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Isolation, Identification and Degrading Characteristic of a Cyhalofop-Butyl-Degrading Bacteria, and Cloning, Expression of a Cyhalofop-Butyl-Hydrolyzing Gene chbH

Author: NieZhiJuan
Tutor: HeJian
School: Nanjing Agricultural College
Course: Microbiology
Keywords: Cyhalofop Pseudomonas sp.QDZ-1 Microbial degradation Gene cloning and expression The Cyhalofop hydrolase gene chbH
CLC: X172
Type: Master's thesis
Year: 2011
Downloads: 14
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Abstract


Cyhalofop-butyl is a widely used aryloxyphenoxy propanoate (AOPP) herbicide. Great concerns have been raised about the migration, transformation, degradation of cyhalofop-butyl in the environment and its potential danger to human body.In this study, five cyhalofop-butyl-degrading strains were isolated from rice field soil and designated as Strain QDZ-1, QDZ-A, QDZ-B, QDZ-C and QDZ-D. Based on its physiological and biochemical characters and the result of the 16S rRNA homologue sequence, Five cyhalofop-butyl-degrading strains were preliminarily identified as identified preliminary as Agromyces sp. (strain QDZ-A), Stenotrophomonas sp. (strain QDZ-B), Aquamicrobium sp. (strain QDZ-C), Microbacterium sp. (strain QDZ-D) and Pseudomonas sp. (strain QDZ-1). QDZ-1 was selected for further study due to it relatively high degrading efficiency and the fact that isolates in genus Pseudomonas play very important role in degradation of a wide variety of xenobiotic pollutants.Strain QDZ-1 is a non-spore-forming, Gram-negative, short rod shaped (approximately 1.5-2.2μm in length and 0.6-0.8μm in width), and motile with polar flagella. Colonies grown on LB agar for 1-2 days are circular, convex with entire margins and pale yellow. Growth is observed over a temperature range of 15-37℃(optimum 25~30℃), a pH range of 5.5~10.0 (optimum7.0) and a salinity range of 0-3.5% NaCl (optimum 0.5%NaCl). Growth of strain QDZ-1 suffered obvious inhibition when concentrations of NaCl are more than 3.5%.The strain QDZ-1 could utilize cyhalofop-butyl as the sole carbon source for growth, Pseudomonas sp. QDZ-1, degraded 84.5% of 100 mgL-1 cyhalofop-butyl in 5 d of incubation in flask, When other AOPP herbicides were used as substrates, strain QDZ-1 could degrade 68.9% diclofop-methyl,64.6% haloxyfop-P-methyl,92.6%quizalofop-P-ethyl,90.8% fenoxaprop-P-ethyl and 79.6% of fluazifop-P-butyl, and used these AOPP herbicides as carbon sources for growth. The optimum temperature for cyhalofop-butyl degradation by strain QDZ-1 was 30℃, The optimal pH was observed to be approximately 7.0. The effect of cyhalofop-butyl concentration on degradation was also examined. The strain QDZ-1 and cell-free extract produced a visible transparent halo of cyhalofop-butyl degradation on agar plates supplemented with 150 mg/L cyhalofop-butyl. During the degradation of cyhalofop-butyl, some major metabolites were analyzed by MS. Structural core of cyhalofop-butyl-Ester key was firstly broken and cyhalofop acid generated. Cleavage of ether key and decarboxylation, famidation of cyano group when cyhalofop-butyl is degraded by strain QDZ-1.A novel esterase gene, chbH, encoding a cyhalofop-butyl-hydrolyzing carboxyles-terase was cloned from Pseudomonas sp. QDZ-1, Sequence analysis indicated that chbH gene consisted of 996 bp encoding a protein of 332 amino acids. The molecular mass of the denatured enzyme was approximately 36 KDa. The deduced protein was compared with the known enzymes available from the Protein Data Bank (NCBI database). ChbH showed the highest similarity with some hypothesis or putative esterase or hydrolase, e.g., a lactone-specific esterase from Pseudomonas fluorescents (82% identity), a hydrolase or acyltransferase from Ralstonia eutropha H16 (50% identity) and an alpha/beta hydrolase from Burkholderia multivorans ATCC 17616 (48% identity).Gene chbH was expressed in Escherichia coli BL21 (DE3) and the ChbH was purified using Ni-nitrilotriacetic acid affinity chromatography. The catalytic efficiency of ChbH toward different AOPP herbicides followed the order of quizalofop-P-ethyl fenoxaprop-P-ethyl> cyhalofop-butyl≈fluazifop-P-butyl> diclofop-methyl haloxyfop-P-methyl; the results indicated that the chain length of the alcohol moiety strongly affected the biodegradability of the AOPP herbicides, while the substitutions in the aromatic ring had only slight influence.

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