疏绵状嗜热丝孢菌热稳定几丁质酶的纯化及其性质研究  

Purification and properties of a thermostable chitinase from thermophilic fungus Thermomyces lanuginosus

作　　者：郭润芳[1] 李多川[1] 王荣[1] 

机构地区：[1]山东农业大学环境生物系,泰安271018

出　　处：《微生物学报》2005年第2期270-274,共5页Acta Microbiologica Sinica

基　　金：国家自然科学基金 (3 0 2 70 0 13 ;3 0 170 0 13 );国家"863计划"(2 0 0 3AA2 41162 )~~

摘　　要：采用硫酸铵沉淀、DEAE SepharoseFastFlow阴离子层析、Phenyl Sepharose疏水层析等步骤获得了凝胶电泳均一的疏绵状嗜热丝孢菌 (Thermomyceslanuginosus)几丁质酶。经SDS PAGE和凝胶过滤层析测得纯酶蛋白的分子量在 4 8～ 4 9 .8kD之间。该酶反应的最适温度和最适pH分别为 5 5℃和 4 5 ,在pH4 5条件下 ,该酶在 5 0℃以下稳定 ;6 5℃的半衰期为 2 5min ;70℃保温 2 0min后 ,仍保留 2 4 %的酶活性。其N 端氨基酸序列为AQGYLSVQYFVNWAI。金属离子对几丁质酶的活性影响较大 ,Ca2 + 、Na+ 、K+ 、Ba2 + 对酶有激活作用 ;Ag+ 、Fe2 + 、Cu2 + 、Hg2 + 对酶有显著的抑制作用 ;以胶体几丁质为底物的Km 和Vmax值分别为 9 .5 6mg mL和 2 2 . 12 μmol min。抗菌活性显示 ,该酶对供试病原菌有不同程度的抑制作用。A thermostable extracellular chitinase from culture supernatant of a thermophilic fungus Thermomyces lanuginosus was purified to SDS-PAGE homogeneity, by using ammonium sulfate fraction, DEAE-Sepharose Fast flow chromatography, Pheny1-Sepharose Fast Flow chromatography. A molecular mass of the purified enzyme was between 48～49.8kD determined by SDS-PAGE and gel filtration chromatography. The chitinase exhibited optimum catalytic activity at pH 4.5 and 55℃ respectively. It was thermostable at 50℃ and retained 24 activity after 20min at 70℃. The half life time of the enzyme at 65℃ was 25min. Different metal ions showed different effects on the chitinase activity. Ca 2+,Ba 2+,Na+,K+ enhanced the enzyme activity , whereas Fe 2+,Ag+, Hg 2+,Cu 2+ caused obvious inhibition. The K m and V max values of chitinase on colloidal chitin were 9.56mg/mL and 22.12μmol/min respectively. The chitinase showed antifungal activity aginst tested fungi to different degree.

关 键 词：嗜热真菌 THERMOMYCES LANUGINOSUS 几丁质酶 纯化 性质 

分 类 号：Q946[生物学—植物学]