| 巴斯德梭菌甘油脱水酶基因的克隆表达、纯化及酶学性质的研究 |
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| 引用本文： | 唐悦,孟晓蕾,齐向辉,韦宇拓,黄日波.巴斯德梭菌甘油脱水酶基因的克隆表达、纯化及酶学性质的研究[J].精细化工,2006,23(11):1056-1059. |
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| 作者姓名： | 唐悦 孟晓蕾 齐向辉 韦宇拓 黄日波 |
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| 作者单位： | 1. 广西大学,生命科学与技术学院,广西,南宁,530005
2. 广西大学,生命科学与技术学院,广西,南宁,530005;南宁中诺生物工程公司,广西,南宁,530005 |
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| 基金项目： | 国家高技术研究发展计划(863计划) |
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| 摘 要： | 用聚合酶链反应(PCR)技术,从巴斯德梭菌(C lostridium pasteurianum)扩增出甘油脱水酶基因(dhaBCE),在大肠杆菌中高效表达,SDS-PAGE(聚丙烯酰胺凝胶)电泳结果显示,分别有相对分子质量为66、21、16 kD三条特异性蛋白表达条带。用金属镍亲和层析及S-300H凝胶层析将重组蛋白进行分离纯化,所得纯酶的比活为4.26 U/mg。重组酶的最适反应温度42~44℃,最适作用pH=9.10~9.50,它对三个底物结构类似物的米氏常数(Km)值分别是:1,2-丙二醇0.38 mmol/L,甘油0.29 mmol/L,1,2-乙二醇2.0 mmol/L;对辅酶B12的Km值为0.22μmol/L。
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| 关 键 词： | 甘油脱水酶 基因表达 重组酶纯化 酶学性质 |
| 文章编号： | 1003-5214（2006）11-1056-04 |
| 收稿时间： | 2006-04-04 |
| 修稿时间： | 2006-04-042006-05-22 |
Expression, Purification and Characterization of Glycerol Dehydratase from Clostridium Pasteurianum |
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| TANG Yue,MENG Xiao-lei,QI Xiang-hui,WEI Yu-tuo,HUANG Ri-bo.Expression, Purification and Characterization of Glycerol Dehydratase from Clostridium Pasteurianum[J].Fine Chemicals,2006,23(11):1056-1059. |
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| Authors: | TANG Yue MENG Xiao-lei QI Xiang-hui WEI Yu-tuo HUANG Ri-bo |
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| Abstract: | The gene encoding glycerol dehydratase from Clostridium pasteurianum was amplified by PCR and expressed in Rosetta(DE3).The recombinant protein was purified by immobilized metal affinity chromatography and gel filtration chromatography.The purified enzyme presented as three protein bands on SDS-PAGE with molecular weight of 66,21 and 16 kD.The specific activity of glycerol dehydratase was 4.26 U/mg.The optimum activity of glycerol dehydratase was at pH 9.10～9.50 and 42～44 ℃.Km for the three main substrates were 0.29 mmol/L for glycerol,0.38 mmol/L for 1,2-propanediol and 2.0 mmol/L for 1,2-ethanediol.Km for adenosylcobalamin coenzyme was 0.22 μmol /L. |
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| Keywords: | glycerol dehydratase gene expression purification characterization |
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