Study On The Expression And Activity Of Alkane Hydroxylase Gene Of Hydrocarbon Degenerating Bacteria TY22

In recent years,all kinds of oil spill accidents also occur frequently with the continuous mining of oil.The problem of soil pollution caused by oil leakage is becoming more and more serious,which has attracted the attention of scholars at home and abroad.Therefore,people are beginning to look for a high efficient and low cost management method.Among them,microbial soil remediation has gained more and more attention with its unique advantages.Purification,separation and identification of microorganisms that can efficiently degrade petroleum hydrocarbons from petroleum contaminated areas have been studied.And the mechanism of degrading hydrocarbon microbes has become a hot topic in this field.The intracellular alkane hydroxylase plays a key role in the process of the degradation of petroleum hydrocarbon.Studying the alkane hydroxylase gene and its related regulatory expression will help to explore the degradation mechanism of alkane degrading bacteria to petroleum hydrocarbon.This provides a certain practical basis for the remediation of microbial oil pollution..The research group collected samples from the oil-contaminated soil near the refinery of Nanchong,and screened a petroleum-degrading bacteria TY22 that can efficiently degrade medium and long-chain alkanes.The strain belonged to Acinetobacter beijerinckii and it can effectively degrade alkanes C₆ to C₃₂.Therefore,the bacteria have the potential to repair petroleum-contaminated soil.The homologous similarity between the alkB sequence of the strain TY22 and the alkB sequence of Acinetobacter sp.M-1 reached 88%.In order to explore the characteristics of the TY22 alkane hydroxylase,the upstream and downstream primers of the alkB gene were designed by consulting a large number of literature.The encoding sequence of the hydroxylase gene for degrading petroleum hydrocarbons was amplified by polymerase chain reaction,using the genomic DNA of the TY22 as a template.The target gene was ligated with the vector pET28a?+?to construct a recombinant plasmid,and the protein was expressed in E.coli BL21?DE3?.It was found that the expression of the target protein was higher with 0.1 mM IPTG,180 rpm,37?culture 4 hours.Electrophoretic detection showed that the molecular weight of the target protein was about 45 kDa,which was the same as expected.In order to further explore the reaction conditions of the enzyme activity of the hydrolyzate,the recombinant protein was purified by nickel affinity chromatography.The concentration of the purified recombinant protein was 1.475 mg/ml,measured by Coomassie brilliant blue.The optimum conditions for the activity of the crude extract of the enzyme were tested.And the optimum temperature was between 30?and40?,and the optimum pH was about 7.5.Under the conditions of pH 7.5 and 37?,the activity of the enzyme was 0.076 U/mg.The functional complementation experiments were carried out in the strain Pseudomonas fluorescens KOB2?1 and E.coli GEc137.The results show that the alkB gene of the TY22 has the function of degrading alkanes C₁₂2 to C₁₆.The results provide a certain theoretical basis for the further use of the strain in the biological treatment of oil wastewater.