| 题名 | 专一性脱硫微生物的菌种筛选、代谢调控及相关基因研究 |
| 作者 | 缑仲轩 |
| 学位类别 | 博士 |
| 答辩日期 | 2004 |
| 授予单位 | 中国科学院过程工程研究所 |
| 授予地点 | 中国科学院过程工程研究所 |
| 导师 | 刘会洲 |
| 关键词 | 生物脱硫 二苯并唆吩 选择性脱硫 脱硫基因 红平红球菌LSSES一1 核糖体结合位点 基因重组 |
| 其他题名 | Screening of Specific Desulfurization Bacteria Strains and Study of Their Metabolism Regulation and Related Gene Fragments |
| 学位专业 | 化学工艺 |
| 中文摘要 | 专一性脱硫菌能选择性地脱除化石燃料模型含硫化合物二苯并鹰吩（DBT）中的硫元素，整个碳链骨架以2-羟基联苯（HBP）的形式保存下来，有望在石油和煤炭的脱硫过程中发挥作用，引起了人们的广泛关注。 经过选择性培养和代谢产物分析，从我国贵州省赤水气田被油气污染过的土壤中筛选分离出24株脱硫菌，其中有4株为专一性脱硫菌。对脱硫活性最强的一株专一性脱硫菌进行了菌种鉴定分析。该菌形态特征，生理生化反应、细胞壁化学组分及16SrDNA序列表明，该菌属于红球菌进化分枝，与红平红球菌同源性最高，故命名为Rhodococcus erythropolis LSSES-1。该菌具有卡那霉素抗性，属室温菌，在25-32℃的环境下均能生长，适宜的生长温度是28一30℃。最适的生长pH值为6.5-9。该菌能利用多种碳源，其中以甘油最佳，适宜的一甘油浓度为89／L；菌体生长和脱硫的最适氮源是乙酸钱（最适浓度2g／L）。该菌能在 10mmol几以下的DBT培养基中生长，其生长最佳的DBT浓度是1 mmol／L，脱硫的最佳DBT浓度是0.5mmol／L。二甲基亚矾是细胞生长与脱硫的有效硫源。该菌还能选择性地脱除苯并嚷吩、苯硫醚和4，6一二甲基DBT的硫。与DBT的对照实验表明，DBTO2（DBT Sulfone）对LSSES-1的生长有较大的抑制作用。在LSSE8-1脱硫时，补给中间产物DBTOZ，会有DBT和HBP产生。表明在细胞水平上，从DBT到DBTO2这一步反应是可逆的。HBP的存在对细胞的生长和脱硫活性均有影响。当水相中HBP的浓度达到0.4mmol／L时，细胞生长明显地被抑制。以LSSE8-1的质粒DNA做模板进行PCR反应，扩增得到三个脱硫结构基因片段，测序后发现，A基因和B基因与Rhodococcuse尽throPolis IGTSS的相关基因的同源性为100％，C基因的同源性为99％，说明红球菌脱硫结构基因是高度保守的。利用大肠杆菌表达质粒PinPoint Xa-T载体分别构建了三个脱硫基因的重组菌。PinPoint-dszC重组菌能将DBT转化成为DBTO2；PinPoint-dszA重组菌能将DBTO2转化成为一种水溶性物质；PinPtrint-dszA重组菌与PinPoint-dszB一起能将DBTO2转化为HBP。Pililioint-dszB重组菌加入100μmol／L异丙基-β-D-硫代半乳糖营（I：PTG）在30℃，200 r／min诱导表达4小时，全细胞蛋白电泳分析发现在52kd有一蛋白带产生，推测是Pi口oint一DszB融合蛋白。分别提取红平红球菌LSSES一1、戈登氏菌LSSEJ一1和球形诺卡氏菌R一9的质粒DNA，以此为模板，PCR扩增并克隆了包括整个脱硫操纵子在内的4．skb的DNA片段，测序发现，该序列高度保守，同源性高达99．5％。序列比较发现LSSES一1在其脱硫操纵子之外有较多的单核普酸插入突变，甚至于在调控区附近也有一处插入突变：进一步的分析表明，每段结构基因的上游一12一一15或一9一一12范围内存在一段保守序列AGGA，该段序列所转录的mRNA中的AGGA序列正好能与165］丑NA末端的序列UCCU互补结合，是mRNA与核搪体结合位点。A基因和B基因的核糖体结合位点AGGAC比C基因的结合位点多一个碱基，可能会导致A酶和B酶翻译的机会更多。这可以从基因水平上解释戈登氏菌LSSEJ一1脱硫时DBTOZ不积累的现象利用嵌合染料叮l建橙消除了LSSES一1的内源性质粒，得到了一株失去脱硫活力的LSSEO菌株，证明LSSES一1的脱硫基因也位于质粒上。利用穿梭质粒pBS3OS将整个脱硫操纵子片段转入LSSEo，脱硫活性恢复，证明重组质粒pBS3OS一dsz能在红平红球菌LssES一1中表达。将重组质粒pBs305一dsz转入LssES一1中得到一株脱硫活性可能更高的菌株LSSES一2。在没有任何选择性压力的情况下培养2天，LSSES一2的质粒稳定性达51．8％，具有较高的稳定性。本论文工作为化石燃料生物脱硫技术（B DS）的工业化应用提供了必要的菌株和基础数据，也为进一步的研究工作奠定了技术基础。 |
| 英文摘要 | Specific bacteria desulfurization can selectively remove the sulfur in model sulfur-containing compounds such as dibenzothiphene (DBT) in fossil fuel and DBT is recalcitrant to traditional hydrodesulfurization process. The carbon skeleton of DBT is kept intact during the process and the end product is 2-hydroxybiphenyl (HBP). These strains are expected for production of ultra-low sulfur fuel from petroleum and possibly coal. The soil sample contaminated with natural gas and petroleum from Shishui county, Gui Zhou province, was collected. By selective growth and analysis of the metabolites, 24 desulfurization strains were isolated, and among them 4 strains can be used in desulfurization process. Then, the most competent strain was identified. The morphological, physical and biochemical properties, cell wall analysis and 16SrDNA sequencing suggested that the isolated strain belonged to the evolution branch of Rhodococcus with highest homology to Rhodococcus erythropolis. The strain was named as Rhodococcus erythropolis LSSE8-1. The strain is kanamycin resistant, and belongs to room temperature bacteria which can be grown between 25~32°C, with the optimal growth temperature at 28~30°C. The optimal pH value for the growth was between 6.5 ~9> with certain tolerance of enviroment at change of pH. Glycerol was the optimal carbon source at the optimal concentration of 8g/L; Ammonium acerate was the optimal nitrogen source for both growth and desulfurization process, with the optimal concentration of 2g/L. The strain can grow well in the culture with DBT concentration below 10 mmol/L; however, the optimal DBT concentration for the growth was 1 mmol/L, and the optimal concentration for the desulfurization was 0.5 mmol/L. Dimethyl sulfide was an effective sulfur source for cell growth and desulfurization. Besides DBT, the strain can also desulfurize benzothiophene, phenyl sulfide and 4, 6-dimethyl dibenzothiophene (4, 6-DMDBT). Taking DBT as control, DBTO2 (DBT sulfone) was more harmful to the growth of LSSE8 — 1. By adding DBTO2 to the culture, both DBT and HBP accumulated in the media, indicating that the reaction from DBT to DBTO2 was reversible in the cells. HBP was harmful to both the growth and biodesulfurization activity of cells. When the concentration of HBP in the aqueous phase was 0.4 mmol/L or more, the growth of LSSE8—1 was inhibited distinctly. Plasmid DNA was extracted and used as template for PCR amplification; three structural gene fragments were amplified by PCR and isolated. They are 1.3, 1.0 and 1.2 kb in length respectively. These gene fragments were cloned and sequenced. Data showed that the homology of both dszA and dszB between LSSE8-1 and IGTS8 was 100%. The homology of dszC between LSSE8-1 and IGTS8 was 99%. The results showed that the structural genes were highly conservative. Pinpoint Xa-T is an expressing vector for E.coli. Three recombinant E.coli strains were constructed. Metabolites analysis showed that the strain with PinPoint-cfczC can convert DBT to DBTO2. The strain with PinPoint-cfazA can convert DBTO2 into a soluble material. With both strains which contained PinPoint-cfczA and PinPoint-ckzB respectively, DBTO2 was converted into HBP. The strain with PinPoinWszB plasmid was activated over night and grew for lh, 100/imol/L isopropyl- P -D-thiogalactoside (IPTG) was added and induced at 30 °C with 200r/min to express DszB for 4 hours. Whole cell protein electrophoresis analysis showed clear protein band of 52kd was the fused protein of PinPoint-DszB. Rhodococcus erythropolis LSSE8-1, Gordonia nitida LSSEJ — 1 and Nocardia globerula R—9 are all actinomycetales. Their plasmid DNA was extracted and acted as reaction plate. A large DNA fragment about 4.5 kb including the whole dsz operon was amplified by PCR. After ligated with pGEM-T vector the fragment was cloned in E. coli and then sequenced. The sequence information revealed that the related dsz sequences of these trains were highly conservative with sequence homology as high as 99.5%.There were several inserted base pairs beyond the dsz operon as compared with others, and one inserted base was even near the regulatory area. Further analysis indicated that in the upstream about—12 15 or —9~ —12 area beyond the three structural genes, there was a highly conservative sequence AGGA. The corresponding mRNA sequence can combine with sequence (1463) UCCU of 16SrRNA, which was located near the 3'end. This was the ribosomal binding site (RBS). There was a little difference of RBS between dszQ and dsz A or B, because besides AGGA, both dszA and dszB have another base cytosine which can also bind with 16SrRNA too. This provides more chance to translate DszB and DszA than DszC. This can explain why there was no accumulation of DBTO2 at all during desulfurization process of LSSEJ-1. With the addition of intercalating dye acridine orange, the endogenesis plasmid was eliminated, then a new strain LSSE0 without the ability of desulfurization was obtained. The experimental phenomena suggested that the dsz gene of LSSE8-1 was located in the plasmid. With shuttle vector pBS305, the whole dsz operon was transformed into LSSE0, and then the desulfurization activity was recovered. This indicted that the recombinant plasmid pBS305-dsz can express in Rhodococcus erythropolis LSSE8 — 1 The recombinant plasmid was transformed into LSSE8 —1, and the new strain LSSE8-2 may possess enhanced desulfurization ability to that of LSSE8-1. Without any selective pressure and growing in LB culture for 2 days, the stability of the recombinant plasmid in LSSE8-2 was as high as 81.8%.This showed that the LSSE8-2 was stable. |
| 语种 | 中文 |
| 公开日期 | 2013-09-16 |
| 页码 | 134 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1409]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 缑仲轩. 专一性脱硫微生物的菌种筛选、代谢调控及相关基因研究[D]. 中国科学院过程工程研究所. 中国科学院过程工程研究所. 2004. |
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