The Construction Of Ibuprofen Degradable Subclone Strains And The Gene Location Of Regulators

Ibuprofen is the typical phenylpropionic acid like and non-steroidal anti-inflammatorydrugs (NSAIDs). It belongs to the most important pharmaceuticals and personal care products(PPCPs). The research about reducing and eliminating its harm and potential risks to publichealth and aquatic environment focuses on the issue of microbial degradation of ibuprofen.All these procedures such as screening the ibuprofen degradable strains; Cloning theibuprofen degradable genes and illustrating the possible ibuprofen degradable mechanism atthe molecular level will provide strains guarantee for the ibuprofen degradable microbialinoculums’ exploitation and the ibuprofen elimination in the urban sewage during earlierprocessing period as well as in source of drinking water protectorate. Ibuprofen subclonestrains were constructed based on Sphingomonas sp. strains Ibu-2. The molecularidentification of meta-cleavage activity differences between ibuprofen degradable fosmidlibrary strains3G7and4F6were exposited. Major findings and conclusions were summarizedin the following:(1)3G7was the sole positive clone strain which accumulated brown catechol polymersin the constructed Ibu-2fosmid library. When catechol was added to the liquid culture ofIbu-2fosmid library, the other clone strain4F6accumulated yellow products. Those2fosmidlibrary clone strains3G7and4F6almost had the same degradation genes backgroundaccording to the PCR amplified results.4F6had higher meta-cleavage activity than3G7during the ibuprofrn and non-ibuprofen treatments. The Tn5insertion site of the mutants G9and H5that were derived from transposon insertion mutant library of3G7was located at theC23O gene.(2) Tn5transposon was electroporated from fosmid library clone3G7to4F6mediatedwith λ-red homologous recombinase in the pKD46plasmid. The Tn5mutants of4F6-G9and4F6-H5were constructed after curing pKD46plasmid at42℃. No significant differencesamong the3G7and4F6Tn5mutants were shown in the MC activity assays. This researchprovides a simple and efficient method to target ipfC230gene with the gene knockoutprocedure. It would be a valuable tool for the further study of ibuprofen degradation regulators.(3) Several subclone strains that were including1.2kb,1.6kb,2.5kb,4.0kb and8.0kbgene fragments respectively were constructed. The fosmid plasmids of strain3G7and4F6were extracted. After using1%agarose gel to concentrate and Kit to purify those plasmids,the samples of3G7and4F6were digested with ApaI, BamHI, ClaI, EcoRI and HindIIIrespectively. The sole bands that were only existed in3G7or4F6were cut from those enzymedigested plasmids agarose gel after comparing their gel picture. The vector pBBR1MCS-4was digested with same enzyme. The target gene fragment and the correspondence vectorfragment were ligated with T4DNA ligase. Those transformant were electroporated to JM109.The white positive transformant were selected on X-gal plates and were confirmed with PCRby using M13R and T7F primers. These recombination plasmids from positive subclonestrains were extracted and sequenced. There was a catechol degradation regulator in thesubclone strain that was including1.2kb gene fragment digested with HindIII.(4) Those restriction enzymes digested fragments were electroporated to the oppositecompetent cell. Recombination strains were screened after over-express of subclone plasmidto the opposite cell. The MC activities were measured among ibuprofen clone stains, subclonestrains and recombination strains. Degradation regulator was located comparing thoseplasmids sequencing. When4kb fragment that was digested with ApaI from3G7was put to4F6, the MC activity was dramatically decreased. It proved that over-express this4kbfragment in4F6will decrease the MC activity. There was a negative regulator(down-regulated gene) in this4kb fragment. The Tn5transposon mutants A1of3G7hadhigher MC activity than original clone strains3G7and4F6, i.e. the insertion of Tn5enhancedMC activity. There was a negative regulator in this Tn5insertion locus.