| 3-PBA is known to be non-specific and frequently detected metabolite of various pyrethroids(a class of insecticides, which is globally used for agricultural and consumer applications) such as cypermethrin, deltamethrin, permethrin, cyhalothrin, etc. It has been used as a marker for pyrethroids exposure, and it possess anti-estrogenic activity due to which it is also considered as endocrine disruptors.During biodegradation study of various pyrethroids,there are not any metabolite found,so prediction research analyzes 4-D could also use 3-PBA as its carbon source. Post-study on degradation research shows that the degradation rate of 3-PBA(250mg/L) by 4-D is 50%. In the paper,to find an efficient and advanced method which can analyze the content of 3-PBA,research improves the traditional method by HPLC and has a good linear relation.With the construction of genomic library from(Acinetobacter sp.)4-D,experiment screens the recombinant 4D-3-4 who could degrades 3-PBA as sole carbon source.Sequenced the insertion of 4D-3-4, analyzing from the BLAST result, we can conclude that the degradation ability owe to the gene D34 with the activity of catechol 1,2-dioxygenase.Research also analyzes amino acid composition,secondary structure composition and protein tertiary structure diagram of this gene.Amino acid Sequence length is306,the number of aligned proteins is 47,the numberof matched PDB structures is13.Analyzing the gene sequence by SWISS-MODEL,the potein doesn’t have disulfide bonds and has two domains.The active site is the first domain,the other is relevant to folding intermediate and Transmembrane.The protein folding doesn’t need molecular chaperons but needs the participation of Ca2+.The primer is designed on the authority of opening reading frames(ORF) with Primer Premier 5.0 and creat e restriction sites of Nde I and Hind III restriction endonuclease. The recombinant expression vector pET-21b-D34 plasmid is constructed and transformed into competent cell BL21(DE3). The research also investigates SDS-PAGE analysis during which the specific expression of 46 kDa protein can be found.3-PBA degrading protein has catechol response feature and the best reacting condition is pH 7.0 and 30oC,which is in consonance with 4-D. |
