产气肠杆菌几丁质酶的分离纯化及性质研究

PURIFICATION AND PROPERTIES OF CHITINASE FROM ENTEROBACTER AEROGENES

从自然罹病死亡的草原毛虫 (Gynephorapruoergnesis)体内分离到一株产气肠杆菌(Enterobacteraerogenes) ,它在几丁质的诱导下能产生较高活性的几丁质酶。发酵液经硫酸铵盐析、DEAE纤维素柱层析和SephadexG - 1 0 0柱层析分离出几丁质酶。用SDS PAGE测得该酶的分子量为 42 5kD。水解几丁质的Km 值为 2 88mg/mL- 1 。酶反应的最适温度为55℃ ,最适pH值为 6 0 ,金属离子对几丁质酶活性影响较大 ,其中Zn2 +、Ba2 +、Ca2 +和Mn2 +对酶有较强的激活作用 ,而Hg2 +、Co2 +和Mg2 +则有较强的抑制作用。

A bacterium producing chitinase was isolated from the dead body of Gymephorap ruoergensis . A chitinase was isolated from the culture of E.aerogenes and purified by means of ammonium sulfate precipitation, DEAE cellulose column chromatography, and Sephadex G 100 column gel filtration. The purified chitinase showed homogeneity on the native polyacrylamide gel electrophoresis. Its molecular weight was estimated to be about 42.5kD by SDS PAGE. The optimum pH and temperature for hydrolysis of chitin were 6.0 and 55℃ respectively. Michaelis constant was 2.88mg/mL. Different metal ions showed different effects on the chitinase activity, The chitinase activity was enhanced by Zn 2+ ,Ba 2+ ,Ca 2+ ,Mn 2+ and was strongly inhibited by Hg 2+ ,Co 2+ ,Mg 2+ .