黏质沙雷氏菌胞外几丁质酶的纯化及特性

Purification and Characterization of Extracellular Chitinase from Serratia marcescens

黏质沙雷氏菌几丁质酶发酵上清液经硫酸铵沉淀、透析、DEAE-琼脂糖凝胶阴离子交换层析和苯基-琼脂糖凝胶疏水层析,得到电泳纯的几丁质酶和几丁质结合蛋白CBP21。该几丁质酶和CBP21分子质量分别约为58 ku和21 ku,CBP21对该几丁质酶水解几丁质增效明显。几丁质酶反应最适温度为50℃,最适pH约为6.5～7.0。该酶在55℃以下、pH 4.5～8.0范围内稳定。酶的Km值为0.22 mg/mL,Vm为1.26μmol/(min.mg)。金属离子K+、Sn2+、Mn2+对酶有一定激活作用,而Pb2+、Hg2+和Cu2+则强烈抑制其活性。该几丁质酶的糖基含量约为3.3%。EDTA和2-ME可分别提高酶活力65%和105%。H2O2强烈抑制酶活力,提示其活性中心可能存在硫氢基。

An extracellular chitinase and a chitin-binding protein （CBP21） were isolated from the culture of Serratia marcescens and purified to electrophoretic homogeneity by ordinal procedures containing ammonium sulfate precipitation, DEAE-Sepharose and Phenyl-Sepharose chromatography. Their relative molecular masses were estimated to be respectively about 58kD and 21kD by SDS-PAGE. CBP21 had great synergistic effect with the chitinase on chitin hydrolysis. The optimum temperature and pH for the enzyme activity were 50℃ and 6.5 respectively. The enzyme activity was stable under 55℃ and in the pH range of 4.5 - 8.0. Michaelis constants of the enzyme were Km 0.22 mg/ mL and Vm 1.26 ixmol/（min·mg） respectively. The activity was enhanced by K＋, Sn2＋ and Mn2＋ and was strong- ly inhibited by Pb2＋ , Hg2＋ and Cu2＋. EDTA and 2-mercaptoethanol （2-ME） enhanced the activity by 65% and 105% respectively. H2O2 strongly inhibited chitinase activity, which indicated that hydrosulfide group was the possi- ble essential residue for enzyme activity.