Purification and Characteristics of Endo-Polygalacturonase from Korean Tomato

한국산 토마토의 Endo-Polygalacturonase 정제 및 성질

Endo-polygalacturonase was purified from tomato, Lycopersicon esculentum L. The purification procedures included gel filtration on Sephadex G-150 and DEAE-cellulose ion exchange chromatography. Yield of the enzyme purification was 12.74 %. Purified enzyme was confirmed as a active single band by the SDS-polyacrylamide gel electrophoresis. When the purified enzyme was applied to SDS-PAGE, the molecular weight was estimated about 50,000. The optimum pH for the enzyme activity was 5.0 and the range of its stability to the pH was 4.0 to 5.0. The optimum temperature was $50^{\circ}C$, while the enzyme was abruptly inactivated above $50^{\circ}C$. From the result of the study on the effects of metals ion, it was found that $Ag^+$, $Zn^{++}$ and $Mg^{++}$ increased on the enzyme activity. In contrast, $Ba^{++}$, $Hg^{++}$, $Pb^{++}$, $Ca^{++}$, $Mn{++}$, $Cu^{++}$, $Fe^{+++}$, $Na^+$ and $K^+$ decreased it. the reaction catalyzed by this enzyme followed typical Michaelis-Menten kinetics with the Km value of $1.43{\times}10^{-1}\;mol/l$.

한국산 토마토로부터 endo-polygalacturonase를 gel filtration과 이온교환크로마토그라피에 의하여 약 24배 정제할 수 있었고, 최대 효소활성을 위한 PH는 5.0, 최적온도는 $50^{\circ}C$ 였으며 pH안정범위는 $4.0{\sim}5.0$, 열안정성은 $50^{\circ}C$에서 1시간 열처리 하였을 때 약 45 % 실활 되었다. 정제된 이 효소는 SDS-polyacrylamide gel 전기영동에 의하여 단일밴드로 확인되었으며, 그 분자량은 50,000정도였고, Km값은 $1.43{\times}10^{-1}\;mol/l이었다. 금속이온중 $Ag^+$, $Zn^{++}$이온이 효소의 활성을 촉진시켰으며, $Na^+$, $K^+$등의 이온에 의해서는 활성이 저해되었다.