绿僵菌分解昆虫外壳蛋白酶MAP-21的纯化与特性

PURIFICATION AND CHARACTERIZATION OF CUTICLE-DEGRADING PROTEASE FROM ENTOMOPATHOGENIC FUNGUS, METARHIZIUM ANISOPLIAE

以蝉蜕为底物诱导绿僵菌产生分解昆虫外壳蛋白酶。发酵液经超滤、UltrogelAcA54凝胶层析、制备IEF电泳 ,纯化了一种蛋白酶MAP - 2 1 ,SDS -PAGE电泳后经银染色呈单带。该酶的Mr为 2 7kD左右 ,pI为 7 6。它的特异识别氨基酸为Arg,其活性可被PMSF和TLCK抑制 ,表明其活性中心有Ser和His残基。它还可被胰蛋白酶的典型抑制剂Leu peptin、Antipain及STI等所抑制 ,而胰凝乳蛋白酶抑制剂TPCK和胰凝乳弹性蛋白酶抑制剂TEI对其活性无影响。专一底物和抑制剂特性试验结果表明MAP - 2 1是类胰蛋白酶。此外 ,该酶还可被EDTA所抑制 ,表明金属离子为其活性所必需。另外还研究了MAP - 2 1的最适作用温度和 pH ,以及温度耐受性等特性。

The cuticle degrading proteases from entomopathogensis fungus, Melarhizium anisopliae ,were induced by adding cicada exuviae, colloidal chitin, shrimp cuticle, maggot cuticle, horsefly cuticle and silkworm chrysalis cuticle into minimal medium. After ultrafiltration, Ultrogel AcA 54 column and IEF, a protease designated as MAP 21 with Mr 27 kD, and pI 7.6 were purified. It was shown that the recognition site of MAP 21 was Arg, PMSF and TLCK could inhibited the activity of this protease, indicating that there were Ser and His residues in the active center. The inhibitors to trypsin, leupeptin antipain and STI also repressed the activity of MAP 21, while chymostatin, TPCK and elastatinal TEI were shown no inhibition to its activity, demonstrating that, MAP 21 was a trypsin like protease. Other properties of MAP 21 were also reported.