State Key Laboratory of Integrated Management of Pest Insects & Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
J Biotechnol. 2010 Apr 1;146(3):114-9. doi: 10.1016/j.jbiotec.2010.01.020. Epub 2010 Feb 2.
Biodegradation or bioremediation is a more efficient and environmental friendly method for detoxification of hexachlorocyclohexane (HCH) residues compared to physical and chemical methods. Here, we report the functional expression of dehydrochlorinase (LinA) and enhanced green fluorescent protein (EGFP) in Pseudomonas nitroreducens for efficient biodegradation of gamma-HCH. The broad-host-range plasmid pVAG33, harboring dehydrochlorinase gene (linA) and enhanced green fluorescent protein gene (egfp), was constructed and transformed into the soil-isolated 1,2,4-trichlorobenzene (1,2,4-TCB)-degrading strain P. nitroreducens J5-1. Functional expression of LinA and EGFP was confirmed in the recombinant strain by Western-blotting analysis and by determining their enzymatic activities and fluorescence intensity. The recombinant strain could rapidly degrade 10microg ml(-1) gamma-HCH in 28h determined by GC-ECD analysis. It could completely mineralize gamma-HCH via gamma-HCH through 1,2,4-TCB and 3,4,6-trichlorocatechol and eventually entered the TCA cycle as determined by GC-MS analysis. The engineered strain can be applied in the form of a biocatalyst in a bioreactor for rapid degradation of HCH and chlorobenzene residues. Meanwhile, it can be easily monitored on-line by fluorescence of EGFP for its activity and fate.
与物理和化学方法相比，生物降解或生物修复是一种更有效和环保的六氯环己烷（HCH）残留解毒方法。在这里，我们报告了脱氯化氢酶（LinA）和增强型绿色荧光蛋白（EGFP）在假单胞菌硝化还原菌中的功能表达，用于高效生物降解γ-HCH。构建了携带脱氯化氢酶基因（linA）和增强型绿色荧光蛋白基因（egfp）的广谱质粒 pVAG33，并将其转化到土壤分离的 1,2,4-三氯苯（1,2,4-TCB）降解菌株 P. nitroreducens J5-1 中。通过 Western-blotting 分析和测定酶活性和荧光强度，在重组菌株中证实了 LinA 和 EGFP 的功能表达。通过 GC-ECD 分析确定，重组菌株可在 28h 内快速降解 10μg ml(-1)的γ-HCH。通过 GC-MS 分析确定，它可以通过 1,2,4-TCB 和 3,4,6-三氯邻苯二酚将γ-HCH 完全矿化，并最终进入 TCA 循环。该工程菌株可作为生物催化剂在生物反应器中用于快速降解 HCH 和氯苯残留。同时，它的活性和命运可以通过 EGFP 的荧光很容易地在线监测。
