Isolation And Identification Of Propyzamide-degrading Bacteria And Cloning And Expression Of Amidohydrolase Gene

Propyzamide was deveioped by Rohm&Haas in 1969.It is a widely used benzamide herbicide for controlling weeds in crops alfalfa,soybeans,peas,lettuce,cotton and sunflower.It has a good effect on annual and perennial weeds and certain broadleaf weeds.Propyzamide is widely used because of its high herbicidal activity and low toxicity to mammals.However,the excessive and frequent application of propyzamide may result in some environmental problems.Therefore,it is necessary to develop some efficient strategies to eliminate propyzamide residues from the environment and agricultural products.Biodegradation involves the use of living microorganisms to degrade pollutants,and is generally considered to be a major factor determining the fate of pollutants in the environment.Many microorganisms that can effectively degrade organic pollutants have been isolated successfully,but strains capable of degrading propyzamide have not been reported.An efficient propyzamide-degrading strain W1 was isolated from the long-term pesticide-contaminated soil and identified as Comamonas testosteroni based on morphology,physiological and biochemical characteristics as well as 16S rDNA sequence analysis.The optimum growth temperature and pH of strain W1 were 30 ? and 7.0.Strain W1 is aerobic growth and the cell growth is good when the NaCl concentration is less than 2%.The metabolite 3,5-dichlorobenzoic acid was identified by MS/MS analysis and this strain converted propyzamide by cleavage of the amide bond,and it could further degrade 3,5-dichlorobenzoic acid.The optimum temperature and pH were 30 ? and 7.0 for propyzamide degradation,respectively.Under these conditions,strain W1 could degrade 99%of 80 mg L^-1 within 18 h at 5%inoculation.However,it could not degrade a variety of herbicides including propanil,diuron,isoproturon,monuron,acetochlor,alachlor,pretilachlor.The solubility of 3,5-dichlorobenzoic acid is much higher than that of propyzamide in water;therefore,a clear transparent halo will be formed around the colonies due to the transformation of propyzamide to 3,5-dichlorobenzoic acid in LB agar containing 300 mg L^-1 ppropyzamide.Using this approach,propyzamide amide hydrolase gene camH was cloned from the library.The ORF is 1452 bp long and encodes a protein composed of 483-amino acids.The G+C content of pamh gene sequence is 59%.The sequence of camH was compared with other known enzymes available in the NCBI database.CamH shows the highest identity?41%identity?with an amidase from Mesorhizobium sp.L2C084A000.The highly conserved catalytic triad of amidase signature?AS?enzyme family,Ser-Ser-Lys,was found in the amino acid sequence of CamH.Thus,we concluded that CamH from Comamonas testosterone W1 was a new member of the amidase signature family.The gene expression vector pET29a-carmH was constructed.The camH could express in E.coli BL21?DE3?and the pure recombinant enzyme CamH was obtained after purifying by Ni-NAT.The optimum temperature and pH value of CamH is 50 ? and 9.5,which with a good pH stability and thermal stability.K_m and V_max of CamH for propyzamide were 0.889 mM and 0.161mM min^-1,respectively.