Screening Of Oil Degrading Bacteria And Cloning Of Key Genes

Animal and vegetable fats are widely present in the natural environment,in form of triglycerides.Large amounts of fats and oils discharged into the environment will cause soil and water oil pollution.At present,the amount of household garbage and other domestic waste is increasing year by year.Therefore,more and more attention has been paid to the degradation of catering waste oil and the corresponding environmental problems.In some organisms,triglycerides are degraded by esterases into fatty acids and glycerol.Fatty acids are converted to succinic acid through the glyoxylic acid pathway.Succinic acid can enter the metabolic pathways of other biological macromolecules.Therefore,the key step in the construction of a single carbon source efficient degradation strain for oils and fats is to obtain the key enzyme genes of the glyoxylate cycle.In this study,we enriched and selected a strain with the ability to degrade oil from the sludge at the sewage discharge site of Shanxi slaughterhouse.The strain was identified as a Gram-negative bacterium after physiological and biochemical identification and 16 SrRNA gene sequence homology analysis,belonging to Achromobacter sp.We used UV spectrophotometer and liquid chromatography to determine the degradation characteristics of the strains.The test results show that:(1)The degradation rate of the olive oil reached85% after 72 hours of growth.(2)The optimum p H value of the strain to degrade oil and fat is 7.5,and the optimum temperature is 35 ?.(3)The strain maintains a high degradation rate in an alkaline environment with a pH value below 9,and at the same time,the strain can tolerate a salt concentration below 40 g/L.We obtain the gene sequences of isocitrate lyase and malate synthase by homologous cloning.The results of bioinformatics analysis show that:(1)The open reading frames of citrate lyase and malate synthase gene sequences are 816 bp and 492 bp,encoding 272 and 164 amino acid residues,respectively.(2)The citrate lyase prediction model contains abundant ?-helix structures and a protein binding site,and the sequence does not contain signal peptides and disulfide bonds.The tertiary structure is in the form of homotetramers and does not contain ligands.(3)The malate synthase prediction model contains9 ?-helices and 3 ?-sheet structures and 4 protein binding site sequences,and does not contain signal peptides and disulfide bonds.The tertiary structure is in the form of monomers and does not contain ligands.In this study,the oil-degrading strains isolated are not only of great significance to the study of the microbial degradation of oils and fats,but also enrich the microbial resources of oil-degrading bacteria,and provide the theoretical basis and technical support for the comprehensive utilization of oil degradation and water pollution.The energy reuse of grease waste has significant economic benefits.