嗜热毛壳菌内切β-葡聚糖酶的分离纯化及特性

Purification and Properties of An Endocellulase from The Thermophilic Fungus Chaetomium thermophile

探讨了液体发酵嗜热毛壳菌 (Chaetomiumthermophile)产生的内切 β 葡聚糖酶的分离纯化及特性。粗酶液经硫酸铵分级沉淀、DEAE SepharoseFastFlow阴离子层析、Pheny1 Sepha rose疏水层析、Sephacry1S 1 0 0分子筛层析等步骤便可获得凝胶电泳均一的内切 β 葡聚糖酶。经 1 2 5 %SDS PAGE和凝胶过滤层析法分别测得所分离纯化酶蛋白的分子量约为 6 7 8kD和6 9 8kD。该酶反应的最适温度和pH分别为 6 0℃和 4 0～ 4 5在pH5 0条件下 ,该酶在 6 0℃下稳定 ;70℃保温 1h后 ,仍保留 3 0 %的活性 ;在 80℃的半衰期为 2 5min。金属离子对内切β 葡聚糖酶的活性影响较大 ,其中Na+ 对酶有激活作用 ;Fe2 + 、Ag+ 、Cu2 + 、Ba2 + 、Zn2 + 等对酶有抑制作用。该酶对结晶纤维素没有水解能力。

An endocellulase from culture supernatant of a thermophilic fungus Chaetomium thermophile was purifided to homogeneity, by using ammonium sulfate fraction, DEAE\|Sepharose Fast\|flow chromatography, Pheny1\|Sepharose Fast Flow chromatography and Sephacry1 S\|100 chromatography. The enzyme was a glycoprotein with an apparent molecular weight of 67 800 and 69 800, as determinded by 12 5% SDS\|PAGE and gel filtration respectively. The endocellulase was optimally active at pH 4 0～4 5 and 60℃. It was thermostable at 60℃ and retained 30% activity after 60min at 70℃. The half life time of the enzyme at 80℃ was 25min. Different metal ions showed different effects on the endocellulase activity. Na + enhanced the enzyme activity, whereas Fe 2+ , Ag +, Cu 2+ , Ba 2+ and Zn 2+ cause obvious inhibition. But it didn't work on crystalline cellulose.