Enzyme activity involved in bacterial degradation of octylphenol polyethoxylate：alcohol dehydrogenase, aldehyde dehydrogenase, acetyl-CoA synthetase, isocitrate lyase and malate synthase

• Main Authors: Chen-Wei Yeh, 葉辰瑋
• Other Authors: Shir-Ly Huang
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/11740071350870060319

碩士 === 國立中央大學 === 生命科學系 === 104 === Alkylphenol polyethoxylates (APEOn) that include octylphenol polyethoxylate (OPEOn) and nonylphenol polyethoxylate (NPEOn) are non-ionic surfactants and widly used in industy, agriculture and domestic purposes. APEOn are often found as pollutants in natural aquatic environments and in raw municipal wastewater. Ethyleoxylate chain of APEOn is degraded by microorganisms in environment to produce estrogenic metabolites, such as nonylphenol and octylphenol. They accumulate in environments and cause chronic ecotoxicity as endocrine disrupters to aquatic organisms, wildlife, and humans. Pseudomonas nitroreducens TX1 was found from sediment of irrigation farmland and was capable to use a wide range concentrations of APEOn, as a sole carbon source. By proteomics and transcriptome RNA seq analysis, alcohol dehydrogenases, aldehyde dehydrogenases and acetyl-CoA synthetase have higher expression level when growth in MSB medium containing 0.5% OPEOn as a sole carbon source than that in 0.5% succinate. Therefore, crude extract of P. nitroreducens TX1 growth in 0.5% OPEOn was used to optimize the activity assay for three enzyme. The enzyme activities of alcohol dehydrogenase, aldehyde dehydrogenase and acetyl-CoA synthetase have been shown 4.1, 6.9 and 2.1 fold increase than those in 0.5% succinate. The substract specificty of OPEOn-dependent alcohol dehydrogenases was demonstrated that APEOn related compounds are the enzyme substrates but not using polyethoxyl glycol (PEG) and ethanol. Due to the draft genome sequence of P. nitroreducens TX1 was already published and provides basal informations in investigating gene loci and sequence related to significant roles in alkylphenol polyethoxylate degradation. Transposon mutagenesis was used to create mutant library of P. nitroreducens TX1 for the screening of growth defected mutants on OPEOn. Over 30,000 mutants have been isolated after triparental mating between transposon DNA containg E.coli and P. nitroreducens TX1. 145 mutants grew slowly or did not grow on OPEOn medium. The mutated genes in 130 mutants have been identified. Among these mutants, six transposon insertion mutants and eight mutants were idendifed in isocitrate enzyme and malate synthase, respectively. Therefore, the two enzyme were selected for further enzymatical activity study. The two enzymes have 8.9 fold and 1.7 fold increased in their activities in crude extracts from growth in OPEOn MSB medium than in succinate MSB medium. Furthermore, isocitrate lyase was cloned and expressed in E.coli. The isocitrate lyase has high enzyme activity for its putative target, isocitrate, but has no activity for OPEOn and its derivatives. Therefore, according to these results, the isocitrate lyase is not directly involved in the biodegrading of OPEOn. The isocitrate lyase may paly an important role at the downstream metabolic step, converting to glyoxylate cycle. Based on current evidences by proteomics, metabolites, transposon mutagenesis mutants and enzyme activities in this study, we proposed the OPEOn metabolic pathway in P. nitroreducens TX1 that the OPEOn biotransformation follows the proposed polyethoxylate chain biodegrading mechanism that OPEOn processes the terminal end of polyethoxylatic alcohol oxidation by alcohol dehydrogenases to form terminal aldehyde and further oxidizes to form carboxylic acid, named OPEC, by aldehyde dehydrogenase. The two carbon source, acetate, was somehow produced from OPEC, and catalyzed by acetyl-CoA synthetase to form acetyl-CoA. It goes into glyoxylate cycle via isocitrate lyase and malate sythase to produce OAA and succinate in TCA cycle to support P. nitroreducens TX1 growth.