Study On Construction Of Pyrene-degrading Engineered Microorganisms And Characterization Of Pyrene Degradation

Aromatic hydrocarbons,especially polycyclic aromatic hydrocarbons?PAHs?,have mutagenic and carcinogenic effects on humans and animals,which are difficult to degrade under natural conditions,and it is more difficult to degrade high molecular weight polycyclic aromatic hydrocarbons containing three or more benzene rings,which is more harmful to the environment and human body.For example,pyrene,a typical polycyclic aromatic hydrocarbon containing four benzene rings,mainly exists in the distillates of coal tar pitch,and requires a variety of oxidase to be degraded gradually.The key enzyme that plays the role of ring opening cracking is catechol-2,3-dioxygenase?C23O?.The catechol dioxygenase gene was cloned from different microorganisms and the suitable host was selected to express it in order to improve the degradation efficiency of polycyclic aromatic hydrocarbons.Therefore,in this study,two types of promoters,SPOI-II and P2,were used to regulate the expression of exogenous C23O gene,and Escherichia coli was used as host strain to explore the growth and pyrene degradation of engineering bacteria.It provides a new idea for the construction of recombinant bacteria which can efficiently degrade aromatic compounds in the future.In conclusion,two genetically engineered microorganisms were constructed based on Over-lap PCR technique.The main results are as follows:?1?Construction of PET-28a-T7+C23O expression vector,heterologous expression of catechol-2,3-dioxygenase and study of enzymatic properties,the results showed that:the optimum reaction temperature and pH value were 30?and 7.0,respectively.Mn²⁺promoted the enzyme activity,while other metal ions such as Fe²⁺,Cu²⁺,Ni²⁺,Zn²⁺and Co²⁺inhibited the enzyme activity,among which Zn²⁺and Co²⁺showed stronger inhibition.To use bioinformatics software to predict the secondary and tertiary structure of C23O,the results show that:C23O is an acidic hydrophilic protein with no signal peptide.It is composed of 17.9%?-helix,25.8%?-fold and56.3%irregular crimp,and contains Fe³⁺and HPX binding sites.?2?Amplification of catechol-2,3-dioxygenase sequence with two promoters by Over-lap PCR,then two recombinant plasmids,PMD-19T-SPOI-II+C23O and PMD-19T-P2+C23O,were obtained by ligating them into PMD-19T vector.The two recombinant plasmids was transduced to the E.coil Bl21 for constructed genetically engineered microorganisms,the results of SDS-PAGE showed that:the two genetically engineered microorganisms could express the target protein normally,and the size was about 44.3 kDa,which indicated that the genetically engineered microorganisms were successfully constructed.?3?Determinationofgrowthcurveoftwogeneticallyengineered microorganisms in pyrene medium by dilution method of plate counting,the results showed that:when genetically engineered microorganisms were added to pyrene inorganic salt medium alone,the number of SPOI-II-C23O and P2-C23O genetically engineered microorganisms were zero on the third day.When pyrene degrade wild-type bacteria XJ-6 was added simultaneously,the genetically engineered microorganisms could grow,and on the fifth day,the highest number of bacteria was38×10⁶ CFU and 35×10⁶ CFU,respectively.Determination of pyrene degradation rate in each experimental group by HPLC,the results showed that:after 7 days,the degradation rates of pyrene by XJ-6,XJ-6+SPOI-II-C23O and XJ-6+P2-C23O were35.76%,12.37%and 12.3%,respectively.The addition of glucose can increase the degradation rate of pyrene in each experimental group.The results of indoor simulated restoration experiments show that:when genetically engineered microorganisms were added to soil alone,SPOI-II-C23O and P2-C23O could grow,and after 42 days,the highest number of bacteria was 55×10⁴ CFU and 68×10⁴ CFU,respectively.Determination of pyrene degradation rate in each experimental group by fluorescence spectrophotometer,the results showed that:after 48 days,the degradation rates of pyrene by XJ-6,XJ-6+SPOI-II-C23O and XJ-6+P2-C23O were21.6%,25.12%and 26.4%,respectively.