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 μmol/(min·mg) respectively.The activity was enhanced by K+,Sn2+ and Mn2+ and was strongly 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 possible essential residue for enzyme activity.