SDS-Chitin-PAGE电泳法分析大葱叶几丁质酶特性

Characterization of chitinase isoenzymes in Allium Fistulosum L leaves by Sodium dodecyl sulfate-Chitin-Polyacrylamide gel electrophoresis

摘要: 使用Sodium dodecyl sulfate-几丁质-聚丙烯胺酰胺凝胶电泳（SDS-Chitin-PAGE）方法分析了大葱葱叶几丁质酶的特性。在大葱叶组织中共检测到三种几丁质酶同工酶,分别命名为CH-A、CH-B、CH-C。分析结果表明,这三种几丁质酶具有SDS抗性,β-巯基乙醇对其活性也无显著影响。在SDS-Chitin-PAGE前,样品缓冲溶液与样品蛋白粗提液孵化处理的最适温度为24℃、时间为10～20min。凝胶电泳酶活分析表明,大葱葱叶中CH-A、CH-B的耐热温度可达60℃,CH-C的耐热温度达到70℃。葱叶中CH-A的最适pH为5.2～5.6,CH-B的最适pH为4.0～4.4,CH-C的最适pH为6.6～8.0。

Abstract: Allium Fistulosum L leaves was representatively selected to test their chitinase characteristics by the optimized SDS-Chitin-PAGE.The results showed that three chitinase isozymes, CH-A, CH-B and CH-C, were detected in Allium Fistulosum L leaves.Allium Fistulosum L had the very special characteristic of SDS resistance.β-mercaptoethanol had no significant effect on the chitinase activity.The optimal incubating temperature and time of the crude enzymes with the sample buffer were 24℃, 10～ 20min before the electrophoresis.The activity of CH-A and CH-B could be observed up to 60℃, the activity of CH-C could be observed up to 70℃.The optimal pH ranges for CH-A, CH-B and CH-C were pH 5.2～ 5.6, pH 4.0～ 4.4, and pH 6.6～ 8.0, respectively.