来源于嗜热古细菌Pyrococcus furiosus乳糖酶基因的原核表达及酶学性质分析

›› 2008, Vol. 10 ›› Issue (2): 76-81.

来源于嗜热古细菌Pyrococcus furiosus乳糖酶基因的原核表达及酶学性质分析

段文娟[1] 杨娇艳[1,2] 李卓夫[1,3] 张伟[1] 张志芳[1]

[1]中国农业科学院生物技术研究所,北京100081 [2]华中师范大学生命科学学院,武汉430079 [3]长春理工大学生命科学技术学院,长春130022

收稿日期:2008-01-28 修回日期:2008-03-12 出版日期:2008-04-15 发布日期:2009-07-29
通讯作者: 张伟，副研究员，博士，从事微生物工程研究。Tel：010-62136470：E-mail：zwcafls@hotmail．com
基金资助:
国家“863”计划（2006AA020201）和中央级公益性科研院所基本科研业务费专项资金资助.段文娟,硕士研究生,主要从事微生物分子生物学研究.

Expression of β-Galactosidase from Pyrococcus furiosus |in E. coli and Analysis of Lactase Properties

DUAN Wen-juan, YANG Jiao-yan, LI Zhuo-fu, ZHANG Wei| ZHANG Zhi-fang

1. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081 ; |2. College of Life Sciences, Huazhong Normal University, Wuhan 430079 |3. College of Life Sciences Technology, Changchun University
of Science and Technology, Changchun 130022, China

Received:2008-01-28 Revised:2008-03-12 Online:2008-04-15 Published:2009-07-29

摘要/Abstract

摘要：

以嗜热古细菌Pyrococcus furiosus的基因组DNA为模板，通过PCR克隆获得乳糖酶基因celB。将celB基因插入到表达载体pET．30a（＋）上构建原核重组表达质粒pET-celB，转化大肠杆菌B121，阳性转化子在28％下经IPTG诱导4h后进行SDS．PAGE电泳和酶活性测定，结果表明celB基因在大肠杆菌中获得高效表达，乳“糖酶基因celB表达的乳糖酶蛋白CELB分子质量约为58kDa。CELB是耐高温酶，其酶促反应最适温度为105％，在95％至110％之间热稳定性较好；pH5．0时该乳糖酶水解活力最高，在pH5．0～10．0之间，pH稳定性较好，该酶对金属离子依赖性不强。

关键词: 乳糖酶嗜热古细菌酶学性质原核表达

Abstract:

The lactase gene from Pyrococcusfuriosus was studied in this experiment. The cloned gene was expressed in Escherichia coli and the properties of lactase were determinated, celB gene was amplified by PCR and subsequently cloned, then ligated to the pET-30a（＋） vector. The recombinant plasmid pET-celBB was transformed into E. coli BL21. The positive transformants were induced at 28~C for four hours by IPTG . SDS-PAGE analysis and lactase activity assay showed that celB gene was over-expressed in E. coll. The molecular weight of the recombinant expressed lactase CELB was about 58 kDa. CELB is a thermostable lactase. The optimum temperature was 105% and better heat stability was achieved between 90℃ to 110℃. The optimum pH value was 5.0 and better pH stability was gained from pH 5.0 to 10.0. It is concluded that the lactase has a very weak dependence on metal ions.

Key words: lactase , Pyrococcus furiosus , lactase properties , prokaryotic expression

中图分类号:

Q786

段文娟,杨娇艳,李卓夫,张伟,张志芳. 来源于嗜热古细菌Pyrococcus furiosus乳糖酶基因的原核表达及酶学性质分析[J]. , 2008, 10(2): 76-81.

DUAN Wen-juan, YANG Jiao-yan, LI Zhuo-fu, ZHANG Wei| ZHANG Zhi-fang . Expression of β-Galactosidase from Pyrococcus furiosus |in E. coli and Analysis of Lactase Properties[J]. , 2008, 10(2): 76-81.

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