β-Fructofuranosidase, an hydrolyzing β-1,6-fructosidic linkage, was immobilized covalently on the oxidized microbial cell wall of a Penicillium species (PS-8), which is totally different from the conventional whole cell immobilization.
The microbial cells of the PS-8 strain, which has a fluffy characteristic during cultivation, was treated with sodium metaperiodate to form active aldehyde groups on them. The periodate treated cells were reacted with the β-fructofuranosidase. This mixture of cell and the enzyme was again treated with 0.5% glutaraldehyde for 1 hr and finally spinned with a syringe (diameter 1 mm) to form pellets, and dried at air.
By determining the effects of periodate concentration on the aldehyde formation of microbial cells, the optimal treating concentration of periodate was 1.2 grams per gram of the dry weight and the formation of the aldehyde was 35% as the % dialdehyde units. The reaction temperature did not give any effect on the formation of aldehyde of the microbial cells at this conditions.
The concentrations and treating time of glutaraldehyde on the immobilization of the β-fructofuranosidase on dialdehyde microbial cells were very important factors in the enzyme immobilization. The highest enzyme activity was observed to be immobilized for 30 minute at 0.5% (w/v) concentration of glutaraldehyde.
The final pellet products (1×2mm) of the immobilized β-fructofuranosidase showed good mechanical strength and was not powderized in shape when they were soaked even for long periods of time. The flow rate was also satisfied when the pellets were packed in the column. Overall recovery of β-fructofuranosidase activity of the final immobilized product showed 26% of the total activity applied.
Kinetic parameters of the soluble and the immobilized β-fructofuranosidase were compared. The results showed that the optimal pH of the immobilized β-fructofuranosidase was PH5, which was same as that of the soluble enzyme. The optimal temperature of the immobilized β-fructofuranosidase was 55℃ and that of the soluble enzyme was 65℃. The activation energy was 19 KJ $mol^{-1}$. Thermal stability of the β-fructofuranosidase was increased by this immobilization.
The operational stability of the immobilized β-fructofuranosidase in the column reactor was observed to obey the first order kinetics. By determination of the half life by the decay constant, the half life of the immobilized β-fructofuranosidase was 37 days.
미생물 세포를 효소 고정 지지체로 사용하고 여기에 periodate 와 Glutaraldehyde 를 사용하여 β-fructofuranosidase를 고정화 시키는 새로운 방법이 시도 되었다. Penicillium species(PS-8)으로 명명 된 곰팡이를 배양한뒤, 세포 벽에 존재하는 다당체를 periodate 를 사용하여 활성화 된 알데 히드기를 얻을수 있었다. 이때 건조세포 1 그램당 periodate 1.2 그램이 최적처리 조건임을 알수 있었고 이 최적 처리 조건에서 온도가 알데하이드 형성에 미치는 영향은 거의 없었다. periodate 에 형성된 활성화 알데히드기는 효소의 아미노기와 반응하여 Schiff base 를 만들어 공유적으로 세포지지체에 고정화 되었다.
더 높은 효소의 고정화를 위하여 glutaraldehyde 가 처리되었다. Glutaraldehyde 는 0.5%(w/v) 의 농도와 1 시간의 반응시간에서 최대 효소 고정화율을 나타냈으며 이때 고정화율은 26% 이었다.
최적 반응 조건에 의해 제조된 고정화 효소는 55℃ 에서 최적 온도를 나타내 가용 효소에 비해 10℃ 가 낮아졌으나 열에 대한 안전성은 증가되었다. 최적 pH 는 가용효소와 변화가 없이 pH 5 였고 K'm 는 55 mM 로서 가용효소보다 약간 증가 되었으며 활성화 에너지는 $19KJ mol^{-1}$이었다.
회분식 반응기와 충진식 연속 반응기에서의 몇가지 실험이 고정화 효소를 사용하여 행해졌다. 회분식 반응기 내에서 6 번의 반복된 반응기간 동안 고정화 효소활성에 약간의 감소가 있었으나 좋은 안전성을 보여 주었다. 충직식 연속 반응기로부터는 효소의 활성 감퇴속도가 일차 반응을 따르며, 감퇴 상수는 $7.77×10^{-4} hr^{-1}$ 이고, 활성 반감기는 37일이었다.
