Gene Cloning And Characterization Of The Key Enzymes Involved In Acetochlor-Degrading Pathway

Chloroacetanilide herbicides are one of the most widely used herbicide families. Members of this family share a molecular core consisting of 2-chloroacetanilide and differ only in the type and arrangement of substitutions. Acetochlor (2-chloro-iV-(ethoxymethyl)-N-(2-ethyl-6-methylphenyl)-acetamide) is an important chloroacet anilide herbicide. Chloroacetanilide herbicides are selective pre-emergent herbicides which used to control broadleaf weeds and annual grasses in corn fields.The main acetochlor and. its biodegradation products, which cause serious negative impacts of the environment.and agricultural ecosystems according to the researches, reporte, included 2-ethyl-6-methylaniline (MEA), chloroacetyl-indoline,2’-methyl-6’-ethyl-2-chloroacetanilide (CMEPA).An efficient CMEPA-hydrolyzing strain Delftia sp. T3-6 and a strain Rhodococcus sp. T3-1 which could transform acetochlor into CMEPA, were isolated from acetochlor-polluted soil previously. Acetochlor could be mineralized completely with these isolated strains. In this study, an amide hydrolase that hydrolyzed CMEPA into MEA was purified from Delftia sp. T3-6, and its encoding gene was cloned and expressed in a heterologous manner in E. coli. A three-component acetochlor N-deethoxymethylase (N-dealkylase) from Rhodococcus sp. T3-1 was purified and its encoding gene was cloned.Fast and effective enzyme assay method is the precondition for protein purification, a new method for rapid determination of MEA hydrolyzed from CMEPA was established in this study. This method was based on the color reaction between aromatic amines and 4-aminoantipyrine, and it showed a good linear relationship between absorption at 535nm wavelength and concentration of MEA in 20 mM Tris-HCl (pH 7.0) buffer, CMEPA, SDS, methanol and other organic reagents,1 mM metal ions have no significant effect on the reaction. The reaction was significantly affected by temperature and pH. The method is also applicable to the determination of other derivatives of aniline and phenol.A highly active amide hydrolase (DamH) was purified from Delftia sp. T3-6 using ammonium sulfate precipitation, DEAE anion exchange, hydrophobic interaction chromatography, and Superdex G-200 gel filtration. After the four-step purification process, DamH was purified by 17.7 folds, with a 0.29%recovery. The molecular mass of the purified enzyme was estimated to be 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The N-terminal 15-residue amino acid sequence of DamH was determined to be NH2-GTSPQSDFLRALFQS. Peptide fingerprint analysis also identified two additional peptide fragments of DamH as NH2-APEADDELRR and NH2-TNPLANPLKASYQGFPR.Based on the N-terminal sequence and results of peptide mass fingerprints, oligonucleotide primers were designed to amplified the damH gene using chromosome walking techniques in Delftia sp. T3-6. The cloned damH gene was 903 base pairs in length, encoding a 300-amino acid protein with a calculated molecular mass of 32 kDa. The corresponding amino acid sequence was searched against the Genbank database. The results revealed that DamH shares 67% identity with ChnC which involved in cyclohexane degradation gene cluster from Acinetobacter sp. SE19. The gene (damH) was expressed in Escherichia coli BL21(DE3). DamH was a bifunctional hydrolase showing activity to amide and ester bonds. The specific activities of recombinant DamH were 5036 U/mg for CMEPA (amide hydrolase function) and 612 U/mg for 4-nitrophenyl acetate (esterase function). The optimum substrate of DamH was CMEPA, with Km and kcat values of 0.197 mM and 2804.32 s", respectively. DamH could also hydrolyze esters such as 4-nitrophenyl acetate, glycerol tributyrate, and caprolactone. The optimal pH and temperature for recombinant DamH were 6.5 and 35℃, respectively; the enzyme was activated by Mn2+ and inhibited by Cu2+, Zn2+, Ni2+, and Fe2+. DamH was inhibited strongly by phenylmethylsulfonyl and SDS, inhibited weakly by EDTA and DMSO.The sitedirected mutagenesis experiments confirmed S149-E244-H274 was the catalytic site of DamH. DamH contained a catalytic triad Ser-Glu-His (Ser149, Glu244 and His274) that is highly conserved in ester 3FAK. Through homology modeling and sequence comparing of DamH and 3FAK, the results revealed that DamH possessed the common G-X-S-X-G (amino acid residues 147 to 151) esterase motif, and an HGX motif (79HGG81), indicated that it belongs to the "GX-type" hydrolases. The recombinant DamH expressed in E. coli BL21(DE3) was purified by Ni2+-NTA and dialysed. Crystals in crystallization conditions was screened out at 293 K using sitting-drop vapor diffusion technique. In the conditions, the protein at the concentration of 15 mg/mL, the mother liquor components are: 0.2 M ammonium acetate,20% w/v polyethylene glycol 3350 and pH 7.1. Crystals grown from condition crystallization diffracted to 3.4 A.The first step in degradation of the acetochlor was considered to be the process of N-deethoxymethylation with the degradation product of CMEPA from acetochlor, and then CMEPA would be hydrolyzed to MEA by DamH. The N-dealkylase that involved in the catalytic reaction from Rhodococcus sp. T3-1 was very unstable. The prepared N-dealkylase with common crushing method lost the catalyze activity. In this paper, the method of cell-breaking and the prepare of crude enzyme from Rhodococcus sp. T3-1 were studied, and a simple and rapid method for detection of N-dealkylase activity was developed. The crude enzyme of Rhodococcus sp. T3-1 was prepared with the method of grinding by liquid nitrogen (solution buffer:1 mM NADH+H*,40% sucrose,0.1 M NaCl, 10 mM MgCl2,2 mM β-mercaptoethanol 20 mM Tris-HCl (pH 7.0)). The activity of acetochlor N-dealkylase was detected indirectly by two step catalytic chromogenic method.The ferredoxin reductase EthA (45 kDa) and ferredoxin EthD (11 kDa) and cytochrome P450 monooxygenase EthB (43 kDa) were purified from the extraction enzyme of Rhodococcus sp. T3-1 by fraction precipitation of ammonium sulfateand hydrophobic interaction chromatography. Based on the results of peptide mass fingerprints, the primers were designed and the gene cluster (ethABCD) were cloned by PCR amplification and expressed in E. coli BL21(DE3). The activity of N-dealkylase which convert acetochlor to CMEPA has been detected in the case of the ethABD expressed in E. coli BL21(DE3). The three-component enzyme system (EthA-EthD-EthB) were identified to be involved in the reaction progress of acetochlor N-dealkylation.