than the other. For the production of fructose, the batch reactor and packed bed reactor were employed. In the batch reactor, total reducing sugar produced from Jerusalem artichoke tuber extract (50 mg/ml) by the immobilized enzyme on aminoethyl cellulose (20 units) for 23 hr was 42 mg/ml. From this reaction mixture, 32 mg of fructose per ml was obtained (88% conversion). In the packed bed reactor, the immobilized enzyme activity was affected by the superficial velocity. In this respect the column with H/D ratio, 10.3, yielded higher conversion and was accepted as an optimum considering pressure drop also. In the plug flow reactor with H/D ratio, 10.3, the following conditions were accepted as preliminary optimum ; enzyme loading, 104 units ; space time, 3.8 hr ; temperature, 40℃ ; 7% inulin concentration. In operation with these conditions, the conversion with 7% inulin was 90% and the productivity was 1.7 mole/l/min.
To examine the operational stability, the reactor packed with the immobilized inulase on aminoethyl cellulose was operated at the preliminary optimum conditions. The activity decay constants of the enzyme immobilized on aminoethy1 cellulose were found to be 0.05 $day^{-1}$ with 7% inulin as feeding substrate and 0.04 $day^{-1}$ with the Jerusalem artichoke tuber extract. In the operation with high concentration of substrate, significant substrate protection of the immobilized enzyme against heat inactivation was observed.
돼지감자내에 존재하는 이눌린으로부터 과당을 연속적으로 생산 하기 위하여 Sepharose 6B 로 정제한 $\underline{K. fragilis}$ 의 이눌라아제를 Tygon tube 와 aminoethylcellulose 에 고정화하였다. Tygon tube 에 고정화하는 경우에 용매로 클로로포름을 사용하여 65℃ 로 3-aminopropyltriethoxysilane 을 처리하는 반응과 10 % glutaraldehyde 농도가 최적의 조건으로 밝혀졌다. Aminoethylcellulose 의 경우에는 2 % glutaraldehyde 농도가 최적이었다. 최적의 반응조건에 의해 제조된 고정화 효소의 성질을 전보 (남백희, 효소 가수분해에 의한 돼지감자로 부터의 과당생산, 석사논문, 한국과학원 생물공학과 (1977))의 가용성 효소와 비교해본 결과는 다음과 같다.
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고정화효소에서는 가용성 효소에 비해 Km' 과 인베르 타아제의 이눌라아제 역가에 대한 비가 증가하였고 반면에 활성화 에너지는 감소되었다. 이러한 현상은 내부 물질 전달의 한계에 의한 것이며 Tygon tube 에 고정화한 효소에서 이눌린을 기질로 사용할 경우에 두드러지게 나타났다.
Aminoethylcellulose 에 고정화한 이눌라아제는 기질을 사용 하지 않았을 경우에는 열 안정성이 좋지 않았으나 놀라웁게도 실제 가동중의 열 안정성은 증가 되었다. 이로 미루어보아 높은 농도의 이눌린 용액이 고정화 효소의 열 안정성에 기여하는 것으로 추측되었다.
회분식 반응기와 충진식 연속 반응기에서의 일련의 실험을 높은 역가와 수율을 가진 aminoethylcellulose 에 고정화한 효소를 사용하여 행하였다. 전보의 가용성 효소의 경우에는 200 unit 의 효소를 사용하여 ml 당 50 mg 의 당이 포함된 돼지감자 추출물로부터 50℃ 반응 온도에서 2시간만에 33 mg 의 과당을 얻을 수 있었으며 환원당으로서의 수율은 90 % 이었다. 본 실험에서 20unit 의 고정화 효소를 사용한 회분식 반응의 경우 같은 농도의 돼지감자 추출물로부터 45℃의 반응 온도에서 23 시간만에 32 mg의 과당을 얻을 수 있었으며 수율은 83 % 이었다. 회분식 반응과는 달리 충진식 반응에서는 반응기 체재시간이 6 시간 이상일 경우 100% 의 수율을 나타내었다. 충진식 반응에서 7 % 이눌린을 사용하여 3.8 시간의 반응기 체재시간을 최적으로 결정했으며 수율은 90 % 이었고 분당 1.7 몰 농도의 과당 생산성을 보여 주었다.
