Molecular Mechanisms Of Two Newly-isolated Bacterial Strains During Biodegradation Of Typical Brominated Flame Retandants

Tetrabromobisphenol-A（TBBPA）,as one of the most frequently used Brominated flame retardants（BFRs）,has been used extensively to prevent fire in the products including electronic equipment,construction materials,plastics,furnishing foam and textiles.Unfortunately,it has been found in air,dust,water,sewage sludge,sediment,soil,and human and animal tissues because it could release to the environment during its production,use,disposal,and recycling processes.TBBPA has given rise to people’s attention around the world due to potential harmful effect on the environment and human beings.To date,most of works on the biodegradation of TBBPA were mainly focused on the isolation and characterization of the degradation bacterial strains,the debromination and biodegradation kinetics as well as biodegradation mechanism of the TBBPA.The reports on the biochemistry and the molecular genetic mechanism as well as the key enzyme encoding gene analysis and application,genetic modified strain construction were very limited.Thus,in this thesis,two pure bacterial strains,Ochrobactrum sp.T and Bacillus sp.GZT,with higher ability to degrade TBBPA and 2,4,6-tribromophenol（TBP）,respectively,were selected as our target strains.The identification of TBBPA and TBP degrading genes and catalyzed enzymes from these two strains were carried out by combing whole-genome sequencing with gene cloning and expression.Furthermore,a progressive purification protocol including crude extract preparation,ammonium sulfate precipitate,DEAE-cellulose,and gel-filtration was used to isolate the degradation enzyme from the wild type Bacillus sp.GZT.Finally,the main produced intermediates were identified,and the fate of TBP and TBBPA by the corresponding degrading enzyme under oxic conditions were explored,respectively.Main results of this thesis are listed as follows:（1）By genome sequencing of Ochrobactrum sp.T,a total of 24 open reading frames（ORFs）related to the dehalogenases were achieved.Then these genes were systematically cloned and expressed and one constructed strain containing inserted genes,designated as tbbpa was found to be able to degrade TBBPA.Sequence alignments analysis of the amino acid sequences of the recombinant enzyme showed that it shared 100%identity with haloacid dehalogenases.Like the original strain,the constructed strain could fully remove TBBPA（6 mg/L）with 37.8%mineralization efficiency and 78%of debromination efficiency within 96 h under of 30°C,pH 7.0.Gene expression study also revealed that,tbbpa gene expression can be inducted and was up-regulated in the presence of TBBPA.The co-substrate TBP promoted the TBBPA biodegradation at the reaction beginning by the constructed strain,while the addition of bisphenol A（BPA）could promote the TBBPA degradation.（2）A TBBPA-reducing enzyme from the cytoplasm fraction of the above aerobe recombinant strain was purified using nickel-affinity chromatography.A total of 10.2-fold enrichment with a yield of 58.5%was achieved and the specific activity of the enzyme increased from 0.3 to 3.06 units/mg protein after purification.The molecular mass of the purified bromophenol dehalogenase was determined to be 117 kDa by SDS-PAGE and MALDI-TOF/TOF.The recombinant enzyme possessed an optimal pH of 6.5 and an optimal temperature of 30°C,and manifested a K_m of 26.6μM and a V_max of 0.133μM/min/mg.In addition,the present of the cofactor NADPH and methyl viologen could strongly increase the enzyme activity of TBBPA dehalogenation,whereas the addition of Cu²⁺,ascorbic acid,cytochrome C to the enzyme mixtures showed strongly inhibition effects.Overall,the enzyme displays wide substrate specificity towards brominated compounds and particularly high activity towards 2,6-dibromophenol（2,6-DBP）and 4-bromophenol（4-BP）.Based on the three identified metabolites,the biodegradation mechanism of TBBPA by this enzyme can be described as followed:with the help of oxygen,TBBPA was initially oxidized by dehalogenase to deprivate an electron and proceeded via beta-cleavage to form 4-propenyl-2,6-dibromophenol and 2,6-dibromophenol,Then,4-propenyl-2,6-dibromophenol was further converted to TBP,which was further dibrominated to form 4-BP.After that,sequentially reductive debromination of bis（4-bromophenoyl）ether to 4-bromodiphenyl ether and diphenyl ether by replacement of a Br atom with a H atom were also deduced.（3）After a progressive purification protocol,a purified dehalogenase with TBP degrading activity was achieved from TBP degradation strain Bacillus sp.GZT.The results showed that as the purification progress,the target enzyme was purified 47-fold and the specific TBP dehalogenase activity increased from 0.4 to 18.8 U/mg of protein.Approximately 28.6%of the activity of TBP dehalogenase in the crude enzyme was recovered.SDS-PAGE analysis revealed a single band with a molecular mass of 63.4 kDa.Through MALDI-TOF/TOF analysis,the dehalogenase showed high similarity with oligopeptide ABC transporter oligopeptide-binding protein and peptide ABC transporter substrate-binding protein.Under optimal conditions of pH=6.5,35 ^oC,up to 80%degradation efficiencies were achieved within 120 min.The measured K_m value and V_max were 78μM and 0.65μM/min/mg/protein,respectively.Besides,the enzyme showed activity toward 2-bromophenol（2-BP）,2,4-dibromophenol（2,4-DBP）,2,6-DBP,3-bromophenol（3-BP）and 4-BP.By cloning and expression of the purified enzyme encoding gene,the purified enzyme and encoding gene was characterized.The metabolism mechanism of TBP by this dehalogenase showed that three bromine atoms on the TBP molecule were substituted in a stepwise progression.The first step of the reaction is producing 2,4-DBP and 2,6-DBP by removing either an ortho or para bromine atom,then these two dibromophenol products further metabolize into benzoic acid and penylacetic acid through 2-BP.This mechasnism is very similar with the TBP biodegradation mechanism by the Bacillus sp.GZT,suggesting that the dehalogenase we purified is the major enzyme in this strain.（4）A 5.18-Mb genomic DNA containing 21 putatively TBP degradation-related opening reading frames（ORFs）was obtained by draft genome sequencing.After cloning and expression of these genes into E.coli BL21（DE3）,a total of five recombinant strains containing the functional genes,designated as tbpA,tbpB,tbpD,tbpE and tbpC,recovered the ability to grow on the mineral medium amended with TBP,2,4-DBP,2-BP,phenol and 2,6-dibromo-4-methylphenol,respectively,and the corresponding enzyme was designated as tbpA,tbpB,tbpD,tbpE and tbpC.The tbpA gene encodes a cytochrome P450 reductase that can catalyze the initial step of TBP degradation and able to remove 98.8%of TBP.Reverse transcription-PCR analysis showed the relative transcript levels of these genes were significantly up-regulated and they were inducible by their specific substrate.The proposed biodegradation mechanism inferred that the tbpB,tbpC and tbpD encodes similar halophenol dehalogenases,which catalyzes the convert of 2,4-DBP,2,6-dibromo-4-methylphenol and 2-BP,respectively.Finally,the debrominated products phenol is transformed by tbpE to form CO₂ and H₂O.