Abstract
To produce ${\beta}-cyclodextrin({\beta}-CD)$, a cyclodextrin glucanotransferase(CGTase) producing Aspergillus sp. CC-2-1 was isolated from soil. The enzyme was purified and its enzymological characteristics were investigated. It was found that production of CGTase reached to the maximum when the wheat bran medium containing 0.1% albumin, 2% $(NH_4)_2S_2O_8$, 2% soluble starch and 0.2% $KH_2PO_4$ was cultured for 5 days at $37^{\circ}C$. The purity of CGTase was increased by 13.14 folds after DEAE-cellulose ion exchange chromatography and Sephadex G-100, G-150 gel filtration and the specific activity was 172.14 unit/mg. Purified enzyme was confirmed as a single band by the polyacrylamide gel electrophoresis. The molecular weight of CGTase was estimated to be 27,800 by Sephadex G-100 gel filtration and SDS-polyacrylamide gel electrophoresis. The optimum pH and temperature for the CGTase activity were 9.0 and $80^{\circ}C$, respectively. The enzyme was stable in pH $8.0{\sim}11.0$ at $60{\sim}80^{\circ}C$. The activity of purified enzyme was activated by $K^+,\;Cu^{2+}$ and $Zn^{2+}$. The activity of the CGTase was inhibited by the treatment with 2,4-dinitrophenol and iodine. The result suggests that the purified enzyme has phenolic hydroxyl group of tyrosine, histidine imidazole group and terminal amino group at active site. The reaction of this enzyme followed typical Michaelis-Menten kinetics with the $K_m$ value of 18.182 g/L with the $V_{max}$ of 188.68 ${\mu}mole/min$. The activation energy for the CGTase was calculated by Arrhenius equation was 1.548 kcal/mol.
{\beta}-CD를 생산하기 위하여 CGTase를 생성하는 Aspergillus sp. CC-2-1 균주를 토양으로부터 분리하였으며, CGTase생성을 위하여 0.1% albumin, 2% $(NH_4)_2S_2O_8$, 2% soluble starch, 0.2% $KH_2PO_4$를 밀기울 배지에 첨가하여 $37^{\circ}C$에서 5일간 배양 시 최대의 활성을 나타내었다. Sephadex G-100과 G-150을 사용한 gel filtration과 DEAE-cellulose를 이용한 이온 교환크로마토그래피로 13.14배 정제하였으며, specific activity는 172.14 unit/mg이었다. 정제효소는 poly-acrylamide gel 전기영동에 의하여 단일밴드로 확인되었으며, 분자량은 gel filtration과 SDS-polyacryl amide 전기영동으로 측정한 결과 27,800정도로 측정되었다. CGTase의 효소학적 특성은 최적 pH, 최적 온도는 pH 9.0과 $80^{\circ}C$였으며, pH $8.0{\sim}11.0$과 $60{\sim}80^{\circ}C$에서 안정하였다. 금속이온 중 $K^+,\;Cu^{++},\;Zn^{++}$에서 효소활성이 증대하였고, 효소활성 저해제 중 iodine과 DNP에 의해서 저해가 나타나 효소분자 중 tyrosine의 phenolic hydroxyl group과 histidine imidazole group과 말단아미노기가 효소구조에서 활성중심에 존재한다고 판단되었다. 효소의 $K_m$값과 $V_{max}$값은 18.182 g/L, 188.68 ${\mu}mol/min$이며, 활성화 에너지는 1.548 kcal/mol이였다.
(Department of Food Engineering, Sangju National University)