Abstract
A chitinase-producing bacterium, Serratia marcescens JM, was isolated from a seashore muds. A chitinase was purified by ammonium sulfate precipitation, affinity adsorption, hydroxylapatite and sephadex G-200 column chromatography. The chitinase obtained from Serratia marcescens JM was purified 42.2 folds with the overall yield of 7.1%. The purified chitinase showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular weight of the enzyme was 59,000 and the apparent kinetic parameters $K_m\;and\;V_{max}$ for the purified chitinase were 5.17 mg/mL and 39.8 unit/mL, respectively. The optimum pH and temperature of the purified chitinase were 7.0 and 50$^{\circ}C$, respectively and the purified enzyme was stable on pH 7.0 up to 50$^{\circ}C$. The enzyme were activated by $Cu^{2+},\;Ca^{2+}\;and\;Mg^{2+}$ and inhibited by $Hg^{2+}$ respectively. In addition, Cysteine increased the chitinase activity and EDTA, MIA, PCMB and SDS inhibited enzyme activities. Major cations, $MG^{2+},\;Ca^{2+},\;K^+\;and\; Na^+$ present in seawater slightly stimulated the chitinase activity.
Chitinase를 생성하는 세균인 serratia marcescens JM을 해안 갯벌 시료로부터 분리하여, ammonium sulfate precipitation, affinity adsorption, hydroxylapatite와 Sephadex G-200 column chromatography를 통하여 정제하였다. 정제된 chitinase는 7.1% 회수율과 4.22의 정제도를 나타내었으며, 전기영동시 단일밴드를 얻을 수 있었고, SDS-PAGE에 의해 측정된 분량은 59,000으로 나타났다. 정제된 chitinase의 $K_m$과 $V_{max}$는 5.71mg/mL과 39.8 unit/mL로 나타났다. Chitinase의 최적활성 pH와 온도는 7과 50$^{\circ}C$였고 최적안정pH는 7.0이며 50$^{\circ}C$이하에서는 안정하였다. $Cu^{2+}\;Ca^{2+}$ 및 $Mg^{2+}$는 효소활성을 증가시켰으나 $Hg^{2+}$와 $I_2$는 효소 활성을 억제시켰다. 또한 cysteine은 효소활성을 증가시키나 EDTA, MIA, PCMB, 및 SDS는 효소활성을 억제시켰다. 해수 음이온 중 $MG^{2+},\;Ca^{2+},\;K^+$는 효소활성을 약간 증가시켰으나 $Na^{2+}$ 이온은 1mM이상농도에서 활성이 억제되었다. 본 논문에서 정제된 chitinase는 여러가지 특이점이 있는 serratia효소였다.