| 乳酸克鲁维酵母乳糖酶基因在大肠杆菌中的表达及酶学性质 |
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| 引用本文： | 徐顺清,陈杏洲,崔罗生,梁运祥,张忠明.乳酸克鲁维酵母乳糖酶基因在大肠杆菌中的表达及酶学性质[J].华中农业大学学报,2010,29(2):175-180. |
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| 作者姓名： | 徐顺清 陈杏洲 崔罗生 梁运祥 张忠明 |
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| 作者单位： | 华中农业大学生农业微生物学国家重点实验室,武汉,430070 |
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| 基金项目： | 浙江省重大科技攻关项目(2004C12010)资助 |
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| 摘 要： | 通过PCR扩增从乳酸克鲁维酵母基因组获得乳糖酶基因lac4,将其克隆至大肠杆菌表达载体pET-28a(+),并在大肠杆菌BL21中获得表达,酶活性达(44.78±2.84)U/mL。利用Ni-ResinHP亲和层析技术纯化该酶,SDS-PAGE分析表明,重组酶分子质量约为122ku。酶学性质研究表明,该酶最适反应温度为43℃,37℃时半衰期为30min,最适反应pH为7.0,在pH4.0～10.0范围内有良好的稳定性,Ca2+、Zn2+、Fe2+、Co2+、Mg2+、Mn2+对酶有非常明显的激活作用,比酶活性为(255.55±2.32)U/mg,酶反应常数Km为3.49mmol/L,最大反应速率vmax为4.22mmol/(min.mg)。
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| 关 键 词： | 乳酸克鲁维酵母 乳糖酶 基因表达 酶学性质 |
| 收稿时间： | 2009/4/15 0:00:00 |
| 修稿时间： | 2009/6/28 0:00:00 |
Expression and Enzymatic Properties of Lactase Gene from Kluyveromyces lactis in Escherichia coli |
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| XU Shun-qing,CHEN Xing-zhou,CUI Luo-sheng,LIANG Yun-xiang and ZHANG Zhong-ming.Expression and Enzymatic Properties of Lactase Gene from Kluyveromyces lactis in Escherichia coli[J].Journal of Huazhong Agricultural University,2010,29(2):175-180. |
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| Authors: | XU Shun-qing CHEN Xing-zhou CUI Luo-sheng LIANG Yun-xiang and ZHANG Zhong-ming |
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| Affiliation: | State Key Laboratory of Agricultural Microbiology,Huazhong Agricultural University,Wuhan 430070,China;State Key Laboratory of Agricultural Microbiology,Huazhong Agricultural University,Wuhan 430070,China;State Key Laboratory of Agricultural Microbiology,Huazhong Agricultural University,Wuhan 430070,China;State Key Laboratory of Agricultural Microbiology,Huazhong Agricultural University,Wuhan 430070,China;State Key Laboratory of Agricultural Microbiology,Huazhong Agricultural University,Wuhan 430070,China |
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| Abstract: | The lactase gene(lac4) was amplified from the genome of Kluyveromyces lactis through polymerase chain reaction(PCR) and then cloned into Escherichia coli expression vector pET-28a(+),and the lac4 gene was successfully expressed in Escherichia coli 21,enzyme activity reached (44.78±2.84) U/mL.The recombinant lactase was purified with affinity chromatography technology,then analyzed by SDS-PAGE,its molecular weight was approximate 122 ku.The optimal reaction conditions of lactase such as temperate,pH were 43 ℃ and 7.0,its half-life was 30 min at 37℃.It was very stable on the condition of pH 4.0 to10.0,and can be greatly activated by Ca~(2+),Zn~(2+),Fe~(2+),Co~(2+),Mg~(2+),Mn~(2+),its specific activity was (255.55±2.32) U/mg,the response constant Km was 3.49 mmol/L,the maximum reaction velocity v_(max) was 4.22 mmol/(min·mg). |
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| Keywords: | Kluyveromyces lactis lactase gene expression enzymatic properties |
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