Isolation And Identification Of DBP And DEHP-degrading Strain And Complete Genome Sequence Analysis

Di-?2-ethylhexyl?phthalate?DEHP?and di-n-butyl phthalate?DBP?were common plastic plasticizers in phthalate esters?PAEs?.PAEs were widely used in the architecture industry,household goods,and medical equipment,and so on.Because there is no covalent bond among DBP,DEHP and plastic,they are easy to release from the plastic products and migrate into the surrounding environment,such as surface water,soil and atmosphere.In order to eliminate DBP and DEHP in the environment,biodegradation is one of the widely studied methods.The preliminary studies on the microbial degradation of DBP and DEHP were about the screening,domestication and degradation characteristics of degrading bacteria.However,the research on degrading functional genes was not very thorough.Therefore,it is necessary to increase the research input on the molecular level of degrading bacteria.The enrichment of the gene bank data had a certain theoretical and practical significance for the exploration of different bacteria in the future.In this study,a plant endophyte that could efficiently degrade DBP and DEHP was isolated from a plant near a polluted canal in Shaoguan City.Based on morphological identification,physiological and 16S rRNA gene sequence analysis,it was identified as Bacillus subtilis.?1?Using PAEs?DBP and DEHP?as degradation substrates,the optimum pH,temperature for growth and biodegradation by Bacillus subtilis was 7.0 and 30°C,respectively.PAEs?50 mg/L?could be degraded by the strain within 3-5 days incubation,and the DBP degradation rate was 90%,and the DEHP was about 40%.?2?At first,bacillus subtilis acted on an ester bond in DEHP,causing it to break down to form monoethylhexyl phthalate?MEHP?,then hydrolyze the other ester bond to form phthalates?Phthalic Aicd,PA?,finally formed the simple compound CO₂ and H₂O under the action of a series of enzymes.?3?The whole genome sequencing of this strain was performed by Illumina Hiseq third-generation sequencing technology produce 1473727800 bp filtered sequence,representing a 100-fold coverage of the genome.The SOAP denovo software was used to initially assemble the sequencing data and the blasR ratio was used to correct the Pacbio data,followed by subsequent assembly,and finally the genomic map of the Bacillus subtilis strain was completed.?4?The whole genome size of the strain was 4149636 bp with the GC content of43.70%.There was a CRISPs?clustered,regularly spaced short palindromic repeats?in the strain.Bacillus subtilis also contained 24 rRNAs,81 tRNAs,and 11 sRNA operons,25gene islands with a total length of 343635 bp.A total of 5 prophages with an average length of 25100 bp,354 interspersed repeated sequences and 70 short tandem repeats.?5?Bacillus subtilis was whole genome scaned and obtained 4344 open reading frames?ORF?.In order to obtain functional annotations of predicted genes,the predicted protein sequences were compared with NR database,Swiss-Prot database,COG,and GO database,respectively.Through GO functional analysis,it was found that the main function of Unigene was catalytic function,binding function and transport activity.The KEGG annotation results indicated that the Unigene enrichment pathway was mainly concentrated on metabolic pathways,biosynthesis of secondary metabolites,and microbial metabolism in different environments.?6?Combined with gene annotation results,enzyme genes involved in PAEs degradationwerepredictedandincluded3loop-cleavingenzymes,1carboxymuconolactone decarboxylase,3 decarboxylase enzymes,and 4 monooxygenase enzymes.The above research provided a theoretical basis for the next transcriptome sequencing.The above results provided the theoretical basis for the next transcriptome sequencing.At the same time,it provided a new research direction for the bioremediation of DBP and DEHP in the environment.