菜豆鲜荚中菜豆凝集素的提取与分离纯化工艺研究

Extraction,Separation and Purification of Phaseolus vulgaris Agglutinin(PHA)from Common Bean Fresh Pods

以BeanSaintespritaoeilrouge菜豆鲜荚为试验材料，优化了菜豆凝集素的提取和分离纯化工艺，研究了提取剂、料液比、pH值和提取时间对菜豆凝集素活性的影响，建立了硫酸铵分级沉淀、DEAE-52阴离子交换层析和Superdex-200凝胶过滤层析的组合方法用于分离纯化菜豆凝集素。研究结果表明：以磷酸盐缓冲溶液为提取剂，料液比1∶25，pH值6～8，浸提时间8h，菜豆植物凝集素的提取效果适宜。菜豆凝集素浸提液经硫酸铵分级沉淀，DEAE-52阴离子交换层析，Superdex-200凝胶过滤层析，冷冻干燥后可获得纯度较高的菜豆凝集素。纯化的样品经非变性聚丙烯酰氨凝胶电泳检测为单一条带，血凝法检测有血凝活性。

In this paper,the processes of extraction,separation and purification of Phaseolus vulgaris agglutinin(PHA)from the common bean "Bean Saint esprit a oeil rouge" fresh pods were optimized.The effects of extracting agent,material-liquid ratio,pH and extracting time on PHA were studied,and the combined method of ammonium sulfate precipitation, DEAE- 52 anion exchange chromatography and Superdex-200 gel filtration chromatography for extracting,isolating and purifing the PHA from fresh pods of common bean has been established. The main results were as follow:1)Theoptimized technical parameters for common bean extraction process were material-liquid ratio 1∶25,pH 6-8,extracting time 8 h,PBS as extracting agent;2)The high purity PHA was obtained with ammonium sulfate precipitation,dialysis,DEAE-52 anion exchange chromatography,concentrated by ultrafiltration and Superdex-200 gel filtration chromatography. The obtained PHA shows one band with electrophoresis test and has blood clotting activity.