The Identification And Enzymology Analysis Of Triazine Hydrolase From Atrazine-degrading Bacterias

Atrazine, or weiqujin （Chinese name of commodity）, are widely used as ameans of weed control in agriculture overall the world. Because of its stabilityexistence in the environmental, not degradation, resulting in the destruction ofthe ecological environment, which will threaten the security of human foodand drinking water. Bacterias degrading atrazines can be divided into threecategories by their different triazine degrading genes:atzABCDEF,trzN-atzBC and atzABC-trzD. trzN-atzBC encodes triazinehydrolase, the first enzyme in the process of atrazine degrade. Besides,trzN-atzBC gene seems to be more common than atzA in atrazine-degradingbacteria and its product enzyme has a broad substrate range. Considering thesecharacters, the biologic functions of trzN-atzBC gene in atrazine-degradingbacteria has a good prospect of research and development.Objective:To understand the biologic function of triazine hydrolase gene in depthand provide evidences for the development of bio-repair technology throughthe study of triazine hydrolase gene from atrazine-degrading bacteria.Method:1The expression and purification of triazine hydrolase1.1Construction of expression vector and strains: PCR amplification wasperformed to acquire the DNA fragments of triazine hydrolase gene trzN, thenthe coding region of trzN was inserted into the efficient expression vectorpET-28b（+）. Plasmid and proper chaperones was transformed into E. coliBL21（DE3）.1.2Optimization of expression conditions: Selection suitable of L-（+）-Arabinose concentration, growth temperature and IPTG concentration,optimal expression condition, increase the proportion of soluble protein. 1.3Preservation condition optimization: Choose optimum buffer solution,optimum pH and optimum buffer concentration to keep the best biologicactivity of the protein. Test the impact of temperature, pH and theconcentration of metal ion on the enzymic activity.1.4Purification conditions optimization: According to affinitychromatography method, with different concentrations of midazolam elutionbuffer elution of TrzN protein, purified target protein.2Comparative study of triazine hydrolase for enzymatic properties2.1Study on the optimum conditions: Test the impact of temperature, pH andthe concentration of metal ion on the enzymic activity.2.2The study of the substrate specificity: Study on the ability of TrzNdegradation for different substrates.3Determination of enzyme kinetic constants: Test and calculate their K_mvalueand V_maxvalue.4The distribution of triazine hydrolase in atrazine degradation strains: Inorder to verify the protein expression of triazine hydrolase encoded by trzNfrom atrazine degrading bacteria, western blot detection was performed. Thepurified protein product was separated from E. coli BL21（DE3） transformedby efficient expression carrier of pET-21b（+）-trzN and primaryantibody（rabbit polyclonal antibody） was prepared. Then this primaryantibody to TrzN and secondary antibody（horseradish peroxidase labeledsheep anti rabbit antibody） were used in western blot experiments to test theexpression of triazine hydrolase in different atrazine degrading bacterias.Results:1The expression and purification of triazine hydrolase1.1Construction of expression vector and strains: According to the doubledigestion and sequencing results show efficient expression carrier ofpET-28b（+）-trzN was constructed successfully.1.2The optimal expression conditions of TrzN: Addition L-（+）-Arabinose to afinal concentration of0.015%when the OD₆₀₀reach0.5to inductionexpression for1.5h provides the best culture condition for the TrzN expression strain. Addition IPTG to a final concentration of1.5μmol·L^-1toinduction expression in15℃for16h product the most soluble protein.1.3The optimum storage conditions of TrzN: TrzN crude enzyme solution atpH8.0,50mmol L^-1potassium phosphate buffer keep the greatest enzymicactivity.1.4Purification conditions optimization: Target protein is eluted by the200mmol L^-1of imidazole concentration, and contains no impurity protein.2Comparative study of triazine hydrolase for enzymatic properties2.1Study on the optimum conditions: Purified TrzN protein has a optimum pHof8.0, it can keep stable in a neutral condition of pH8.0; Purified TrzNprotein is stable in range of room temperature to30℃and enzymic activityreduction after30min is small, enzymic activity reduction is significant after30min in40⁵0℃, and TrzN protein is totally deactivated after30min in70℃.Co²⁺, Mn²⁺, Fe²⁺, Ca²⁺and Mg²⁺can activate TrzN, especially Co²⁺. Cu²⁺andNi²⁺have small inhibition effect on TrzN and cause a reduction of enzymicactivity.2.2The study of the substrate specificity: TrzN has a broad substrate range, itshowed highest degrading activity for ametryn, second higher degradingactivity for dipropetry, and TrzN had small degrading activity for prometryne,atratone and atrazine.3Determination of enzyme kinetic constants: The K_mvalue and V_maxvalue ofthe four atrazine degrading bacterias are close. The K_mvalue is42±10μmol·L^-1, and the V_maxvalue is172±10μmol·L^-1min^-1.4The distribution of triazine hydrolase in atrazine degradation strains: ALLatrazine degrading bacterias but Pseudomonas sp. ADP can generate immunefluorescent reaction with TrzN antibody.Conclusion:1The TrzN protein from the four atrazine degrading bacterias are similar intheir degrading activity.2ALL atrazine degrading bacterias but Pseudomonas sp. ADP can generateimmune fluorescent reaction with TrzN antibody.