枯草芽孢杆菌B034拮抗蛋白的分离纯化及特性分析

PURIFICATION AND PARTIAL CHARACTERIZATION OF ANTAGONISTIC PROTEINS FROM BACILLUS SUBTILIS B034 *

枯草芽孢杆菌（Ｂａｃｉｌｕｓｓｕｂｔｉｌｉｓ）Ｂ０３４分离自水稻叶面，对水稻白叶枯病菌具有较强的拮抗能力。除去菌体培养液以７０％饱和度硫酸铵沉淀所得的拮抗物粗提液对热稳定，对胰蛋白酶不敏感，对蛋白酶Ｋ、链霉蛋白酶Ｅ部分敏感，对氯仿部分敏感，其作用的活性ｐＨ范围低至４，高至１２以上，比较耐碱性。粗提液经ＰｈｅｎｙｌＳｅｐｈａｒｏｓｅＣＬ４Ｂ柱层析、ＤＥＡＥＳｅｐｈａｃｅｌ柱层析和ＨＰＬＣ的Ｓｕｐｅｒｄｅｘ７５ＨＲ１０／３０柱层析，得到二个拮抗活性峰：Ｐ１和Ｐ２。Ｐ２经ＳＤＳＰＡＧＥ和ＰＡＧＥＩＥＦ电泳显示为单一蛋白带，分子量５０３ｋＤ，等电点６２５。

Bacillus subtilis B034, an antagonistic bacteri a isolated from the phyllosphere of rice, strongly inhibits the growth of Xanthomonas oryzae pv. oryzae which causes the rice bacterial blight. Crude extract was obtained by precipitation of the cell free culture of B034 with 70% saturated (NH 4) 2SO 4. The suspension of the precipitate strongly inhibits Xanthomonas oryzae pv. oryzae , and it is thermostable, resistant to trypsin, partial sensitive to proteinase K, pronase E and chloroform. Its active pH range is wide, from 4.0 to more than 12.0, relative more stable in high pH. Two antagonistic peaks were obtained from the crude extract of B034 after Phenyl Sepharose CL 4B, DEAE Sephacel and FPLC Superdex 75 HR 10/30 column chromatography. One of these two peaks, named P2, was shoed only single band with 50.3kD and pI6.25 in SDS PAGE and PAGE IEF, respectively. The N terminal partial sequence of protein P2 was analyzed.