番茄离体花柄脱落区多聚半乳糖醛酸酶提取纯化体系的优化

Purification System of A Polygalacturonase from Tomato Pedicel Abscission Zone

多聚半乳糖醛酸酶是植物器官脱落过程中的重要水解酶,实验以20μL.L-1乙烯处理的离体番茄花柄为试材,建立了与脱落相关的多聚半乳糖醛酸酶提取与纯化体系:以50 mmol.L-1乙酸缓冲液(pH=5.5)为提取液,加入0.1 mol.L-1NaCl、1 mmol.L-1DTT提取效果较好;将酶的粗提液低温浓缩后,经Sephadex G-75凝胶层析分离纯化,最佳流速为0.2 mL.min-1,适宜上样量为3.5 mL;再将凝胶层析分离的活性部分低温浓缩后,经CM Sepharose CL-6B离子交换层析再次纯化,流速为0.3 mL.min-1、洗脱液pH值5.5纯化效果最好。经上述提取纯化过程,得到了分子质量为30.2 kD的多聚半乳糖醛酸酶蛋白。该提取纯化体系为探讨与脱落相关酶的性质及其活性调控提供了参考。

Polygalaturonase is an important hydrolase during abscission process. In this experiment,the pedicels explants of tomato in vitro treated with 20 μL ·L^-1 ethylene were used to extract and purify the polygalacturonase which related to abscission. The extraction and purification system was as follows.50 mmol · L^-1 acetate buffer （pH=5.5） was the extract buffer by adding 0. 1 mol · L^-1 NaCl and 1 mmol·L^-1 DTT;the enzyme extracts then concentrated and purified by Sephadex G-75 chromatography gel with the optimum condition of 0.2 mL · min^-1 flow rate and 3.5 mL sample volume;then the part with high polygalacturonase activity was concentrated and CM Sepharose CL-6B ion exchange chromatography was used with 0.3 mL · min^-1 flow rate and elute pH 5.5. The molecular masses of the purified polygalacturonase was 30.2 kD. The experiment provides the reference for the study of character and activity regulation of polygalacturonase.