Cloning, Expression, Purification, and Characterization of an Organophosphate-Degrading Enzyme in Escherichia Coli

Abstract:

OpdA is one of organic phosphorus degradation enzyme gene from Agrobacterium radiobacter that may be the most promising targets for the digestion of digestion. In this study, we describe for the cloning and expression in Escherichia coli (E. coli) of plasmid pET28b-opdA, followed by purification by NTA-Ni2+ agarose affinity chromatography. Protein expression and purification were evaluated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).The Results showed that the optimal value of inoculum OD600 before induction, inducing time, final IPTG concentration and inducing temperature respectively were 0.5,5h,1 mmol/L,37°C. We obtained the concentration of renatured protein was 18.312mg / L. The Km was 4.26μmol/L at 37 °C, and the maximum reaction velocity (Vmax) was 3.2669μmol/L • min.

• Periodical: Advanced Materials Research (Volumes 610-613)
• Pages: 203-209
• DOI: https://doi.org/10.4028/www.scientific.net/AMR.610-613.203
• Online since: December 2012
• Authors: Jin Bin Wang, Da Chao Chen, Dong Xiao Hu, Xue Feng Su, Xue Ming Tang
• Keywords: Characterization, Expression, Organophosphate-Degrading Enzyme, Purification

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