萝卜CBPs的分离及部分酶学特性研究

A Study on the Purification and Properties of Chitin-binding Proteins from Raphannuns Sativus

通过脱氨再生几丁质凝胶亲和层析和 CM-纤维素离子交换层析从萝卜中分离出一组CBPs,并对 CBP1和 CBP2溶菌酶酶学特性作了研究。活力分析表明 :CBP1和 CBP2均为溶菌酶 /几丁质酶双功能酶 ;CBP3和 CBP4只有几丁质酶活力。 SDS- PAGE纯度分析表明 :CBP1和 CBP2已达到电泳纯 ,相对分子质量分别为 2 690 0和 2 4 80 0 ;CBP3仍具有两个组分 ,CBP4则由于含量过低 ,在凝胶上无蛋白带。 CBP1和 CBP2部分酶学特性分析表明 :CBP1的最适反应条件为 p H 5 .8,5 5℃ ,0 .4g/L溶壁微球菌 ;CBP2的最适反应条件为 p H6.8,45℃ ,0 .4g/L溶壁微球菌 ;CBP1在 p H3.4～ 1 0 .6,0～ 5 5℃较稳定 ,CBP2在 p H2 .0～ 1 0 .6,0～ 60℃较稳定。

A set of chitin-binding proteins are purified from Raphannuns Sativus by the method of affinity chromatography on a crab deaminated regenerated chitin column, and further purified by the method of cation exchange chromatography on CM-cellulose column. Four fractions, namely CBP1, CBP2, CBP3 and CBP4, are obtained. The analysis of activity suggests that both CBP1 and CBP2 are lysozyme/chitinase, and that CBP3 and CBP4 only have chitinase activity. The results of SDS-polyacrylamide gel electrophoresis show that both CBP1 and CBP2 are purified, the relative molecular mass of which are 26 900 and 24 800 respectively. CBP3 has two fractions while CBP4 has no band on SDS-polyacrylamide gel because of its low concentration. Furthermore, the lysozyme activity properties of both CBP1 and CBP2 have been studied. The optimum conduction of CBP1 is pH 5.8, 55 ℃ and 0.4 g/L Micrococcus Lysodeikticus, and that of CBP2 is pH 6.8, 45 ℃ and 0.4 g/L Micrococcus Lysodeikticus. CBP1 is stable in pH 3.4-10.6, 0-55 ℃, and CBP2, in pH 2.0-10.6 and 0-60 ℃.