| Summary: | 碩士 === 國立雲林科技大學 === 環境與安全工程系碩士班 === 92 === Quinoline, an aromatic N-heterocyclic chemical compound produced in coal tar and other petroleum producing processes, is a known toxicity and carcinogen to the human body. In the past, the research of quinoline biodegrading bacterial strain in Taiwan usually focused on the physiological mechanisms studies, such as enzyme activity test, optimal cultivation, and metabolism process. Although many achievements have been accumulated and a deeper understanding of biodegradation of quinoline and it’s biochemical characteristic has been accomplished, little has been done in the field of molecular biology. Therefore, the purpose of this study is to build a basic tool and selecting system for gene cloning and expression. The bacterial strain used in the experiment, Pseudomonas stutzeri RMRC PAH5, is provided by Chiayi Refining and Manufacturing Research Institute that can treat quinoline as sole substrate. The degradation rate of quinoline can reach over 97 % that has been confirmed by HPLC.
The highlight of this study is to clone the quinoline 2-oxidoreductase (Qor) gene from the Pseudomonas stutzeri RMRC PAH5 where the former play an important role in degrading quinoline. Four pairs of sense/antisense oligonucleotide (S1/AS1), (S2/AS2), (Start/S1 up) and (AS2 down/Stop) were designed and synthesized as primers for PCR method. The resulting products are (S1/AS1 ): 1.3 kb, (S2/AS2): 1.2 kb, (Start/S1 up): 0.34 Kb and (AS2 down/Stop): 1.8 kb separately. After purification, all the previous DNA fragment were inserted into the subcarrier, yT&A vector, by T-A cloning method. The positive colony after screening that include Qor as well as flanking regions were denominated as pHy-S1/AS1, pHy-S2/AS2, pHy-SS and pHy-AdS. It was shown that the total length of P.s.Qor is 3756 bp after gene sequence analysis.
The gene specific primers (GSP), Qor start and Qor stop, were properly designed in order to construct the complete full length of P.s. Qor. The expectative DNA fragment can be obtained via PCR method and be inserted into a subcloned pGEM-T easy vector to proceed fast screening. The result proves that insertion is P.s. Qor gene. The complete gene-coding region were identified through the gene sequence analysis. It was found the complete P.s. Qor gene is composed of three subunits with transcriptional order as qorM, qorS and qorL. By comparing the sequence of P.s. Qor with P. putida 86 Qor shows their significant homology is above 99%, which means abrupt change of Qor gene is not very often during the evolution. It is postulated this gene is a highly conservative area and it’s corresponding enzyme activation might be reduced or disappeared once it’s sequence has been changed.
The amino acid sequence of P.s. Qor is compared with various molybdenum containing hydroxylases by GCG command mode of NHRI. It was found the three subunits of P.s. Qor amino acid sequence contains highly conserved areas and different binding motifs. The open reading frame (ORF) of P.s. QorM has 867 bp and it’s corresponding protein consists of 288 amino acid. The P.s. QorM probably may harbor the FAD cofactor, but no conserved area was found. The P.s. QorS has 513 bp ORF and it’s corresponding protein consists of 168 amino acid. The P.s. QorS includes two distinct motifs and each one of them contains four cysteines that arranged in a specific structure. The four cysteines of the first motif locate in the positions of 48, 53, 56, and 68 aa with a structure as (C-X4-C-G-X-C-Xn-C). The second motif has a structure of (C-G-X-C-X31-C-X-C) where it’s four cysteines locate in the positions of 107, 110, 142 and 144 aa. This latter motif presumably is the binding site of the typical plant-type 【2Fe-2S】ferredoxins. The 2368 bp coding region of the P.s QorL was translated to a 788 amino acid sequence and probably contains the binding sites of the pterin molybdenum cofactor. The putative binding sites locate at position of 252~258, 369~380, 506~509, 542~548 and 741~746 aa.
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