| 引用本文： |
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杜丽琴,杨键,朱绮霞,杨登峰,韦宇拓,黄日波.酿酒酵母蔗糖酶基因的克隆、异源表达及重组酶学性质研究[J].广西科学,2008,15(2):184-188. [点击复制]
- DU Li-qin,YANG Jian,ZHU Qi-xia,YANG Deng-feng,WEI Yu-tuo,HUANG Ri-bo.Cloning, Expression and Characterization of Gene Encoding Invertase from Saccharomyces cerevisiae[J].Guangxi Sciences,2008,15(2):184-188. [点击复制]
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| 摘要: |
| 以酿酒酵母(Saccharomyces cerevisiae)基因组DNA为模板,通过PCR扩增得到蔗糖酶基因(suc 2),并将其克隆到表达载体pSE380中,将得到的重组质粒pSE-suc 2转化进E.coli BL21中,利用镍金属螯合层析方法分析测定其酶学性质。重组菌株的SDS-PAGE结果显示重组蔗糖酶基因(suc 2)有60kDa目的蛋白出现,纯化的SDS-PAGE分析得到均一的蛋白条带;重组蔗糖酶的Km值为47.73mmol/L,最大反应速率为79.59mg还原糖mg-1蛋白min-1,最适温度为42℃,最适pH值为5.5,Zn2+、Cu2+对重组蔗糖酶酶活有较强的抑制作用,Ba2+、Mg2+、Mn2+对重组蔗糖酶酶活稍有激活作用。 |
| 关键词: 蔗糖酶 克隆 表达 酶学性质 |
| DOI： |
| 投稿时间：2007-11-12 |
| 基金项目:广西科技攻关项目(桂科攻0630003A2);广西科学基金项目(桂科青0640006);广西大学科研基金项目(X061060)资助 |
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| Cloning, Expression and Characterization of Gene Encoding Invertase from Saccharomyces cerevisiae |
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DU Li-qin1, YANG Jian1, ZHU Qi-xia1, YANG Deng-feng2, WEI Yu-tuo1, HUANG Ri-bo2
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| (1.College of Life Science and Technology, Guangxi University, Nanning, Guangxi, 530004, China;2.Guangxi Academy of Sciences, Nanning, Guangxi, 530007, China) |
| Abstract: |
| The gene encoded invertase from Saccharomyces cerevisiae was amplified by PCR, and inserted into the expression vector pSE380, then introduced into E.coli BL21.The recombinant protein was purified by immobilized metal affinity chromatography.It was presented as a single protein band on SDS-PAGE with mol ecular weight of 60kD.The recombinant enzyme followed typical Michaelis-Menten kinetics with an apparent Km of 47.73mmol/L.Its Vmax value was 79.59mg reducing sugar mg-1 protein min-1.The optimum activity of the recombinant invertase was found to be at pH value 5.5 and 42℃.The invertase activity was increased slightly by addition of Ba2+、Mg2+、Mn2+, while inhibited by Zn2+、Cu2+. |
| Key words: invertase cloning expression characterization |
