对硝基苯酚降解菌Pseudomonas sp. PDS-7的降解特性及 其降解相关基因的克隆

Characterization of a p-nitrophenol degrading bacterium Pseudomonas sp. PDS-7 and cloning of degradation relevant genes

摘要:

摘要：【目的】本研究的目的是分离对硝基苯酚(PNP)降解菌，研究其对PNP的降解特性；克隆其降解相关基因，并进行表达。【方法】本研究通过富集培养法和系列稀释平板涂布法分离PNP降解菌株；采用形态观察、生理生化特征测定和16S rDNA分析对菌株进行初步鉴定；通过摇瓶试验研究菌株降解特性；利用SEFA-PCR技术克隆降解相关基因，并亚克隆到表达载体pET29a 中，构建重组表达质粒pETpnpC，再转入受体菌E.coli BL21 (DE3)中进行诱导表达；通过分光光度法测定表达产物的酶活力。【结果】分离到一株PNP降解菌PDS-7，将该菌株鉴定为假单胞菌属（Pseudomonas sp.）；该菌株能够以PNP作为唯一碳源、氮源和能源生长，菌株对PNP的最高耐受浓度为80 mg/L，最适降解温度为30°C，偏碱性条件有利于菌株对PNP的降解；克隆了PNP降解过程中的偏苯三酚1,2-双加氧酶基因pnpC及马来酰醋酸还原酶基因pnpD (GenBank登陆号EU233791)；将pnpC在E.coli BL21 (DE3)菌株进行了诱导表达，表达产物对偏苯三酚和邻苯二酚均有邻位开环活性，比活力分别为0.45 U/mg protein 和 0.37 U/mg protein，表明偏苯三酚1,2-双加氧酶基因pnpC得到了活性表达。【结论】分离鉴定了一株PNP降解菌Pseudomonas sp. PDS-7，研究了该菌株的降解特性，克隆和表达了降解相关基因。

关键词:关键词：对硝基苯酚;Pseudomonas sp. PDS-7;降解特性;基因克隆

Abstract:

Abstract: [Objective] The aim of this study was to (i) isolate and characterize bacteria capable of degrading p- nitrophe-nol (PNP); (ii) determine the kinetics of biodegradation, (iii) clone and express the PNP-degrading related genes. [Meth-ods] Enrichment method and serial dilution spread-plate method were employed to isolate PNP-degrading strain. Mor-phological, physiological & biochemical tests and 16S rDNA sequence analysis were used to identify the isolate. Degra-dation kinetics was studied by flask test. PNP-degrading related genes were cloned by SEFA-PCR method. Hydroxyquinol 1,2-dioxygenase encoding gene pnpC was cloned into pET29a to construct the recombinant plasmid pETpnpC and ex-pressed in E. coli BL21 (DE3). The activity of the expressed product was determined by spectrophotometric method. [Results] Strain PDS-7 capable of utilizing PNP as the sole carbon, nitrogen and energy source was isolated and identified as Pseudomonas sp. It could tolerate the PNP concentration up to 80 mg/L, the optimal temperature for degradation was about 30°C and alkaline pH benefited PNP degradation. Hydroxyquinol 1,2-dioxygenase and maleylacetate reductase en-coding gene pnpC and pnpD were cloned and sequenced respectively, the sequence was deposited in GenBank with the accession number EU233791. pnpC was expressed in E. coli BL21 (DE3), the expressed product in cell-free crude extracts showed ortho ring cleavage activity to hydroxyquinol and catechol, with the special activity 0.45 U/mg protein and 0.37 U/mg protein, respectively, indicating pnpC gene encoding hydroxyquinol 1,2-dioxygenase was actively expressed. [Conclusion] One PNP-degrading strain Pseudomonas sp. PDS-7 was isolated and identified. Its degradation kinetics was studied. Its degradation relevant genes were cloned and expressed.

Key words:Keywords: p-nitrophenol; Pseudomonas sp. PDS-7; degradation kinetics; gene cloning