The Bioinformatics Analysis Of Acinetobacter DSSKY-A-001 Estrogen-degrading Enzymes

In recent years,the environmental estrogen pollution problem has aroused the concern of domestic and foreign scholars.Biodegradation is an effective way to control estrogen pollution in the environment.The study on the expression regulation of estrogen degrading enzyme and its gene is an important basis for the construction of the engineering bacteria for the degradation of estrogen.Therefore,it is important to study the basic properties of estrogen-degrading bacteria and the analysis of the structure and function of estrogen-degrading enzymes.In this paper,a strain of estrogen-degrading bacteria was isolated from the soil samples collected from a pharmaceutical factory in Beijing,and its basic characteristics,metabolic mechanism and characterization of degradation enzyme genes were studied.The results are as follows:1.Isolation and identification of estrogen-degrading bacteriaThrough the morphological observation,physiological and biochemical analysis and 16 S rDNA sequence analysis and identification,the results showthe strain is Acinetobacter,named DSSKY-A-001.2.The Whole genome sequencing and functional gene annotation of Acinetobacter DSSKY-A-001Single molecule realtime was used to perform the whole gene sequencing.The results show that the strain DSSKY-A-001 genome size is 3132860 bp,the content of GC is 41.61%,the CDS note number 2963,the gene coding sequence accounted for 85.99% of the total length of the genome.A total of 76 tRNAs,21 rRNAs,1 sRNA,6 random repeats,and 3 tandem repeats were obtained.2174 genes,1770 genes,2036 genes,1347 genes and 2877 genes were obtained using COG,KEGG,GO,Swiss-Prot and NR annotations.Through the functional gene annotation,the results show that a total of seven genes associated with estrogen-degrading enzymes were obtained.In this paper,we analyzed the estrogen-degrading enzyme-related genes and estrogen metabolic pathways in strain DSSKY-A-001.3.Study on estrogen degradation ability and prediction of metabolic pathway of AcinetobacterDSSKY-A-001The estrogen degradation rate and the growth density of the strain DSSKY-A-001 were determined by high performance liquid chromatography and microplate reader.The results show that the degradation rate of E2 increased with the increase of culture time,and the degradation rate reached 76% at the 3th day.The degradation rate of E2 reached 89% at the 6th day.Three kinds of estrogen metabolic intermediates were detected by high performance liquid chromatography and mass spectrometry,and further predict the metabolic pathway of estrogen and the enzyme may be involved in the estrogen-degradation.4.Expression and bioinformatics analysis of estrogen-degrading enzymeBased on the strain DSSKY-A-001 genome functional gene annotation and sequence homology comparisongene analysis three kinds of estrogen degradation related enzymes.Research on cloning of gene and expression of mRNA,Catechol 1,2 dioxygenase gene RspwpGM001668,dioxygenase gene RspwpGM002188 and 7?-hydroxysteroid dehydrogenase gene RspwpGM001333 using PCR and RT-PCR technology.BLAST,ExPASy-Translate,ExPASy-Protparam,DNAMAN,SOPMA,tool and SWISS-MODEL were used to analyze and predict the structure and function of 3 kinds of estrogen-degrading enzymes,results show,Catechol 1,2 dioxygenase gene RspwpGM001668,dioxygenase gene RspwpGM002188 and 7?-hydroxysteroid dehydrogenase gene RspwpGM001333 consists of 921,813 and 774 nucleotides and encodes a total of 306,270,and 257 amino acids,respectively.Catechol 1,2 di oxygenase RspwpGM001668 and dioxygenase RspwpGM002188 may be hydrophilic proteins,7?-hydroxysteroid dehydrogenase RspwpGM001333 may be a hydrophobic protein.The two level structure is mainly composed of alpha helix,beta fold and random coil,among them,the percentage of the three groups of catechol 1,2 dioxygenase RspwpGM001668 were alpha helix(37.25%),beta fold(7.84%)and random coil(34.64%);Dioxygenase RspwpGM002188 percentage for alpha helix(35.19%),beta(11.11%),random coil(33.33%);7?-hydroxysteroid dehydrogenase RspwpGM001333 percentage for alpha helix(42.80%),beta(8.95%),random coil(30.35%).Catechol 1,2 dioxygenase RspwpGM001668 using 2xsr.1.A as template,dioxygenase RspwpGM002188 using 3wrb.1.A as template,7?-hydroxysteroid dehydrogenase RspwpGM001333 using 3gaf.1.A as template to construct three stage structure.Through the analysis of bioinformatics of the three enzymes,provide a theoretical basis for further research of structure and function for estrogen-degrading enzymes.