For the purpose of continuous hydrolysis of fats and lipids immobilized Candida rugosa lipase was used in packed bed reactor system. So as to broaden the substrate range up to solid fat and to enhance the productivity, isooctane was selected as a solvent.
By the preliminary repeated batch experiment, DEAE-Sephadex was selected as immobilized matrix (data not shown).
The immobilization efficiency was greater than 90% for the loading capacity up to 20㎎ protein/g-wet gel and the maximum loading could be obtained up to 40㎎ /g-wet gel with the immobilization efficiency of 70%. Apparent Km value and optimum pH was 8% (v/v) olive oil in isooctane and 7. 0, respectively.
In order to pack the gel in a column reactor, AOT was used and because of enzyme desorption by AOT, packing method was selected carefully.
To increase the operational stability buffer, buffer-solvent ratio and the amount of loaded enzyme were varied. As the results of these experiments triethanol amine buffer was better than phosphate buffer, and less buffer flow rate and more enzyme-loading gel increased the operational stability. Under last condition the half life of immobilized enzyme was increased over 25 times.
지방의 연속적 가수분해를 위하여 고정화된 Candida rugosa 리파제를 충진 반응기 내에서 사용하였다. 또한 기질에 대한 선택범위를 넓히고 생산성을 높히기 위하여 이소옥탄을 기질에 대한 용매로 사용하였다. 리파제에 대한 담체로서는 DEAE-Sephadex 를 사용하였다.
고정화효율은 20㎎ 단백질/g-함수 담체 가 될때까지 90% 이상이였으며 70% 고정화효율로서 최고 40㎎ 단백질을 1 g 함수 담체에 붙일수 있었다. 겉보기 ㎞ 값은 이소옥탄에 녹아있는 8% (V/V) 올리브유 였으며 적정 pH 는 7.0 이였다.
반응을 오랫동안 유지하기 위하여 완충액, 완충액과 용매의 비, 그리고 단위 담체에 대한 고정화 단백질의 양 을 변화시켜가면서 실험을 수행하였다. 이들 실험에 대한 결과로서 phosphate 완충액 보다는 triethanolamine 완충액이 그리고 적은 완충액의 흐름이, 또한 단백질이 많이 고정화된 경우가 반응을 오랫동안 유지시켰다. 최종 조건에서 고정화된 리파제의 반감기는 처음보다 25 배 이상 증가하였다.