Study On Properties Of Atrazine Degradation Enzyme

The harms caused by atrazine which has the characteristics of long-term residue toenvironment and many kinds of organisms have attracted much more attentions. Recently, thebioremediation has been the main method in the remediation of the atrazine-contaminated due to itsrelatively low cost, effective treatment and no secondary pollution. The biodegradative process isactually enzymatic reaction caused by different kinds of genes encoding the enzyme in thebacterial strain。Therefore, the use of genes encoding the enzymes can effectively degrade atrazinecompared atrazine degradation bacteria. The early stage of laboratory had a deep understanding onthe composition of DNS10and the molecular mechanism of degradation of atrazine, but thedegradation characteristics of the enzyme in the degradation strains DNS10is unclear, which needfor related research. This research aims to purify atrazine degradation enzyme （TrzN、AtzB andAtzC） and study its characterization.The crude extraction of TrzN、 AtzB and AtzC were purified by Ni-NTA affinitychromatography, a highly purified enzyme was obtained exhibiting a single band on SDS-PAGE.The final purification of TrzN resulted in a yield of approximately26.5%with about69.7-foldincrease in purity compared to crude enzyme extract. The final purification of AtzB resulted in ayield of approximately15.2%with about15-fold increase in purity compared to crude enzymeextract. The final purification of AtzC resulted in a yield of approximately22%with about24.2-fold increase in purity compared to crude enzyme extract.Characterization of TrzN was analyzed by taking atrazine as substrate. The optimaltemperature and pH of the enzyme proved at25°C and pH8.0. After measuring, michaelis constantK_mof the enzyme is0.38mmol·L^-1, maximum reaction velocity Vmof the enzyme is2.17μmol·L^-1·min^-1. It is tested that enzyme activity is influenced by some of metallic ion. Zn （II）and Co （II） can slightly activate the enzyme although Cr （III） and Cu （II） strongly inhibited it.TrzN activity in the addition of metal have reached the level of significant differences （P <0.05）compared to that in no addition of metal. With the increase of NaCl concentration, enzyme activitydecreases gradually. The enzyme activity was slightly inhibited by EDTA, and strongly inhibitedby DTT、SDS and β-mercaptoethanol.Characterization of AtzB was analyzed by taking hydroxyatrazine as substrate. The optimaltemperature and pH of the enzyme proved at40°C and pH9.0. After measuring, michaelis constantK_mof the enzyme is0.45mmol·L^-1, maximum reaction velocity Vmof the enzyme is3.17μmol·L^-1·min^-1. It is tested that enzyme activity is influenced by some of metallic ion. Co （II） strongly inhibited the enzyme, which have reached the level of significant differences （P <0.05）compared to that in no addition of metal. Co （II）、Cr （III）、Ca （II）和Ni （II） had no effect on theenzyme. When the NaCl concentration reached1mol·L^-1, AtzB was completely inactive. Theenzyme activity was slightly inhibited by EDTA, and strongly inhibited by DTT、SDS andβ-mercaptoethanol.Characterization of AtzC was analyzed by taking hydroxyatrazine as substrate. The optimaltemperature and pH of the enzyme proved at35°C and pH8.0. After measuring, michaelis constantK_mof the enzyme is1.05mmol·L^-1, maximum reaction velocity Vmof the enzyme is4.85μmol·L^-1·min^-1. It is tested that enzyme activity is influenced by some of metallic ion. Zn （II）and Cu （II） significantly inhibited the enzyme, Cr （III） and Ni（II） slightly inhibited the enzymewhich have reached the level of significant differences （P <0.05） compared to that in no additionof metal. Co （II） andCa （II） had no effect on the enzyme. With the increase of NaCl concentration,enzyme activity decreases gradually. The enzyme activity was slightly inhibited by EDTA, andstrongly inhibited by SDS and β-mercaptoethanol. However, DTT slightly activate the enzyme.