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菌株Pigmentiphagasp.H8中3,5-二溴-4-羥基苯甲酸降解關鍵酶基因odcA的功能驗證

時間:2019-05-25 20:43來源:畢業論文
通過基因敲除和回補以及異源表達實驗證明orf420(odcA)是降解DBHB的關鍵基因,獲得了純的OdcA蛋白。通過體外酶學轉化實驗和LC-MS分析,鑒定OdcA降解DBHB的產物是2,6-二溴對苯二酚

摘要:3,5-二溴-4-羥基苯甲酸(3,5-dibromo-4-hydroxybenzoate, DBHB)是一種溴代芳烴,易存在于化工食品、醫藥工業的廢水中,并且達到可觀的濃度,目前是主要的環境污染物,但對于DBHB的降解研究很少。本實驗室前期從長期受鹵代芳烴污染的土壤中分離篩選到一株高效降解DBHB的菌株Pigmentiphaga sp.H8,經過比較轉錄組學和比較蛋白組學的研究,推斷菌株Pigmentiphaga sp.H8基因組中orf420-426參與DBHB的降解。本文通過基因敲除和回補以及異源表達實驗證明orf420(odcA)是降解DBHB的關鍵基因,獲得了純的OdcA蛋白。通過體外酶學轉化實驗和LC-MS分析,鑒定OdcA降解DBHB的產物是2,6-二溴對苯二酚。通過酶促反應研究了OdcA的酶學特性。35711
關鍵詞:3,5-二溴-4-羥基苯甲酸;Pigmentiphaga sp.H8;odcA;菌株Pigmentiphaga sp. H8中3,5-二溴-4-羥基苯甲酸關鍵降解酶基因odcA的功能驗證
Functional verification of the key gene odcA for 3,5-dibrimo-4-hydroxybenzoate catabolism in strain Pigmentiphaga sp. H8
Abstract:3,5-dibromo-4-hydroxybenzoic acid is a mental type of environmental pollutants belonging to brominated aromatic compound, which can be easily found in medical treatment, chemical industry and food industry. However, few studies on the microbial degradation of DBHB have been done. Pigmentiphaga sp. H8 is a strain isolated from long-term contaminated soil with halogenated aromatic compounds. It can mineralize DBHB quickly and effectively. We can find genes involved in the degradation of DBHB by comparing the differences in mRNA transcription and proteomic expression between DBHB-induced and non-induced strains. The results show that orf420-426 of the strain H8 genome may involved in degradation DBHB. This study uses the methods of gene knockout , complementary experiments and heterologous expression to prove odcA is the key gene of metabolic pathway of DBHB in strain H8. We get the purified OdcA. By in vitro enzymatic transformation experiments and LC-MS analysis, we identified the product of OdcA degradation of DBHB was 2,6-dibromohydroquinone. Studies about the best situation of OdcA’ s enzymatic reactions are also been done.

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Key words: 3,5-dibromo-4-hydroxybenzoate;Pigmentiphaga sp.H8;odcA;
目  錄
摘要.1
關鍵詞.1
Abstract.1
Key words1
引言.2
1材料與方法3
1.1材料3
1.1.1菌株、質粒、因物.3
1.1.2培養基和試劑4
1.2方法.4
1.2.1大腸桿菌一步法感受態的制備與轉化4
1.2.2質粒的提取5
1.2.3細菌總DNA的提取.5
1.2.4靜息細胞法5
1.2.5odcA基因的敲除5
1.2.6odcA基因的回補.5
1.2.7odcA基因的異源表達.6
1.2.8OdcA蛋白的純化.6
1.2.9DBHB的檢測.6
1.2.10 SDS聚丙烯酰胺凝膠電泳(SDS-PAGE).6
1.2.11 OdcA的酶學特性6
2 結果與分析.7
2.1基因odcA缺失菌株H8ΔodcA和回補菌株H8ΔodcA(pMCS-odcA)降解DBHB結果.7
2.2 OdcA異源表達和純化7
2.3 OdcA催化DBHB降解產物的測定8
2.4 OdcA的酶學特性研究結果.8
2.4.1輔因子對OdcA的酶活影響8
2.4.2溫度對OdcA的酶活影響.9
2.4.3pH對OdcA的酶活影響.9
2.4.4金屬離子對OdcA的酶活影響.9
2.4.5OdcA酶促反應動力學常數的測定.10
3討論和總結.10
3.1本實驗研究前提.10
3.2本研究總結.10
3.3本研究創新點與不足.10
致謝11
參考文獻11
引言
鹵代芳烴類化合物具有穩定性好、阻燃、耐疲勞、耐熱等優點,廣泛運用于化工中間體、阻燃劑、農藥、醫藥[1,2]等生產。由于鹵代芳烴具有難降解、高毒性等特點,很多被列為“持久性有機污染物”( Persistent Organic Pollutants, POPs)和“優先控制污染物”(Priority pollutants)[3,4]。鹵素原子的強電子吸附效應使得鹵代芳烴易與生命細胞內的酶系統結合進而對細胞產生毒性。因此,鹵代芳烴造成的環境污染對生態環境和人類健康造成嚴重的威脅[5],鹵代芳烴的微生物降解與污染修復已引起學術界的廣泛關注。研究鹵代芳烴的微生物降解與污染修復具有重要的意義。 菌株Pigmentiphagasp.H8中3,5-二溴-4-羥基苯甲酸降解關鍵酶基因odcA的功能驗證:http://www.aftnzs.live/shengwu/20190525/33789.html
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