曲霉外切菊粉酶基因在大肠杆菌中的表达及其特性。
Expression of an exoinulinase gene from Aspergillus ficuum in Escherichia coli and its characterization.
机构信息
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, PR China.
出版信息
Carbohydr Polym. 2013 Feb 15;92(2):1984-90. doi: 10.1016/j.carbpol.2012.11.087. Epub 2012 Dec 3.
An exoinulinase gene from Aspergillus ficuum JNSP5-06 was overexpressed in Escherichia coli. Two exons of the exoinulinase gene were amplified separately, joined together by an overlap PCR, and expressed in E. coli. The molecular weight of the recombinant exoinulinase was estimated to be 63 kDa. The K(m) and V(max) values for inulin were (7.1±0.2) mM and (1000.0±0.1) μmol/(min mg protein), respectively. The K(m) and V(max) values for sucrose were (347.6±25.9)mM and (12,037.0±801.9) μmol/(min mg protein), respectively. The optimum temperature and pH with inulin as the substrate were 60°C and 4.0, respectively. The optimum temperature and pH with sucrose as the substrate were 55 °C and 5.0, respectively. Its activity was increased by Mn(2+), completely inhibited by Cu(2+), and strongly inhibited by Al(3+), Ag(+), Fe(3+), Fe(2+), Ni(2+), Zn(2+), and Mg(2+). The product of hydrolysis of inulin by the recombinant exoinulinase was fructose.
从aspergillus ficuum JNSP5-06 中过表达了一种外切菊粉酶基因。分别扩增外切菊粉酶基因的两个外显子，通过重叠 PCR 将它们连接在一起，并在大肠杆菌中表达。重组外切菊粉酶的分子量估计为 63 kDa。菊粉的 K(m)和 V(max)值分别为(7.1±0.2)mM 和(1000.0±0.1)μmol/(min mg protein)。蔗糖的 K(m)和 V(max)值分别为(347.6±25.9)mM 和(12037.0±801.9)μmol/(min mg protein)。以菊粉为底物时，最适温度和 pH 值分别为 60°C 和 4.0。以蔗糖为底物时，最适温度和 pH 值分别为 55°C 和 5.0。Mn(2+)能提高其活性，Cu(2+)能完全抑制其活性，Al(3+)、Ag(+)、Fe(3+)、Fe(2+)、Ni(2+)、Zn(2+)和 Mg(2+)能强烈抑制其活性。重组外切菊粉酶水解菊粉的产物是果糖。
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