Clone Of Alkane Hydroxylase Gene And Characterization Of The Petroleum Hydrocarbon Degrading Bacteria TY22

Oil is the world’s most important energy material. But in the course of oil exploration and use, it always cause large amounts of petroleum contaminants leaking into the surrounding environment and cause serious pollution. With the situation becoming more and more serious, bioremediation technology has become a hot research in this field for it is efficient and without secondary pollution, alkane hydroxylase as a key enzyme to start the entire degradation process, has also become the focus of this research. This dissertation studied on a petroleum hydrocarbon degrading bacteria TY22 isolated from petroleum-contaminated soils of Nanchong oil refinery. TY22 was identified as Acinetobacter beijerinckii, its the most appropriate temperature is 30 ℃,optimum pH 7.0, could degrade n-alkanes with chain length C12^C32.Microbial growth and metabolism are closely related to metal ions. This study surveyed the influence of 15 metal ions on petroleum hydrocarbon degrading bacteria TY22. The results showed: Na⁺, Mg²⁺, K⁺, Mn²⁺, Ca²⁺, Co²⁺, Cu²⁺, Zn²⁺ at lower concentration had a promoter action to TY22 growth, but the effect of Ca²⁺, Co²⁺, Cu²⁺,Zn²⁺was relatively small. When they exceed the optimum concentration, they had obvious inhibitory; Fe³⁺, Ti⁴⁺ had certain inhibitory action to TY22, but at low concentration of Fe³⁺ had little effect on it, and when they reached a concentration of 1mmol/L, it had certain growth potential; Al³⁺, Pb²⁺had a strong inhibitory effect on TY22, when their concentration reached 0.8 mmol/L, the biomass had decreased to below 0.1; Ba²⁺, Ag⁺, Ni²⁺ had intense inhibitory action to TY22, when they reached a concentration of 0.1 mmol/L（Ba2 ⁺ reached 0.4 mmol/L）, the biomass was almost zero.This paper also studied the alkane hydroxylase genes alkB of petroleum hydrocarbon degrading bacteria TY22. Genomic DNA of TY22 as a template, we got a partial sequence of the alkane hydroxylase gene by PCR amplification. Then, through the chromosome walking techniques, eventually we got the complete CDS sequence of alkane hydroxylase gene alkB（GenBank Accession: KU867870）, and sequence length is 1236 bp. This gene encodes a stretch of 411 aa protein, relative molecular weight and isoelectric point are 46.7 kDa and 9.22 respectively. This amino acid sequence is closely related to some alkane hydroxylase amino acid sequence of Acinetobacter beijerinckii,homology can reach up to 99%. Predicted it`s secondary structure comprising 37.47%alpha helix, 10.71% beta turn, 35.28% random coil and 16.55% extended strand.Further experiments, we cloned the alkane hydroxylase gene alkB into pET21b（+）vector, and transformed the recombinant plasmid into E.coli BL21（DE3） for expressing.The result showed that the expression strains had the most expression with 0.1 mM IPTG, 30 ℃ culture 6 hours, and relative molecular mass 46 kDa was also in line with expectations. Then cloned the alkane hydroxylase gene alkB into pCom8 vector, and transformed the recombinant plasmid into E.coli GEc137（pGEc47 â–³ B） and Pseudomonas fluorescens KOB2 Δ 1 respectively for gene function complementation test. The results showed: the alkane hydroxylase gene alkB of petroleum hydrocarbon degrading bacteria TY22 could oxidize n-alkanes with chain length C12^C16.