| 苯酚羟化酶基因工程菌表达优化及特性研究 |
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| 引用本文： | 曹湘禹,黄益养,时胜男,马桥,李新亮,孔春雷,沈娥,曲媛媛.苯酚羟化酶基因工程菌表达优化及特性研究[J].环境科学学报,2012,32(7):1549-1556. |
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| 作者姓名： | 曹湘禹 黄益养 时胜男 马桥 李新亮 孔春雷 沈娥 曲媛媛 |
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| 作者单位： | 1. 大连理工大学环境学院工业生态与环境工程教育部重点实验室,大连,116024
2. 哈尔滨工业大学城市水资源与水环境国家重点实验室,哈尔滨,150090 |
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| 基金项目： | 国家自然科学基金(No.51078054, 20923006) |
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| 摘 要： | 利用表面响应法优化苯酚羟化酶基因工程菌(PH_IND)的生长条件(种龄、接种量)及表达条件(诱导OD600、IPTG浓度、诱导时间、诱导温度),并对其粗酶特性进行考察.实验结果表明,菌株PH_IND的最佳生长条件及表达条件为:种龄7.19h,接种量1.39%,诱导OD6000.54,IPTG浓度0.19mmol·L-1,诱导时间2.37h,诱导温度35℃,此时,苯酚羟化酶最大酶活力为0.6167U·mg-1.粗酶特性分析表明,苯酚羟化酶的最适pH和温度分别为8.0和40℃.Ba2+、Fe3+、Hg2+、Pb2+对苯酚羟化酶有明显促进作用,而Ca2+、Co2+、Cu2+、Mg2+、Mn2+对苯酚羟化酶有抑制作用.底物广谱性实验表明,苯酚羟化酶具有极强的芳香化合物降解能力.
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| 关 键 词： | 苯酚 苯酚羟化酶 表面响应法 底物多样性 |
| 收稿时间： | 9/7/2011 12:00:00 AM |
| 修稿时间： | 2011/10/3 0:00:00 |
Optimization of expression conditions and characteristics of gene engineered strain PH_IND |
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| CAO Xiangyu,HUANG Yiyang,SHI Shengnan,MA Qiao,LI Xinliang,KONG Chunlei,SHEN E and QU Yuanyuan.Optimization of expression conditions and characteristics of gene engineered strain PH_IND[J].Acta Scientiae Circumstantiae,2012,32(7):1549-1556. |
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| Authors: | CAO Xiangyu HUANG Yiyang SHI Shengnan MA Qiao LI Xinliang KONG Chunlei SHEN E and QU Yuanyuan |
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| Institution: | Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024;Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024;State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090;Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024;Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024;Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024;Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024;Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024 |
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| Abstract: | Response surface methodology was applied to optimize the culture conditions(seed age and level) and expression conditions(OD600 for inducing,IPTG concentration,induction time and temperature) of phenol hydroxylase,and to study characteristics of crude enzyme.The optimal expression conditions were seed age 7.19 h,level 1.39%,OD600 for inducing 0.54,IPTG concentration 0.19 mmol · L-1,induction time 2.37 h and temperature 35 ℃.Under the optimal conditions,the maximal activity of phenol hydroxylase could reach 0.6167 U · mg-1.The optimal pH and temperature for phenol hydroxylase was 8.0 and 40 ℃,respectively.The enzyme activity was stimulated by Ba2+,Fe3+,Hg2+ and Pb2+,but inhibited by Ca2+,Co2+,Cu2+,Mg2+ and Mn2+.Furthermore,phenol hydroxylase exhibited strong degradation ability of aromatic compounds. |
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| Keywords: | phenol phenol hydroxylase response surface methodology substrate spectra |
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