Differential column reactor에 있어서 고정화페니실린 아미다제의 반응속도론에 관한 연구

Kinetic Study on the Immobilized Penicillin Amidase in a Differential Column Reactor

E. coil ATCC 9637의 균체를 젤라틴과 DEAE-cellulose의 혼합 성형 후 글루트알데히드 가교법으로 제조론 고정화 penicillin amidase의 differential column reactor에서의 반응속도를 논의하였다. 이러한 반응조의 최적 조작조건은 효소충진량 1g, 기질농도30mM(0.1M 인산완충액, pH8.0), 유출속도 4 $m\ell$/min, 온도 4$0^{\circ}C$이었다. 이 최적조건에서 고정화효소의 일반적인 성질을 조사하였다. Km 상수는 4.8mM 이었고 specific activity 308 units/g 고정화 효소이 었다. 또한 고정화효소에서는 기질에 의한 효소반응 저해효과가 보이지 않았다. 이러한 differential column reactor에서는 column내에서의 pH 감소효과 및 외부 화산효과가 없어지기 때문에 이러한 외부적 영향을 받지 않는 고정화효소의 반응 속도론적 연구에 적합함을 알았다.

The penicillin amidase from Escherichia coli (ATCC 9637) was immobilized by entrappment in gelatin and DEAE-cellulose mixture cross-linked with glutaraldehyde, and the kinetics in a differential column reactor was studied. The optimal operating condition of a differential reactor was reasonably met when the enzyme loading was 1g, and 30 mM substrate solution in 0.1 M phosphate buffer (pH 8.0) was fed at flow rate 4$m\ell$/min and 4$0^{\circ}C$. The optimal pH and temperature were found to be 8.0 and 55$^{\circ}C$, respectively. The Michaelis-Menten constant was 4.8 mM while the maximum velocity was 308 units/g of the immobilized enzyme under the condition of the differential reactor. The effect of substrate inhibition disappeared in the immobilized enzyme preparation. The differential reactor was proved to be good for studying the true kinetics since the pH drop and the external diffusional resistance could be eliminated.