Gasoline Biodesulfurization DE-FC07-97ID13570 FINAL REPORT

Pienkos, Philip T.

Enchira Biotechnology Corp. (United States)

关键词: 02 Petroleum;    Oxidoreductases;    Desulfurization;    Biodesulfurization;    Genetic Engineering;   

DOI  :  10.2172/791501
RP-ID  :  DOE/ID/13570
RP-ID  :  FC07-97ID13570
RP-ID  :  791501

美国|英语

来源: UNT Digital Library

【 摘 要 】

Nine strains were identified to grow with gasoline as sole sulfur source. Two different genes were cloned from Gordonia terrae KGB1 and tested for the ability to support gasoline BDS. The first of these, fmoA, was cloned by screening a KGB1 gene library for the ability to convert indole to indigo (a sulfur-regulated capability in KGB1). The fmoA gene was overexpressed in a gasoline tolerant strain of Pseudomonas putida PpG1 and the recombinant strain was shown to convert thiophene to a dimer of thiophene sulfoxide at rates nearly two orders of magnitude higher than KGB1 could catalyze the reaction. Despite this high activity the recombinant PpG1 was unable to demonstrate any activity against gasoline either in shake flask or in bench-scale gasoline BDS bioreactor. A second gene (toeA) was cloned from KGB1 and shown to support growth of Rhodococcus erythropolis JB55 on gasoline. The toeA gene was also identified in another gasoline strain T. wratislaviensis EMT4, and was identified as a homolog of dszA from R. erythropolis IGTS8. Expression of this gene in JB55 supported conversion of DBTO2 (the natural substrate for DszA) to HPBS, but activity against gasoline was low and BDS results were inconsistent. It appeared that activity was directed against C2- and C3-thiophenes. Efforts to increase gene expression by plasmid manipulation, by addition of flavin reductase genes, or by expression in PpG1 were unsuccessful. The DszC protein (DBT monooxygenase) from IGTS8 has very little activity against the sulfur compounds in gasoline, but a mutant enzyme with a substitution of phenylalanine for valine at position 261 was shown to have an altered substrate range. This alteration resulted in increased activity against gasoline, with activity towards mainly C3- and C4-thiophenes and benzothiophene. A mutant library of dszB was constructed by RACHITT (W. C. Coco et al., DNA shuffling method for generating highly recombined genes and evolved enzymes. 2001. Nature Biotech. 19:354-359) method of in vitro recombination. Methods for analysis were developed and a preliminary analysis of the library was performed. A preliminary gasoline process design was constructed and process economics were determined based upon assumptions made from experimental results. The projected cost of gasoline BDS was determined to be competitive with current competing technologies.