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题名	利用双组分调控系统与甲酸脱氢酶构建脱硫工程菌
作者	李海波
学位类别	硕士
答辩日期	2006-06-10
授予单位	中国科学院过程工程研究所
授予地点	过程工程研究所
导师	刘会洲
关键词	红平红球菌 微生物脱硫 双组分调控系统 甲酸脱氢酶
其他题名	Construction of Recombinant Rhodococcus erythropolis for Biodesulfurization by Using “Two-component regulatory system” and Formate Dehydrogenase
学位专业	化学工艺
中文摘要	微生物脱硫具有选择性高﹑反应条件温和﹑设备投资和操作费用低等优点，是实现石油深度脱硫最有效的技术之一。但脱硫微生物的反应速率较慢，要走向工业化应用必须提高已有脱硫微生物的催化活性。本论文针对实验室筛选的专一性脱硫菌红平红球菌LSSE8-1，采用基因工程技术对其进行了改造，构建了两株脱硫工程菌。 1. 双组分调控系统构建工程菌LSSE8-1-PbDGST 采用PCR的方法从红球菌Rhodococcus sp. RHA1中分别克隆了双组分调控系统的结构基因bphST和启动子PbphA1，以大肠杆菌-红球菌穿梭质粒pBS306为载体，以GFP为报告基因，构建了质粒pBS-PbGST；以脱硫基因启动子Pdsz，GFP和pBS306构建了质粒pBS-PdG。结果显示，Pdsz受硫酸盐阻遏，PbphA1不受硫酸盐阻遏。构建了质粒pBS-PbDGST和pBS-DSZ，重组菌LSSE8-1-PbDGST生长细胞脱硫实验表明，芳香化合物对其脱硫活性有诱导作用，甲苯和二甲苯的诱导作用最强，联苯和羟基联苯次之，DBT作用最弱。LSSE8-1-PbDGST静息细胞在6 h内几乎将DBT完全代谢，而LSSE8-1-DSZ的脱硫率仅为72%，LSSE8-1-PbDGST的脱硫活性明显高于LSSE8-1-DSZ。 2. 甲酸脱氢酶构建工程菌LSSE8-1-FDH 从红球菌Rhodococcus sp. RHA1中克隆了编码甲酸脱氢酶的基因(fdh)，构建了重组表达质粒pBS-PFG。2,3,5-三苯基氯化四氮唑显色反应测得重组菌LSSE8-1-FDH的总脱氢酶活为0.4637，高于原始菌LSSE8-1。考察了甲酸钠浓度对LSSE8-1和重组菌LSSE8-1-FDH生长的影响，当甲酸钠浓度为25 mmol/L时，LSSE8-1-FDH的生长最佳。静息细胞脱硫实验表明，LSSE8-1-FDH的脱硫速率比LSSE8-1增加12.5％。表明在LSSE8-1-FDH中辅酶的再生得到了强化。 本论文利用Rhodococcus sp. RHA1中的双组分调控系统和甲酸脱氢酶分别构建了两株工程菌LSSE8-1-PbDGST和LSSE8-1-FDH，解除了硫酸盐对脱硫酶表达的阻遏作用，强化了细胞内辅酶的再生，提高了脱硫活性。
英文摘要	Biodesulfurization (BDS) has the potential to be used in deep-desulfurization with advantages of high selectivity, mild conditions and low operation cost. The limiting factor is the low activity of the biocatalysts for the commercialization of BDS. In this study, two recombinant strains were constructed based on the specific biodesulfurization strain R. erythropolis LSSE8-1. Gene bphST and promoter PbphA1, which were components of “two-component regulatory system”, were amplified from Rhodococcus sp. RHA1. Recombinant plasmid pBS-PbGST was constructed using E. coli-Rhodococcus shuttle vector pBS306, bphST, PbphA1 and GFP. Pdsz, native promoter of desulfurization gene, was amplified from R. erythropolis LSSE8-1. plasmid pBS-PdG was constructed using pBS306, Pdsz and GFP. The recombinant plasmids pBS-PdG and pBS-PbGST were transformed into R. erythropolis LSSE8-1. The fluorescence data showed that GFP expressed under the promoter of PbphA1 more effectively than the expression of gfp driven by the promoter Pdsz in the medium that contain sulfate. Recombinant plasmids pBS-PbDGST and pBS-DSZ were constructed using pBS306, bphST,dszABC, PbphA1 and GFP.and then introduced into R. erythropolis LSSE8-1. The recombinant strains were grown in 0.2×LB medium that contain toluene, xylene and biphenyl, and HPLC analysis that toluene and xylene has the strongest induction effect on promoter PbphA1, biphenyl and hydroxy-biphenyl has second strongest induction effect, DBT also has a slight induction effect. Resting cell desulfurization showed that after 6 h, nearly all of the sulfur in disel oil was removed by the recombinant LSSE8-1-PbDGST, whereas LSSE8-1-DSZ removed only 72% of sulfur in the system, showed that recombinant strain LSSE8-1-PbDGST had higher desulfurization activity than recombinant strain LSSE8-1-DSZ. Formate dehydrogenase gene (fdh) was amplified from Rhodococcus sp. RHA1 and then introduced into R. erythropolis LSSE8-1. TTC reaction showed that the dehydrogenase activity of LSSE8-1-FDH is 0.4637, higher than the dehydrogenase activity of LSSE8-1. Recombinant strain showed an moderately faster growth rate in 25 mmol/L Sodium formate solution. Desulfurization of DBT in the oil-aqueous two phases system by LSSE8-1 and LSSE8-1-FDH was conducted respectively. Compared to the wild strain LSSE8-1, the desulfurization activity of the recombinant strain LSSE8-1-FDH increased 12.5％. In the thesis, two recombinant strains were constructed using Two-component regulatory system and formate dehydrogenase respectively. One of the recombinant strains was not restrained any more by the sulfate, and the cofactor-regeneration was enhanced in the other recombinant strain, both of the strains’s desulfurization activity was enhanced.
语种	中文
公开日期	2013-09-13
页码	89
内容类型	学位论文
源URL	[http://ir.ipe.ac.cn/handle/122111/1218]
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	李海波. 利用双组分调控系统与甲酸脱氢酶构建脱硫工程菌[D]. 过程工程研究所. 中国科学院过程工程研究所. 2006.

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