Study On The 2-Bromonitrobenzene Metabolic Pathway In Pseudomonas Stutzeri ZWLR2-1 And Its Application

Halonitrobenzenes,a group of important intermediates in chemical production,have extensive applications but are toxic to the human beings in the meantime.with the increasing utilization of halonitrobenzenes,the discharge of industrial halonitrobenzenes sewage is bound to increase and accumulate in the environment.It is a serious threat to the ecology and human health.To date,chloronitrobenzenes have been found to be decomposed by several microorganisms and the corresponding degradation pathways have been determined.However,there are no reports on the biodegradation of bromonitrobenzenes.2-Bromonitrobenzene(2BNB)was found to be utilized by 2-chloronitrobenzene(2CNB)utilizer Pseudomonas stutzeri ZWLR2-1 as the sole carbon,nitrogen and energy source for growth,and two key enzymes in its metabolic pathway were investigated.Biotransformation results verified that the nitroaromatic dioxygenase(CnbAaAbAcAd),the key enzyme involved in the first reaction in 2BNB metabolism,could convert 2-bromonitrobenzene into 3-bromocatechol with concomitant nitrite release.Catechol 1,2-dioxygenase,encoded by the cnbC gene,is the second key enzyme catalyzing 3-bromocatechol to form 2-bromomuconate which was confirmed through enzyme activity assay.It was found that CnbC exhibited a broad substrate specificity and high activity to 3-bromocatechol,3-chlorocatechol,catechol and 3-methylcatechol.As stated above,the metabolite 2-bromomuconate was obtained in the degradation process of 2BNB by strain P.stutzeri ZWLR2-1.Muconate is one of significant chemical raw materials,whose physical and chemical properties would be benefiting from the addition of halogen group.Therefore,halogenated muconate has the potential in industrial application.However,there are no reports on the bioproduction of 2-bromomucanate.In this study,the 2-bromomuconate was synthesized from an enzymatic process in which the conversion of 3-bromocatechol to generate 2-bromomucate was catalyzed by catechol 1,2-dioxygenase CnbC.Then,2-bromomucate was separated using HPLC.Finally,its identity was confirmed by LC-MS and nuclear magnetic resonance.Both 2BNB and 2CNB could be transformed by the nitroaromatic dioxygenase CnbAaAbAcAd in P.stutzeri ZWLR2-1 to produce corresponding catechols.Whereas,the enzyme showed no catalytic activity to 3,4-dichloronitrobenzene(34DCNB).It was found there is high degree of similarity in the amino acid sequences of the ? subunit in the dioxygenase family to which CnbAaAbAcAd belong and the changes of some key amino acids of the enzyme might affect its substrate ranges and catalytic activity.So,mutations of four amino acids,including Ile 204 to Leu,Met251 to Ser,Asn258 to Val and Val 350 to Thr were created by site directed mutagenesis,in the hope that the mutant enzymes will have the ability to use 34 DCNB as a substrate.However,the results showed that the catalytic activity of the mutant dioxygenases on nitroaromatic compounds was weakened or even inactivated,and no activity was found to against 34 DCNB.In conclusion,the biodegradation pathway of 2BNB was firstly described in this study,demonstrating that 2CNB degrader P.stutzeri ZWLR2-1 strain could also use 2BNB as a growth substrate.It has provided a new information for the biodegradation of bromonitrobenzenes.At the same time,the systhesis and purification process of 2-bromomuconate was reported for the first time,which provides a possible basis for the future industrial systhesis of 2-bromomuconate through bioproduction.