돼지감자내에 존재하는 이눌린으로부터 과당을 연속적으로 생산 하기 위하여 Sepharose 6B 로 정제한 K. fragilis 의 이눌라아제를 Tygon tube 와 aminoethylcellulose 에 고정화하였다. Tygon tube 에 고정화하는 경우에 용매로 클로로포름을 사용하여 $65\,^\circ\!C$ 로 3-aminopropyltriethoxysilane 을 처리하는 반응과 10 \% glutaraldehyde 농도가 최적의 조건으로 밝혀졌다. Aminoethylcellulose 의 경우에는 2 \% glutaraldehyde 농도가 최적이었다. 최적의 반응조건에 의해 제조된 ...
돼지감자내에 존재하는 이눌린으로부터 과당을 연속적으로 생산 하기 위하여 Sepharose 6B 로 정제한 K. fragilis 의 이눌라아제를 Tygon tube 와 aminoethylcellulose 에 고정화하였다. Tygon tube 에 고정화하는 경우에 용매로 클로로포름을 사용하여 $65\,^\circ\!C$ 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. \begin{center} \begin{tabular}{|cc|ccc|}\hline & 효소 & 가용성 & Tygon tube& aminoethylcellulose\\ 특성 && 효소 & 고정화효소 & 고정화효소\\ \hline \multicolumn{2}{|c|}{설탕에 대한 Km (mM)} & 6.7 & 10 & 7.7 \\ \hline 최적 pH \qquad & 인베르타아제 & 3.0 & 3.5 & 4.0 \\ & 이눌라아제 & 5.5 & 4.5 & 5.5 \\ \hline 최적 온도 & 인베르타아제 & 60 & 60 & 60 \\ $(\,^\circ\!C)$ & 이눌라?팁OE & 55 & 50 & 45 \\ \hline 활성화에너지 & 인베르타아제 & 8.0 & 5.9 & 7.0\\ (kcal/mole) & 이눌라아제 & 6.9 & 3.4 & 5.6\\ \hline \multicolumn{2}{|c|}{인베르타아제/이눌라아제$^{\star}$ & 6.8 & 10 & 7.5\\ \hline \end{tabular} $\star$ 2\% 이눌린 사용 \end{center}이눌린 사용 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 $50\,^\circ\!C$ 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 $45\,^\circ\!C$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.
돼지감자내에 존재하는 이눌린으로부터 과당을 연속적으로 생산 하기 위하여 Sepharose 6B 로 정제한 K. fragilis 의 이눌라아제를 Tygon tube 와 aminoethylcellulose 에 고정화하였다. Tygon tube 에 고정화하는 경우에 용매로 클로로포름을 사용하여 $65\,^\circ\!C$ 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. \begin{center} \begin{tabular}{|cc|ccc|}\hline & 효소 & 가용성 & Tygon tube& aminoethylcellulose\\ 특성 && 효소 & 고정화효소 & 고정화효소\\ \hline \multicolumn{2}{|c|}{설탕에 대한 Km (mM)} & 6.7 & 10 & 7.7 \\ \hline 최적 pH \qquad & 인베르타아제 & 3.0 & 3.5 & 4.0 \\ & 이눌라아제 & 5.5 & 4.5 & 5.5 \\ \hline 최적 온도 & 인베르타아제 & 60 & 60 & 60 \\ $(\,^\circ\!C)$ & 이눌라?팁OE & 55 & 50 & 45 \\ \hline 활성화에너지 & 인베르타아제 & 8.0 & 5.9 & 7.0\\ (kcal/mole) & 이눌라아제 & 6.9 & 3.4 & 5.6\\ \hline \multicolumn{2}{|c|}{인베르타아제/이눌라아제$^{\star}$ & 6.8 & 10 & 7.5\\ \hline \end{tabular} $\star$ 2\% 이눌린 사용 \end{center}이눌린 사용 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 $50\,^\circ\!C$ 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 $45\,^\circ\!C$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.
$\beta$-Fructosidase (inulase) from $\mbox{\underline{Kluyveromyces}}$ fragilis was purified 106 folds using Sepharose 6B column (5.0 X 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 tub...
$\beta$-Fructosidase (inulase) from $\mbox{\underline{Kluyveromyces}}$ fragilis was purified 106 folds using Sepharose 6B column (5.0 X 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 $65\,^\circ\!C$ and 10\% glutaraldehyde treatment were found to be critical points for the immobilization on Tygon tube, while 2\% glutaradehyde was the most 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 activity 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 $60\,^\circ\!C$ and $45\,^\circ\!C$, respectively for the enzyme immobilized on amonoethyl 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 $K_m$ 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 In comparison with soluble enzyme, the optimal pH was not changed, and the overall rate profile became broader with respect to pH range. The temperature effect on enzyme activity was same as that of soluble enzyme below $40\,^\circ\!C$, and showed somewhat increased stability at slightly higher temperature. The kinetic constants obtained by Lineweaver-Burk plot showed somewhat higher values for immobilized enzyme, the $K_m$ value; 4.3 mM and 4.8mM, $K_{ia}$ value; 40mM and 45 mM, Kip value; 22mM and 50mM for the soluble and immobilized enzyme, respectively. Thermal deactivation of immobilized enzyme conformed approximately the first order decay, and the half-life was about 7.8 days. From the information available, a rate equation was derived to predict the performances of penicillin amidase reactors. The progress of batch reaction agreed well with the result of computer simulation for both the immobilized and soluble enzymes, confirming the reliability of the rate equation and the values of kinetic constants. The performance of a single-stage CSTR type enzyme reactor system was simulated by solving the steady-state equation using Newton-Raphson method. The immobilized enzyme reactor system gave higher conversion values than the soluble enzyme reactor for all feed concentrations tested. This observation could be explained by higher values of $K_{ia}$ and $K_{ip}$ for the immobilized enzyme indicating that the inhibitory effect of the products on the immobilized enzyme were less than on the soluble enzyme at the same product concentration. We found that the reactor system with immobilized enzyme gave higher productivity than the one with soluble enzyme, at a given space time or for a level of conversion desired. It showed that 70 to 80\% greater productivity can be achieved using the immobilized enzyme as compared with the soluble enzyme while maintaining 90\% to 95\% conversion of penicillin to 6-APA.
$\beta$-Fructosidase (inulase) from $\mbox{\underline{Kluyveromyces}}$ fragilis was purified 106 folds using Sepharose 6B column (5.0 X 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 $65\,^\circ\!C$ and 10\% glutaraldehyde treatment were found to be critical points for the immobilization on Tygon tube, while 2\% glutaradehyde was the most 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 activity 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 $60\,^\circ\!C$ and $45\,^\circ\!C$, respectively for the enzyme immobilized on amonoethyl 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 $K_m$ 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 In comparison with soluble enzyme, the optimal pH was not changed, and the overall rate profile became broader with respect to pH range. The temperature effect on enzyme activity was same as that of soluble enzyme below $40\,^\circ\!C$, and showed somewhat increased stability at slightly higher temperature. The kinetic constants obtained by Lineweaver-Burk plot showed somewhat higher values for immobilized enzyme, the $K_m$ value; 4.3 mM and 4.8mM, $K_{ia}$ value; 40mM and 45 mM, Kip value; 22mM and 50mM for the soluble and immobilized enzyme, respectively. Thermal deactivation of immobilized enzyme conformed approximately the first order decay, and the half-life was about 7.8 days. From the information available, a rate equation was derived to predict the performances of penicillin amidase reactors. The progress of batch reaction agreed well with the result of computer simulation for both the immobilized and soluble enzymes, confirming the reliability of the rate equation and the values of kinetic constants. The performance of a single-stage CSTR type enzyme reactor system was simulated by solving the steady-state equation using Newton-Raphson method. The immobilized enzyme reactor system gave higher conversion values than the soluble enzyme reactor for all feed concentrations tested. This observation could be explained by higher values of $K_{ia}$ and $K_{ip}$ for the immobilized enzyme indicating that the inhibitory effect of the products on the immobilized enzyme were less than on the soluble enzyme at the same product concentration. We found that the reactor system with immobilized enzyme gave higher productivity than the one with soluble enzyme, at a given space time or for a level of conversion desired. It showed that 70 to 80\% greater productivity can be achieved using the immobilized enzyme as compared with the soluble enzyme while maintaining 90\% to 95\% conversion of penicillin to 6-APA.
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