二苯并噻吩跨膜传递及其强化在红平红球菌LSSE8-1脱硫中的应用
文献类型：学位论文
| 作者 | 王泽龙 |
| 学位类别 | 硕士 |
| 答辩日期 | 2011-05-22 |
| 授予单位 | 中国科学院研究生院 |
| 导师 | 邢建民 |
| 关键词 | 微生物脱硫 二苯并噻吩 细胞裂解液 ABC转运体 HcuABC |
| 其他题名 | The application of the uptake of dibenzothiophene in the biodesulfurization of Rhodococcus erythropolis LSSE8-1 |
| 学位专业 | 生物化工 |
| 中文摘要 | 目前，提高微生物脱硫活性的研究主要集中在提高细胞内脱硫酶的表达量和对二苯并噻吩及其衍生物（CxDBT）的代谢活性上。对于细胞摄取CxDBT的过程则缺少研究。本论文针对本实验室分离的专一性脱硫微生物Rhodococcus erythropolis LSSE8-1，研究其摄取DBT过程对脱硫活性的影响，通过强化其摄取过程提高脱硫活性。考察了R. erythropolis LSSE8-1摄取CxDBT效率对脱硫活性的影响，结果表明：脱硫酶对DBT、4, 6-DMDBT没有选择性，CxDBT跨膜传递严重影响脱硫活性。通过在CxDBT跨膜传递蛋白基因（HcuABC）保守区设计引物扩增其部分片段，证实其在Pseudomonas delafieldii R-8中存在。然后扩增了R-8的HcuABC全基因序列，对比研究发现其与P. aeruginosa NCIMB9571、LESB58、PA01、UCBPP-PA14高度同源，显示了在进化关系上的相关性。其高度同源序列为ABC膜通道蛋白。构建了DBT及其衍生物跨膜传递基因HcuABC表达载体pBSJ-HcuABC，并将其转入专一性脱硫菌R. erythropolis LSSE8-1中进行表达，脱硫活性明显提高。对于DBT脱硫率提高19%，对于4, 6-DMDBT脱硫率提高13%。DBT及其衍生物跨膜传递过程对于微生物脱硫是一个重要甚至是一个限制性因素。因此，pBSJ-HcuABC表达载体对于构建高脱硫活性基因工程菌具有重要意义，必将在微生物脱硫工业化过程中起到重要作用。 |
| 英文摘要 | Biodesulfurization (BDS), with the potential benefits of high selectivity, low energy consumption, and deep desulfurization of fossil fuels, has been concerned worldwide. It will be the effective way of reducing sulfur content of petroleum products in 21 century. However, the flux through the system is still too low for widespread commercial applications. Considerable work has to be done to improve the rate of desulfurization. Most of the work over the past 10 years, has focused on the metabolism of sulfur heterocycles, particularly dibenzothiophenes (CxDBT), by Rhodococcus strains and other relatively closely related species. Few studies on the uptake of CxDBT to inside of cells were carried out. Rhodococcus erythropolis LSSE8-1, a specific desulfurization bacterium, was isolated by our laboratory. In this study, the transfer of DBT and 4, 6-DMDBT to inside of cells and the effect on BDS were studied. Then, the studies of enhancing the process of transfer and improving the biodesulfurization activity were carried out. The impact of the uptake of CxDBT to inside of cells on the biodesulfurization activity was studied. It was shown that the desulfurization enzymes had no priority on DBT over 4, 6-DMDBT. The desulfurization rate of DBT, especially 4, 6-DMDBT in R. erythropolis LSSE8-1 was seriously affected by the transfer into cells. The maximum desulfurization rate of the extracts of the cell lysate was attained when 10 mM NADHNa2 and 20 μM FMN were present. In the first 3 hours, the extracts of the cell lysate showed significantly higher desufurization rate compared to the resting cells and the cell lysate. The desulfurization activity was improved remarkably when Tween80 was present in the system of resting cells. The inhibited effect of NaN3 on desulfurization reactions was studied, which confirmed that the transfer of DBT into cells was an active transport in R. erythropolis LSSE8-1. PCR primers of dibenzothiophene transmembrane uptake genes (HcuA, B, C) of Pseudomonas delafieldii R-8 were designed in the conserved region. Then, the fragments were amplified. Their similarities with P. aeruginosa NCIMB9571-related genes are found to be as high as 99.5%, 98.7%, 98.0%. Primers of HcuABC were designed to amplify the gene cluster. It was found that they are highly conserved among P. aeruginosa NCIMB9571、LESB58、PA01、UCBPP-PA14, which showed the interdependency of evolutionary relationships. The high degree of homology was ABC membrane channel proteins. The uptake-genes of CxDBT (HcuABC) of P. delafieldii R-8 were introduced into the specific desulfurization bacterium, R. erythropolis LSSE8-1. Compared with the wild type, the strains bearing HcuABC genes showed a higher desulfurization activity. The desulfurization ratio of DBT showed a 19% increase, and 13% increase for 4, 6-DMDBT. The uptake of CxDBT into cells is a critical even limited factor of biodesulfurization, so HcuABC expression vector is a favorable approach for developing biocatalysts used in the two-liquid systems, and it might be promotive for BDS in commercial applications. |
| 语种 | 中文 |
| 公开日期 | 2013-09-24 |
| 页码 | 63 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1738]  |
| 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 | 王泽龙. 二苯并噻吩跨膜传递及其强化在红平红球菌LSSE8-1脱硫中的应用[D]. 中国科学院研究生院. 2011. |
入库方式： OAI收割
来源：过程工程研究所
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