Purification And Characterization Of Degrading-enzyme Of Metabolic Intermediate During Prometryn Degradation From Leucobacter Triazinivorans Sp. JW-1

Prometryn is an s-triazine herbicide that is used widely in crop cultivation.Due to its widespread and frequent application,prometryn has posed a risk to soil and water.Bioremediation has become an important method to remove the pollution of triazine herbicides from contaminated sites.Numerous microorganisms capable of degrading chloro-s-triazines such as atrazine have been isolated,and their metabolic characteristics and enzymes involved in metabolic pathways have been extensively documented.However,few studies were studied for microbial degrading-strains of methylthio-s-triazines such as prometryn and their degrading-enzymes concerning metabolic pathways.Molecular mechanism of prometryn degradation needs to be further studied.In our previous study,Leucobacter triazinivorans sp.JW-1 capable of degrading prometryn had been isolated and its metabolic pathways had been demonstrated.The objectives of this study were?1?to purify degrading-enzymes involved in degradation pathway of prometryn in Leucobacter triazinivorans sp.JW-1,?2?to determine enzymatic properties N-isopropylammelide degrading-enzyme?3?to explore substrate specificity.The main results are as follows:1.A quantitative assay was developed for determining intracellular enzyme activities of cell extract in Leucobacter triazinivorans sp.JW-1.Three different degradation enzymes were responsible for degradation of prometryn,2-hydroxypropazine and N-isopropylammelide and these enzymes were precipitated in solution at different concentrations of saturated ammonium sulfate.2.N-isopropylammelide degrading-enzyme was purified 20.4-fold to give a yield of 5.02%after four purification steps including successive ammonium sulfate precipitation,hydrophobic interaction chromatography on phenyl sepharose,gel filtration chromatography on Superdex 200 and anion-exchange chromatography on Q Sefinose.The specific activity of enzyme increased from 0.55 U/mg?crude enzyme?to 11.2 U/mg?purified enzyme?.The amino acid sequences of the N terminus of the enzyme followed the order of SKDFD LIIRN AYLSE KDSVY and the alignment of amino acid sequences indicated that N-isopropylammelide degrading-enzyme had 100%homology with the amidohydrolase AtzC.3.The optimal temperature and pH value for N-isopropylammelide catalytic activity was 42?and 7.0.The purified enzyme was stable in a rage from 4?to42?and retained over 90%of its activity after it was stored at pH 6-8.Fe²⁺,Fe³⁺,Al³⁺,Cu²⁺,Cr³⁺,Zn²⁺,Hg²⁺at concentration of 5 mM were strongly inhibited its enzymatic activity,while the addition of Mg²⁺,Ca²⁺,Ni²⁺at concentration of 5 mM increased its enzymatic activity.SDS was strongly inhibited the N-isopropylammelide degrading-enzyme activity.The EDTA and 1,10-phenanthroline significantly inhibited the activity,indicating that enzyme function is dependent with the metal ion,and the enzymatic activity was inhibited by DEPC,PMSF and iodoacetamide,suggesting that histidine,serine and cysteine residues present in the active site.4.The Michaelis constant?K_m?of catalytic reaction was 0.674 mmol/L,the maximum rate constant(V_max)was 25.1 U/mg at 42?and pH 7.0.Substrate specificity indicated that N-isopropylammelide degrading-enzyme could degrade2,4-dihydroxy-1,3,5-triazines,which exhibited different catalytic efficiencies possibly resulted from substituent groups at the position 6 of 2,4-dihydroxy-1,3,5-triazine compouds and their complexity.