白梨PGIP的提取、纯化及其特征特性研究

Extraction,Purification and Characterization of PGIP from Bai Pear

半乳糖醛酸酶抑制蛋白(Polygalacturonase inhibitoryprotein,PGIP)是位于植物细胞壁的糖蛋白,能够抑制真菌半乳糖醛酸酶(Polygalacturonase,PGs)对细胞壁的降解,从而达到抵御病原菌侵入的目的。经高盐法提取和凝胶过滤层析,获得相对纯度为18.9倍的PGIP,保留活性分别为100%和51.6%;经SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表明,相对纯化的白梨PGIP的分子量为29～42 kDa;用径向辐射法和还原糖法试验表明,PGIP活性受pH影响,但对提取液中KCl的浓度及提取时间不敏感;PGIP抑制活性对温度非常敏感,在85℃处理20 min,PGIP失去85%～90%的抑制活性,在100℃下,PGIP活性全部丧失。

Polygalacturonase inhibitory proteins （PGIP）are glycoproteins located in plant cell walls that specifically inhibit fungal polygalacturonase （PGs）, which could break down cell walls, and make fungi easy to invade into plant cells. The aim of this work is to investigate extraction, purification and its characterization of PGIP from Bai pear （P bretschneideri Rehd）. PGIP from Bai pear fruit tissue was extracted, purified and partially characterized with a high ionic strength extraction buffer and gel filtration chromatography, resulting in a 18.9-fold purification of the PGIP and 51.6% of recovery activity. The following assay by SDS-PAGE eleetrophoresis revealed that the relative purified PGIP has a 29-42 kDa molecular weight. The radial diffusion and reducing sugar assays showed that PGIP inhibition were significantly affected by pH, it was not susceptible to concentration of KCl, nor to extraction time. Heated at 85℃ for 20 min ,the inhibitory activity of PGIP was reduced by 85% - 90%. PGIP activity was completely suppressed at 100℃ for 20 min.