| Phthalic acid esters(PAEs)are widely used as plasticizers in our life,di-n-butyl phthalate(DBP)is one of the mostly used PAEs.With the manufacture,transport and waste of PAEs,they gradually leaked into the environment and posed a serious threat to air,water,soil and organisms.As the limitations of non-biodegradation on PAEs,microbial degradation has become the main pathway to treat PAEs pollution.Therefore,it is very valuable for the bioremediation of environment to screen strains with high-efficiently PAEs-degradating ability and apply them to environment.In our study,an aerobic gram-negative bacterium that could utilize DBP as sole carbon was isolated from the soil heavily contaminated by oil.It was identified to be a strain of Xanthobacter genus and was named YC-JY1 by morphology and phylogeny evolutionary analysis.Physiological and biochemical experiments showed the substances utilized by strain YC-JY1 and the antibiotic genes in it were also preliminary determined.The experiments on degradating characterization of strain YC-JY1 showed that the optimal conditions on DBP degradation by strain YC-JY1 were 30 °C and pH 7.0.Under these conditions,strain YC-JY1 could completely degradate more than 94% of 100-400 mg/L DBP within 5 days.For the higher concentrations of DBP,strain YC-JY1 might need more time to degradate them NaCl could inhibit DBP degradation,metal ions and surfactants had different degrees of promotion or inhibition on DBP degradation.The substrate spectrum experiments revealed that strain YC-JY1 widely utilized other PAEs,the degradation rates of 100 mg/L dipentyl phthalate(DPeP),100 mg/L dihexyl phthalate(DHP)and monobutyl phthalate(MBP)were all above 95%.The intermediates of DBP metabolism were determined by HPLC-MS,they were MBP and phthalic acid(PA)respectively.It could be concluded that DBP was first hydrolyzed to MBP and MBP was further hydrolyzed to yield PA.To investigate the potential application of strain YC-JY1 in real soil,the soil simulation remediation experiment was designed and studied,strain YC-JY1 could repair more than 70% three different kinds of soil contamination by 100 mg/kg DBP within 10 days,indicating great potential application of the strain YC-JY1 in real life.After determining the metabolic pathway of DBP,the key genes acting in this pathway were also investigated.Based on the hydrolase activity and types experiments of PAEs hydrolase in strain YC-JY1,it was determined that the hydrolase in strain YC-JY1 was an intracellular induced hydrolase,which showed the highest hydrolase activity at the end of logarithmic phase.The primers of reported PAEs hydrolase genes were designed to amplify the genome of strain YC-JY1,but no homologous sequences were found.Consequently,a hydrolase gene dphC was cloned from strain YC-JY1 by genomic library construction.Multiple sequences aligning and phylogeny evolutionary analyzing with reported PAEs esterase revealed that the amino acid sequence encoded by dphC containing the "GXSXG" conserved motif,and proposed that DphC might be a new phthalate diesters hydrolase.The recombinant DBP hydrolase DphC reached the highest enzyme activity at 30 °C and pH 8.0.DphC could degradate various dialkyl PAEs,but it could not degradate DPrP and MBP.Kinetic parameters of the dialkyl PAEs that could be degradated were calculated.Intermediates of DBP metabolism were further analyzed by HPLC-MS,only MBP was detected and no PA,so it was confirmed that the hydrolase DphC was a new phthalate diesters hydrolase. |
PDF Full Text Request