Degradation Of Di(2-Ethylhexyl) Phthalate By Rhodococcus Sp.Strain P52 And Cloning And Expression Of The Genes Encoding Dibenzofuran Dioxygenase

Phthalates and dioxins are all environmental hormone Substances, the former of which widely used in plastic Products, and the latter are mainly produced in waste incineration. Due to their constant release to the environment, not only the natural environment is polluted, but also human are facing a huge health threat.In this thesis, Rhodococcus sp. strain p52 was used to degrade bis(2-ethylhexyl) phthalate (DEHP), which is the most representative compound in phthalates. The research Results showed that DEHP can be degraded about 75% as the initial concentration of DEHP is 200mg/L. At the same time, the orthogonal experiment Results indicated that the influence of environmental factors on DEHP degradation rate is as follows:the initial concentration of DEHP> temperature>pH.Rhodococcus sp. strain p52 contains two types of dibenzofuran(DF) dioxygenase systems which can attack DF in angular manner. In srain p52, dbfA gene cluster and dfdA gene cluster encode the DF dioxygenase systems respectively. In this study dbfA1A2, dfdA3, and dfdA4 genes of the dfdA gene cluster were cloned and expressed. SDS-PAGE experiments showed that the expression Product of dbfA1A2 gene contains two protein bands representing two Subunits of terminal oxygenase, DbfAl and DbfA2 respectively. The molecμlar weight of the expression Products are about 65KDa and 22Kda, respectively. The proteins were mainly expressed in the form of inclusion bodies in E. coli BL21 (DE3). The molecμlar weight of dfdA3 expression Product is about 33KDa and was expressed mainly as a soluble protein by E. coli BL21 (DE3). The molecμlar weight of dfdA4 gene expression Product is about 65KDa, which was mainly consisted of inclusions in E. coli BL21 (DE3).Based on the above Results, the function of dbfA1A2 for DF degradation ability in recombinant strain E. coli BL21 (DE3) was also preliminarily discussed in this thesis. The Results showed that the recombinant can degrade DF above 90% in 18h as the initial concentration of DF is 200mg/L. The Results primarily proved that the importance of dbfA1A2 involved in the degradation of DF.In summary, Rhodococcus sp. strain p52 can degrade DEHP significantly and it may have application potential to the remediation of phthalates polluted environment. The cloning and expression of the DF dioxygenase genes provide a basis for the further study of the enzymes activity and application in dioxins removal.