Key Enzymes And Their Roles In The Formaldehyde-degrading Process In Pseudomonas Putida IOFA1

Pseudomonas putida is often used as a model bacterium to study the degradation of sewage and waste because of its complex metabolic system for the degrading of organic waste. The functions of Pseudomonas putida for degradation of many toxic compounds have been widely studied, but few of them focus on the metabolic mechanism during these degradation process. The strain Pseudomonas putida IOFA1showing high formaldehyde-degradation ability was isolated and identified from deep sea sediment in our previous study. In this study, the formaldehyde-degradation mechanism in Pseudomonas putida IOFA1was analyzed by comparing the changes in transcriptome of the formaldehyde-degradation process with different time quantum. We selected three samples in different time quantum during formaldehyde degradation (group A:not added formaldehyde; Group B:20minutes incubation after added formaldehyde; group C:2hours incubation after added formaldehyde). The metabolic network of IOFA1strain changed obviously under the stimulation of formaldehyde by bioinformatic analysis and the classification of different expression gene. Furthermore, the transcriptional changes was discussed at overall level by GO (Gene Ontology) and KEGG classification analysis. The changed genes related to signal, biology, location and other metabolic pathways were studied by genes classification. In this thesis, several key enzymes relataed to the methyl metabolisms that was found in transcriptome were studied to support the illumination of the formaldehyde-degradation mechanism in Pseudomonas putida IOFA1.The main enzymes involved in the formaldehyde-degradation in Pseudomonas putida IOFA1were deterimined by combining the transcriptome analysis and RT-PCR analysis, including formaldehyde dismutase, depend-glutathione formaldehyde dehydrogenase, alcohol dehydrogenase, formate dehydrogenase and formate transporter. Among these enzymes, formaldehyde dismutase (FDM) play an important role in the early stages of formaldehyde-degradation, and it can degrade formaldehyde rapidly into methanol and formate. Alcohol dehydrogenase and glutathione-depend formaldehyde dehydrogenase are necessary to fully degradation of formaldehyde. Other enzymes are related to the degradation of formic acid.The crude enzymes in cell lysate were purified by DEAD-sepharose ion exchange chromatography. One single band was obtained and identified as FDM by mass spectrum analysis. The enzyme had optimal temperature and pH at40℃and7.5, respectively, and was stable at temperature under30℃. Low concentration of Mg2+and Ca2+activated the enzymatic activity. All of these studies lay a foundation for the application of Pseudomonas putida IOFA1in the degradation of formaldehyde pollution.