Abstract
The conditions for immobilization of the partially purified ${\beta}-galactosidase$ form Bacillus subtilis HP4 and the properties of the immobilized enzyme have been investigated. The crude enzyme precipitated with cold acetone was purified about 68-fold through DEAE-cellulose and sephadex G-100 chromatography and its recovery was 19.9% The optimal conditions for Immobilization of enzyme were obtained in 2%(w/v) sodium alginate, 15%(v/v) enzyme solution and 2%(w/v) calcium chloride, and also the optimal stirring thme was 2 hours on the above conditions. The optimum temperature and pH values for immobilized enzyme were $55^{\circ}C$ and 6.5, respectively. Its residual activity was show 25% after heat treatment for an hour at $65^{\circ}C$, and found its high stability in pH 6.0 to 8.0. The enzyme activity was not affected b)· EDTA, 2-mercaptoethanol, KCN, protective agents, and other methal ions except Hg ion and Cu ion. The $K_m\;and\;V_{max}$ values of the immobilized enzyme on ONPG were $1.82{\times}10^{-2}M\;and\;3.57{\times}10^{-8}mole/min$, whereas those on lactose were $2.94{\times}10^{-2}M\;and\;1.68{\times}10^{-7} mole/min$, respectively. The remained enzyme activity for the immobilized enzyme was 95%t of original activity after storage of 40 days at $4^{\circ}C$, and when reused for 5 times was 81%. When skim milk(4.8% lactose) and 5% lactose solution were reacted with the immobilized enzyme(250 units/g) of lactose were 51% and 43%, respectively.
Bacillus subtilis HP-4의 ${\beta}-galactosidase$를 추출, 정제하여 효소의 고정화조건 및 고정화 효소의 특성을 조사하였다. B. subtilis를 배양하여 얻은 조효소액을 acetone분획 DEAE-cellulose에 의한 ion exchange chromatography, Sephadex G-100에 의한 gel filtration과정을 통하여 정제하였던 바 약 68% 정제되었으며, 이때의 회수율은 19.9%이었다. 본 효소의 최적 고정화조건은 2%(w/v)의 sodium alginate농도, 15%(v/v)의 효소농도, 그리고 2%,(w/v)의 $CaCl_2$ 농도에서 2시간 교반하였을 때 이었다. 고정화 효소의 최적온도와 pH값은 각각 $55^{\circ}C$와 6.5이었다. 고정화 효소의 활성은 Hg이온과 Cu이온에 의하여 저해되었으며, EDTA, 2-mercaptoethanol, KCN 등의 금속이온 그리고 보호제에 의한 영향은 없었다. 고정화 효소의 ONPG와 유당에 대한 $K_m$값과 $V_{max}$값은 각각 $1.82{\times}10^{-2}M$과 $2.94{\times}10^{-2}M$ 그리고 $3.57{\times}10^{-8}mole/min$과 $1.68{\times}10^{-7}mole/min$이었다. 고정화 효소를 $4^{\circ}C$에서 40일간 저장 후 잔존활성과 5회 재사용 후 잔존활성을 조사한 결과는 각각 95%와 81%이었으며, 탈지유(4.8% 유당)와 5% 유당용액에서 $50^{\circ}C$, 9시간 반응시켰을 때 유당분해율은 각각 51%와 43%이었다.