Isolation, Degrading Characterization Of A Fenoxaprop-P-ethyl Degrading Strain, And Cloning, Expression Of A Carboxylesterase Gene Feh

Fenoxaprop-p-ethyl (FE) is an aryloxyphenoxy propanoic acid herbicide for controlling of annual grass weeds. It has been applied in China widely. Its residue could cause negative effect on the ecosystem and subsequent rotation of crops. Microorganism plays an important role in the degradation of xenobiotics, and it has received increasing attention from the researchers.One strain, named as JPL-2, capable of degrading FE, was isolated from FE contaminated soil. It was preliminarily identified as Rhodococcus sp. according to its physiological, biochemical characters and the analysis of its16S rRNA gene sequence.Strain JPL-2could grow well between pH5.0and9.0. The optimal pH and temperature for its growth were7.0and30℃, respectively. Fructose and the beef extract were the best carbon and nitrogen source for its growth, respectively. It was resistant to kanamycin.Strain JPL-2could utilize FE as the sole carbon source for its growth, and the degradation efficiency against100mg·L-1FE was94.5%within60h. The optimal degrading temperature and pH were30℃and7.0, respectively, which was in agreement with its optimal growth conditions. The degradation efficiency of FE showed a positive corelation to the inoculation level. With the increasing of initial concentration of FE, the degradation efficiency decreased. However, it could be completely degraed with the prolonged time.Through the construction of genomic DNA library of strain JPL-2, gene feh encoding the Fenoxaprop-p-ethyl-hydrolyzing carboxylesterase was obtained. Sequence analysis indicated that feh gene was consisted of1140bp encoding a protein of380amino acids. According to the analysis of online-software and GenBank database, this sequence had77%similarity to carboxylesterase gene of Rhodococcus sp. T1(GenBank Accession No. AJ605330). The feh gene was ligated into expression vector pET29a, and then transformed into E.coli BL21(DE3) to express. FEH, the expressed product, was purified with Ni-Agarose gel affinity column and subjected to the enzymatic properties study.The investigation on the enzymatic properties showed that the FEH had high stability between10℃and40℃. The optimal catalysis conditions were pH7.5·35℃. Under the concentration of1mmol·L-1, Ni2+, Hg2+, Ag+showed inhibition effect on the activity of FEH, while Fe+, Cu2+, Mg2+, Al3+did not show obvious influence on its activity.