嗜热真菌Thermomyces lanuginosus A_(236)热稳定葡萄糖淀粉酶的纯化及其特性

嗜热真菌ThermomyceslanuginosusA_236在液体培养基中50℃下静止培养14天，粗提酶液经硫酸铵分级沉淀、DEAE-Toyopearl离子交换层析、Butyl－Toyopearl疏水层析、SephacrylS100凝胶过滤和FPLCMonoQ离子交换层析，得到了凝胶电泳均质的葡萄糖淀粉酶。酶促反应产物经TLC分析为葡萄糖，证明纯化的酶为葡萄糖淀粉酶（EC3．2．1．3）。SDS－PAGE测定其分子量为72，000，不具亚基，PI为4．0，富含Val和Leu。酶反应最适温度和pH分别为70℃和5．0。在pH5．0条件下，酶在60℃保温1h，仍具有原酶活性。酶活性在70℃和80℃的半衰期分别为20min和6min。Ca2+对酶有激活作用，Fe3+、Al3+、Hg2+等金属离子对酶活力有一定的抑制作用。纯酶碳水化合物含量为12．4％。纯酶可水解可溶性淀粉、直链淀粉、支链淀粉、糊精、糖原、麦芽三糖和麦芽糖，其中可溶性淀粉为最适底物。

PURIFICATION AND CHARACTERIZATION OF A THERMOSTABLE GLUCOAMYLASE FROM THE THERMOPHILIC FUNGUS THERMOMYCES LANUGINOSUS A_(236)

A thermostable extracellular glucoamylase from the thermophilic fungus Thermomyces lanuginosus A 236 in stahc culture was purified to SDS-PAGE homogenous by ammonium sulfate precipitation, DEAE-Toyopearl chramatography,Sephacryl S100 gel filtration and FPLC Mono Q rechramatography. The molecular weight of the enzyme consisting of a single polypeptide, determined with SDS-polyacrylamide gel electeophoresis was 72 000. The purified glucoamylase was a glycoprotein with the saccharide content of 12.4%. The purified glucoamylase showed maximal activity at pH5.0 and 70℃, and was thermostable at 50℃ and 60℃. The half lifes of the enzyme were 20min at 70℃ and 6min at 80℃. IEF of the enzyme was pH4.0 The enzyme hydrolyzed soluble starch, amylose, amylopectin, dextrin,glycogen, maltotrise, and maltose, The addition of Ca2+ had a stabilizing effect on the glucoamylase, whereas Fe3+, Al3+, Hg2+ ichbited activity of the enzyme.