参环毛蚓肠道内源性纤维素酶的分离纯化及酶学性质分析

通过对参环毛蚓肠道组织提取液进行DEAE阴离子交换及凝胶过滤层析,获得参环毛蚓单一的内源纤维素酶的活性组分.根据AlpHaEaseFCTM软件计算出该组分分子质量约为45 ku,命名为Cx1.羧甲基纤维素钠(so-dium carboxymethylcellulose,CMC)法对Cx1进行酶学性质分析.分析结果显示:对于底物羧甲基纤维素钠,Cx1的米氏常数(Km)为0.289 mg/mL,Vmax为0.446 U.mL-1.min-1,反应最适温度为55℃,最适pH为6.0,在40~60℃、pH5.0~8.0具有较好的热稳定性和pH稳定性,其相对酶活力都能保持在55%以上.Ag+和Ba2+对Cx1起显著激活作用,K+、Zn2+、Mg2+、Ca2+、NH4+、Ni2+、Na+、Pb2+部分抑制酶活性,Cu2+离子对Cx1有完全抑制作用,而Fe2+对酶活力无明显影响.

Seperation, purification and Enzymatic properties of endogenous cellulase from the gut of Pheretima aspergillum

The endogenous cellulase from the gut of Pheretima aspergillum by homogenization was separated by DEAE anion exchanger and gel filtration chromatography, and a single band with cellulase activity was identified by SDS-PAGE and zymography. Its molecular mass was about 45 ku as calculated by AlpHaEaseFC~(TM) software and this cellulase was named Cx1. The results of its enzymatic properties using sodium carboxymethylcellulose (CMC) as the substrate showed that its K_m was 0.289 mg/mL and its V_(max) was 0.446 U·mL~(-1)·min~(-1). Its optimum reaction temperature and pH value were 55 ℃ and pH 6.0, respectively. The enzyme was stable over a broad temperature (40-60 ℃) and pH (5.0-8.0) range. Under these conditions, the levels of enzymatic activity could be retained above 55%. Moreover, it was found that the enzyme was dramatically activated by Ag~+ and Ba~(2+), completely inactivated by Cu~(2+) and partially by many other metal ions, such as K~+, Zn~(2+), Mg~(2+), Ca~(2+), NH~+_4, Ni~(2+), Na~+ and Pb~(2+), while Fe~(2+) showed no effect on the reaction.