| Di-n-octyl ortho-phthalate (DOP) is a kind of ester derivatives of the phthalic acid(PA), which belongs to the phthalic acid esters (PAE). It is widely used in coating, food packaging materials, lubricants, medicine, chemical fertilizer, cosmetics, pesticide, medical equipment, children’s toys and other fields. The bioaccumulation of DOP attracted widespread attention all over the world has harmful effects on human body, causing neurotoxicity, reproductive toxicity, multiple organ cancer and other diseases. US Environmental Protection Agency (EPA) and the Chinese government have identified PAE as priority pollutants. Toxicological experiments showed that DOP has mutagenic, carcinogenic, teratogenic and pseudo/anti-thyroid hormone activity, as well as pseudo/ anti-estrogenic activity of endocrine disrupting propertie to human body. At the same time it is an environment hormone compound. The environmental degradation of DOP could by the ways of biodegradation and photolysis, and the biodegradation is the primary way as photolysis had low efficiency and time-consuming. The current research about DOP microbial degradation includes screening and identification of the degrading bacteria, as well as the analysis of their degradation pathways and degradation characteristics. However, few reports had focus on the main functions about the proteins involved in degradation pathway.In this study, the key enzymes (pehA/B) involved in phthalate degradation were cloned and expressed from Dordonia sp.HS-NH1, and the functions and features of the enzymes were also investigated. Combined with gene knockout technology, the ABC and MFS transporter protein missing mutants were successfully constructed, and the metabolic pathways and degradation characteristics of mutants were analyzed. The main results are as follows:The genome scanning of Gordonia sp. HS-NH1 showed that the length of pehA was 594 bp, encoding 197 amino acids. It was highly homology with Rhodococcus sp. YK2, which is up to 98%. The length of pehB was 852 bp, encoding 283 amina acids, and it has the 99% similarity with Gordonia sp. P8219. Using cloning technology, we successfully constructed recombinant plasmids pET-pehA and pET-peh, and they were highly expressed in E.coli BL21. The SDS-PAGE analysis showed that the molecular weight of pehA and pehB were 27 KDa and 30 KDa, respectively. PehA and pehB has good hydrolytic activity to PAE with long chains, which had a foundation for further study of the functions and structures of these enzymes.The ABC and MFS genes of Gordonia sp. HS-NH1 were successfully knocked out by homologous recombination, which named as HS-NH1-△MFS and HS-NH1-△ABC. The metabolic pathway, gene functions and degradation characteristics of mutant strains were analyzed. The results indicated that the optimum conditions of the HS-NH1 on degradating DOP were 30℃ and pH7.0, and could degrade 80% DOP in 120 hours. Under the same conditions, the DOP degradation efficiency was decreased significantly by HS-NH1-△MFS, and HS-NH1-△ABC could not grow in the medium with DOP as the sole carbon source. Substrate spectrum analysis showed that Gordonia sp. HS-NH1 could use PAE efficiently, as well as its metabolites. The mutabt HS-NH1-△MFS could use PAE with long chains, but the efficiency was lower and it could not use PAE with short-chains. The mutabt HS-NH1-△ABC could not grow in the medium with PAE as sole carbon source. In summary, both MFS and ABC could transport PA in cells, and PA will be further degradated intra-cells. Thus, ABC was the major transport protein to PA, and MFS was an auxiliary transport protein. |
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