高效脱硫工程菌的构建及其脱硫性能研究
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摘要
生物脱硫具有选择性高、反应条件温和等优点,是实现石油及其产品深度脱硫最有效的技术之一。生物脱硫要实现工业化应用必须提高已有脱硫微生物的脱硫活性。本论文以专一性脱硫菌德氏假单胞菌(Pseudomonas delafieldii)R-8为出发菌株,利用基因工程的手段构建脱硫工程菌,以提高其脱硫活性,旨在为脱硫工程菌的应用奠定基础。
根据P. delafieldii R-8菌的脱硫操纵子基因序列设计引物,PCR扩增得到了脱硫操纵子,利用pPR9TT穿梭质粒构建了脱硫操纵子表达载体pPR-dsz,转化原始菌R-8得到了一株多拷贝脱硫基因的脱硫工程菌R-8-1,并对其脱硫性能进行了研究。结果表明,在同样的生物催化脱硫反应条件下,工程菌的脱硫活性达到6.25μmol DBT /g dry cell/h,是原始菌R-8的2倍;柴油的脱硫实验结果表明,在12 h内工程菌静息细胞能将柴油中的硫含量从310.8 mg/L降至100.1 mg/ L,脱硫率达到68%,而原始菌R-8为53%。
R-8菌是一种好氧微生物,为了提高其对氧的利用效率,采用基因工程的手段,将血红蛋白基因在R-8菌中进行异源表达,以期获得生长状况和脱硫活性都得到提高的工程菌。根据透明颤菌(Vitreoscilla)血红蛋白基因(vgb)的序列设计引物,PCR扩增得到vgb,利用pPR9TT穿梭质粒构建血红蛋白基因表达质粒pPR-Pdsz-vgb并电击转化原始菌R-8,获得了重组菌R-8-2。R-8-2菌的CO-差光谱在419 nm处有特征峰出现,表明血红蛋白在脱硫菌中得到了有效表达。在相同培养条件下,工程菌R-8-2和原始菌R-8的生长与脱硫活性比较研究发现,工程菌生长得到了改善,菌体密度比R-8菌提高了20%,脱硫活性达到了原始菌R-8的2.4倍。在柴油脱硫实验中,工程菌能将柴油中的硫含量从320.5 mg/L降至96.6 mg/L,脱硫率达到69.9%,而原始菌R-8为57.2%。
对两株工程菌的遗传稳定性进行了研究。经过对菌株50代的传代培养研究,结果表明重组质粒pPR-dsz和pPR-Pdsz-vgb分别在两株工程菌R-8-1和R-8-2中均具有良好的遗传稳定性。
本论文工作获得了两株生长与脱硫活性都得到提高的脱硫工程菌,为进一步利用基因工程的手段改造脱硫微生物提供了理论依据和技术支持,为脱硫工程菌的应用奠定了基础。
Biodesulfurization is one of the most promising techniques for deep desulfurization of petroleum with advantages of high selectivity and mild reaction conditions. The limiting factor is the low activity of the biocatalysts for the commercialization of BDS. The aim of this thesis is to improve the desulfurization activity of the specific desulfurization strain, Pseudomonas delafieldii R-8, using the approaches of genic engineering and establish the base for further application of desulfurization.
In this study, the dsz operon of Pseudomonas delafieldii R-8 was first cloned into the expressing plasmid (pPR9TT) to construction the recombinant plasmid pPR-dsz, and then reintroduced into strain R-8 to obtain a muti-copy dsz operon engineering strain R-8-1. Compared to the wild-type, strain R-8-1 showed a higher desulfurization activity for dibenzothiophene (DBT). Initial rates of DBT removal by strain R-8-1 were 6.25μmol /g dry cell/h, about 2-fold higher than that for wild-type strain. The recombinant cells were also applied in the desulfurization progress of diesel. It resulted in a 68% reduction of total sulfur from 310.8 mg/L to 100.1 mg/L, whereas only 53% of sulfur was removed by strain R-8. The stability of pPR-dsz in strain R-8-1 had been studied. It indicated that engineering strain has a high genetic stability. These results first obtain a muti-copy dsz operon engineering strain and are helpful for further advance of the biodesulfurization.
To construct an engineering strain of higher desulfurization activity with Vitreoscilla hemoglobin gene (vgb) and study vgb application to the desulfurization of diesel,we constructed the vgb expressing plasmid, pPR-Pdsz-vgb and transformed by electroporation to Pseudomonas delafieldii R-8, then obtained a genetic engineering strain R-8-2. The results of CO-difference spectrum analysis indicated that the Vitreoscilla hemoglobin (VHb) having biological activity was expressed in strain R-8-2. Compared with the wild-type R-8, the density of strain R-8-2 increased by 20%, and its desulfurization activity is also higher in the lower deliquescence oxygen environment. In the end, about 69.9% of total sulfur in diesel oil was removed by strain R-8-2 from 320.5 mg/L to 96.6 mg/L, whereas only 57.2% of sulfur was removed by strain R-8. The results showed that the desulfurization activity of strain R-8-2 was higher than that of R-8 not only under the hypoxic conditions but also under normal aeration.
In the thesis, we construct two engineering strains of higher desulfurization activity of P. delafieldii R-8. Furthermore, the work is helpful for further improvement of desulfurization strains and development in biodesulfurization.
根据P. delafieldii R-8菌的脱硫操纵子基因序列设计引物,PCR扩增得到了脱硫操纵子,利用pPR9TT穿梭质粒构建了脱硫操纵子表达载体pPR-dsz,转化原始菌R-8得到了一株多拷贝脱硫基因的脱硫工程菌R-8-1,并对其脱硫性能进行了研究。结果表明,在同样的生物催化脱硫反应条件下,工程菌的脱硫活性达到6.25μmol DBT /g dry cell/h,是原始菌R-8的2倍;柴油的脱硫实验结果表明,在12 h内工程菌静息细胞能将柴油中的硫含量从310.8 mg/L降至100.1 mg/ L,脱硫率达到68%,而原始菌R-8为53%。
R-8菌是一种好氧微生物,为了提高其对氧的利用效率,采用基因工程的手段,将血红蛋白基因在R-8菌中进行异源表达,以期获得生长状况和脱硫活性都得到提高的工程菌。根据透明颤菌(Vitreoscilla)血红蛋白基因(vgb)的序列设计引物,PCR扩增得到vgb,利用pPR9TT穿梭质粒构建血红蛋白基因表达质粒pPR-Pdsz-vgb并电击转化原始菌R-8,获得了重组菌R-8-2。R-8-2菌的CO-差光谱在419 nm处有特征峰出现,表明血红蛋白在脱硫菌中得到了有效表达。在相同培养条件下,工程菌R-8-2和原始菌R-8的生长与脱硫活性比较研究发现,工程菌生长得到了改善,菌体密度比R-8菌提高了20%,脱硫活性达到了原始菌R-8的2.4倍。在柴油脱硫实验中,工程菌能将柴油中的硫含量从320.5 mg/L降至96.6 mg/L,脱硫率达到69.9%,而原始菌R-8为57.2%。
对两株工程菌的遗传稳定性进行了研究。经过对菌株50代的传代培养研究,结果表明重组质粒pPR-dsz和pPR-Pdsz-vgb分别在两株工程菌R-8-1和R-8-2中均具有良好的遗传稳定性。
本论文工作获得了两株生长与脱硫活性都得到提高的脱硫工程菌,为进一步利用基因工程的手段改造脱硫微生物提供了理论依据和技术支持,为脱硫工程菌的应用奠定了基础。
Biodesulfurization is one of the most promising techniques for deep desulfurization of petroleum with advantages of high selectivity and mild reaction conditions. The limiting factor is the low activity of the biocatalysts for the commercialization of BDS. The aim of this thesis is to improve the desulfurization activity of the specific desulfurization strain, Pseudomonas delafieldii R-8, using the approaches of genic engineering and establish the base for further application of desulfurization.
In this study, the dsz operon of Pseudomonas delafieldii R-8 was first cloned into the expressing plasmid (pPR9TT) to construction the recombinant plasmid pPR-dsz, and then reintroduced into strain R-8 to obtain a muti-copy dsz operon engineering strain R-8-1. Compared to the wild-type, strain R-8-1 showed a higher desulfurization activity for dibenzothiophene (DBT). Initial rates of DBT removal by strain R-8-1 were 6.25μmol /g dry cell/h, about 2-fold higher than that for wild-type strain. The recombinant cells were also applied in the desulfurization progress of diesel. It resulted in a 68% reduction of total sulfur from 310.8 mg/L to 100.1 mg/L, whereas only 53% of sulfur was removed by strain R-8. The stability of pPR-dsz in strain R-8-1 had been studied. It indicated that engineering strain has a high genetic stability. These results first obtain a muti-copy dsz operon engineering strain and are helpful for further advance of the biodesulfurization.
To construct an engineering strain of higher desulfurization activity with Vitreoscilla hemoglobin gene (vgb) and study vgb application to the desulfurization of diesel,we constructed the vgb expressing plasmid, pPR-Pdsz-vgb and transformed by electroporation to Pseudomonas delafieldii R-8, then obtained a genetic engineering strain R-8-2. The results of CO-difference spectrum analysis indicated that the Vitreoscilla hemoglobin (VHb) having biological activity was expressed in strain R-8-2. Compared with the wild-type R-8, the density of strain R-8-2 increased by 20%, and its desulfurization activity is also higher in the lower deliquescence oxygen environment. In the end, about 69.9% of total sulfur in diesel oil was removed by strain R-8-2 from 320.5 mg/L to 96.6 mg/L, whereas only 57.2% of sulfur was removed by strain R-8. The results showed that the desulfurization activity of strain R-8-2 was higher than that of R-8 not only under the hypoxic conditions but also under normal aeration.
In the thesis, we construct two engineering strains of higher desulfurization activity of P. delafieldii R-8. Furthermore, the work is helpful for further improvement of desulfurization strains and development in biodesulfurization.
引文
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