Separation, Purification And Enzymatic Properties Of A Carboxylesterase Responsible For The Hydrolysis Of Metsulfuron-Methyl In Strain Hansschlegelia Zhihuaiae S113

Sulfonylurea herbicides are a class of important herbicides used worldwide for controlling weeds in all major agronomic crops. The herbicides inhibit acetohydroxyacid synthase (AHAS), a key enzyme in the biosynthesis pathway of branched-chain amino acids valine, leucine, and isoleucine in bacteria, fungi and plants. While the herbicides have many advantages such as high efficacies at low dosages, multi-crop selectivities. However, their disadvantages can not be ignored. Most sulfonylurea herbicides are weak acids in neutral to alkaline soils, and thus are degraded at a very slow rate and persist from several months to more than one year. The residues of herbicides in the soil seriously damage subsequent rotation of sulfonylurea sensitive crops like legumes and oilseeds, which can result enormous economic loss. Bioremediation become a kind of significant methods because of its security, economics, convenience and high performance.De-esterification is an important degradation or detoxification mechanism of sulfonylurea herbicide in microbes and plants. In this study, we have demonstrated that the strain Hansschlegelia zhihuaiae S113degrades sulfonylurea herbicides via de-esterification to generate corresponding parent acids, which are herbicidally inactive.So far, the thifensulfuron-methyl’s degrading carboxylesterase activity has been detected in soil, and the pyrazosulfuro’s degrading enzyme activity also has been tested. However, the quantitative determination, enzymology properties and purification about this kind of carboxylesterase are now still not be reported. In this study, the carboxylesterase responsible for the de-esterification of metsulfuron-methyl was purified from the strain S113, and the enzymology properties of the purified enzyme was studied. The main results of this study are as follows:The esterase responsible for metsulfuron-methyl de-esterification was an intracellular enzyme and constitutively expressed in the strain. The metabolite was identified by HPLC-MS/MS as the herbicidally-inactive corresponding parent acids.The esterase was purified from strain S113by ammonium sulfate precipitation, DEAE ion exchange chromatography and PAGE extraction. It is a dimer with a molecular mass of86kDa, a pI of8.4. The optimal pH of the enzyme was observed to be7.5-8.0. The enzyme was stable at the pH range6.0to8.5, retaining more than85%of the original activity after preincubation in the buffer at that pH range for1h. The activity of the enzyme was maximal at40℃. The enzyme was fairly stable up to45℃, retained approximately85%of its activity at45℃for1h, retained20-40%of its residual activity at55℃, and was completely inactivated at65℃. The enzymatic activity was inhibited by more than90%with the metal ions Ag+, Cd2+and Zn2+(1.0mM), the organic phosphorus pesticide methamidophos (2.0mM) and the surfactant SDS (1.0mM), while the metal ions Ni2+(1.0mM) caused40to50%inhibition of the enzyme activity. The chelators EDTA and the surfactant Tween80failed to inhibit the enzyme.The enzyme can de-esterify a wide range of sulfonylurea herbicides with a methyl or ethyl ester. It is interesting that the enzyme has a similar Michael’s constant Km for each of the sulfonylurea herbicides with an ester; however, the catalytic constant kcat for thifensulfuron-methyl is at least10-folds higher than that for the other four sulfonylurea herbicides with an ester that are hydrolyzed at a similar catalytic efficiency.