Lip-sub protein fusion acts as a biological wastewater treatment

Background and Aim : Nowadays Wastewater sufficent and perfect treatment is an important point which attract the attention of all countries .instead of using of chemicals and complicated methods and high expenses .researchers got to use of natural microbial enzymes which works better ,economical,less time and not hazardous for ecosystems .in this study by the use of genetic engineering and molecular methods a new recombinant protein was created with component of Pseudomonas putida and subtilisin protease in order to treat wastewater.Methods : At first Gene sequences of lipase of Pseudomonas putida and subtitlisin of Bacillus subtilis got from Gene bank. The restriction enzymes xhoI at the ۳ʹ and Bamh۱ at the ۵ʹwere chosen. PET۲۲b vector was choosed for the cloning vector.the designed protein was checked by ITASSER,GOR۴,EXPASY online protein prediction software for its structure and function. The calcium chloride method was used for the competence cells of this bacterium. the transformation of pet۲۲b + Lip-Linker-Subtilisin was performed on E.coli BL۲۱DE۳.The confirmatory test of designed structure was done with primers which designed and covered all the sequence and electrophoesis. The expression of recombinant proteins is mediated by the transfer of recombinant vector to host cell and induction of protein production in these cells. In lactose-inducible or IPTG-induced pET system. For confirmation of recombinant protein after ۲۴h induction, SDS-PAGE was performed to observe the expression of recombinant protein. The Western-blot was performed according to the Bumatte method. The purification steps of recombinant protein were performed by the Native method using a histidine tag. The Bradford method used for the protein concentration checking.Results : The bioinformatical results conveyed that the structure is suitable and functional for the aim of the study.for confirmatory test The results of amplifying Amplicon with size ۷۵۰ bp for confirming of gen fusion lipase-subtilisin. The induction reached its optimum within ۲۴ hours. Western blot results indicate protein expression and confirm it. Concentration of recombinant fusion protein LIP-SUB with Nanodrop. The recombinant protein produced with a concentration of ۴.۱۱ mg/ml.Conclusion : According to the results the new recombinant protein has a good and suitable structure for the aim of this study.

کلیدواژه ها:

lipase -subtilisin-protease-wastewater