| Summary: | 碩士 === 國立中央大學 === 系統生物與生物資訊研究所 === 98 === Alkylphenol polyethoxylates (APEOn), including octylphenol polyethoxylates (OPEOn) and nonylphenol polyethoxylates (NPEOn), are non-ionic surfactants and extensively used in industrial, agricultural, and household products. Large quantities of surfactants are often released into the environment and are majorly degraded by microorganisms. These degraded products APEOn, n = 0~3 and alkylphenol (AP), act as environmental hormones which mimic the estrogenic activity that are harmful to the aquatic organisms and human health. The complete metabolic pathways and degrading enzymes still remain unclear. Pseudomonas putida TX2 was isolated from a paddy field in Il-Lan, Taiwan, which shown to grow on 0.05~20% of OPEOn or 0.02% of OP as sole carbon source. From the previous studies by LC-MS analysis, P. putida TX2 was able to degrade OPEOn to OPEOn (n≦3) and the formation of octylphenol (OP) by a whole-cell transformation study. The purpose of this research is to isolate an enzyme which can oxidize OPEOn. In this study, an alcohol dehydrogenase (ADH) from P. putida TX2 was preliminarily demonstrated to react on the substrate and then was partially purified. The molecular weight of the denatured protein was estimated to be 55 kDa. When testing using different cofactors, such as NAD(P)+, FAD+ and PQQ in the enzyme assays, ADH was predicted to be a pyrroloquinoline quinone
(PQQ)-linked ADH. Most of the active ADH (PQQ) was present in the crude extracts of the cell, which takes about 48% of the total activity. Both of the ADH (PQQ) in the crude extracts and partially purified ADH (PQQ) after hydrophobic interaction chromatography showed the highest activity at pH 7 and 40oC. However, the pH and temperature are slightly higher after anionic exchange chromatography, i. e., pH 8 and 45oC, respectively. The Lineweaver-Burk plot showed all the enzymes in the crude extracts and from the partially purified ADH (PQQ) have the highest affinity toward AEO8 follow by NPEOn and OPEOn; however, the maximum rate is highest toward OPEOn to NPEOn and finally to AEO8. The hydrophobic interaction chromatography
was the first purification column used due to higher recovery and purification fold. A strong anionic exchange column was used for the next purification step, but the ADH
did not bind on the column. The catalytic products from partially purified ADH from the flow-through of the anionic exchange column were subjected onto LC-MS. The ethoxylate units of OPEOn were shorten after 80 minutes with the presence of OPEO3 and the disappearance of OPEOn, n = 13 and 14 according to the mass spectrum. Aldehyde was not detected, thus ADH in P. putida TX2 transformation is predicted to simultaneously oxidize the hydroxyl groups and aldehyde moieties (based on the substrate specificity assay) by alcohol dehydrogenase.
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