Isolation,Identification Of Diphenylether Degrading Strain And Its Metabolic Mechanism Of Diphenylether

Diphenyl ether and its derivatives are widely used in agriculture and chemical industry.Despite its important role in ensuring agricultural production and guaranteeing people's life,it caused serious environmental pollution.Previous studies have shown that diphenyl ether compounds are harmful to the ecological environment and health of human and other organisms.Thus,the study of microbial degradation of diphenyl ether and its compounds has great theoretical and practical importance.In this study,a diphenyl ether degrading strain was isolated,which could utilize diphenyl ether as the sole carbon source for growth and the strain was named as XY₁.It was preliminary identified as Sphingobium sp.according to its phenotypic,physiological and biochemical characteristics and 16S rRNA gene sequence analysis.The optimum growth temperature of strain XY₁ was 30 ?.When pH is in the range of 6.0-8.0,the degradation rate of diphenyl ether is higher than 75%and the degradation rate reaches the highest?95%?at pH 7.0.With the increase of inoculation amount,the degradation rate of diphenyl ether was also increased.When the inoculation amount was 20%,strain XY₁ could completely degrade 100 mg · L-1 diphenyl ether in 24 hours.When the initial concentration of diphenyl ether was 200 mg · L-1 or less than 200 mg · L-1,the degradation rate of strain XY₁ was more than 90%,but when the concentration of diphenyl ether reached 300 mg · L-1 or above,the degradation performance of the strain XY₁ to diphenyl ether was decreased,indicating that the high concentration of diphenyl ether could inhibit or poison the growth of XY₁ cells.The metabolites products after degradation of diphenyl ether were detected by HPLC and LC-MS.We found that there was two metabolites which could be detected:2,4-hexadienal phenyl easter and phenol.Here,we proposed the possible metabolic pathway of diphenyl ether degradation by strain XY₁:firstly,through the double oxygenation in the oxygen atoms and oxygen atoms adjacent to the carbon atoms of diphenyl ether to form 2,4-hexadienal phenyl easter;secondly,through ester bond hydrolysis to form phenol and mucelic acid semialdehyde.The strategy used to clone hydrolase gene fragments in strain XY₁ was:diphenyl ether metabolic pathway of the strain XY₁ were consistent with the strain SC₃.They were firstly through the role of dioxygenase to form 2,4-hexadienal phenyl easter,followed by hydrolytic enzyme to hydrolyze ester bonds to produce phenol and mucelic acid semialdehyde.The sequence of the dpeA1A2BC gene which has been cloned is found to have a high homology with the XY₁ genome.Therefore,there are good reasons to judge the two strains was used the same dioxygenase to form 2,4-hexadienal phenyl easter and then hydrolyzed.In addition,our previous strategy for cloning dpeAIA2BC was to download a pool of related genes responsible for the degradation of biphenyls similar to the diphenyl ether structure.There is reason to suspect that the hydrolytic enzymes responsible for the hydrolysis of 2,4-hexadienal phenyl easter and the biodegradation products of biphenyls-2,4-hexadienoic acid hydrolase has a similarity.According to previous studies,we obtained the gene sequence of hydrolase genes which catalyzed the degradation of biphenyl degradation.We compared its amino acid sequence with XY₁ genome using the dot plot function of Omiga 2.0 software to find homology.By comparison analysis,a gene with high homology to the reported bphD gene was found in the genome of XY₁,named as dpeD.Then we introduced this gene into expression vector pET₂8a and induced the expression of its protein production DpeD.Finally,we purified this protein by nickel ion affinity chromatography.