小麦纹枯病菌胞外蛋白酶的纯化与特性

Purification and characterization of an extracellular protease from Rhizoctonia cerealis

小麦纹枯病菌(Rhizoctonia cerealisVanderHoeven)在以小麦细胞壁为唯一碳氮源的液体培养基中可产生多种蛋白酶,培养液经硫酸铵沉淀,SP-Sepharose Fast F low离子交换层析,Pheny l(high-sub)-Sepharose Fast F low疏水层析,SephadexG-75分子筛层析,分离纯化到分子量约40 kD的一种蛋白酶。该酶能水解胰蛋白酶专一性底物Benz-Phe-Val-A rg-NA,对凝乳弹性蛋白酶底物Suc-A la-A la-Pro-Phe-NA和枯草杆菌蛋白酶底物CBZ-G ly-G ly-Leu-NA无活性,能被胰蛋白酶抑制剂aprotinin、leupeptin和soybean trypsin inhib itor强烈抑制,丝氨酸蛋白酶抑制剂PM SF能部分抑制酶的活性,表明该蛋白酶是丝氨酸蛋白酶中的一种类胰蛋白酶。

Rhizoctonia cerealis could secrete several extracellular proteases when grown in liquid medium with wheat cell walls as the sole carbon and nitrogen source. A kind of protease was purified after ammonium sulfate precipitation, SP-Sepharose Fast Flow chromatography, Phenyl （high-sub）-Sepharose Fast Flow chromatography and Sephadex G-75 chromatography. It appeared as a single band corresponding to molecular weight（MW） of approximately 40 kD on SDS-PAGE with silver staining. It showed high activities against the trypsin substrate Benz-Phe-Val-Arg-NA, and also the substrates D-Val-Leu-Arg-NA, Benz-Pro-Phe-Arg-NA and D-Val-Phe-Lys-NA. It was strongly inhibited by aprotinin, leupeptin, soybean trypsin inhibitor and partly inhibited by PMSF, indicating that it is quite probably a trypsin-like protease.