Purification, Characterization of Pullulanase Produced by Aerornonas caviae No. S-76 and Synthesis of Maltosyl-$\beta$-Cyclodextrin

Aeromonas caviae No. S-76이 생산하는 Pullulanase의 정제, 특성 및 Maltosyl-$\beta$-Cyclodextrin의 합성

The crude enzyme solution obtained by shaking culture of Aeromonas caviae No. S-76 isolated from soil as pullulanase producing bacterium was purified by 50 folds with 21% yield by salting out with ammonium sulfate and column chromatography using DEAE-Sephadex A-50 and Sephadex G-150. The purified pullulanase had a molecular weight of 118, 000 approximately by SDS-polyacrylamide slab gel electrophoresis and pI of 4.3 by isoelectric focusing. And optimum reaction temperature and pH for puHulanase were $50^{\circ}C$ and 8.0, respectively. The purified enzyme was relatively stable at pH 6.0~9.0 and below $45^{\circ}C$. This enzyme synthesized maltosyl-$\beta$-cyclodextrin from mixture of $\beta$-cyclodextrin and maltose.

Pullulanase 생산균으로서 토양으로부터 분리한 Aeromonas caviae No.S-76을 진탕배양하여 얻은 조효소액을 ammonium sulfate 침전, DAEA Sephadex A-50 column chromatoraphy, Sephadex G-150 column chromatography에 의하여 정제하였다. 이때 수율은 21이었고 50배의 정제도를 가진 효소단백질을 얻었다. 정제효소는 SDS-polyacrylamide slab gel 정기영동에 의하여 분자량 118,000의 단일단백질이었고, 등전점은 4.3, 작용 최적온도는 $50^{\circ}C$, 작용 최적 pH는 8.0이었다. 또한 이효소는 $45^{\circ}C$ 이하, pH 6.0-90. 범위에서 안정성을 나타내었다. 이 효소를 $\beta$-cyclodextrin과 maltose의 고농도 혼합액에서 작용시켜 maltosyl-$\beta$-cyclodextrin을 합성하였다.