Cloning of a Novel Arylamidase Gene from Paracoccus sp. Strain FLN-7 That Hydrolyzes Amide Pesticides

Abstract

bThe bacterial isolate Paracoccus sp. strain FLN-7 hydrolyzes amide pesticides such as diflubenzuron, propanil, chlorpropham, and dimethoate through amide bond cleavage. A gene, ampA, encoding a novel arylamidase that catalyzes the amide bond cleavage in the amide pesticides was cloned from the strain. ampA contains a 1,395-bp open reading frame that encodes a 465-aminoacid protein. AmpA was expressed in Escherichia coli BL21 and homogenously purified using Ni-nitrilotriacetic acid affinity chromatography. AmpA is a homodimer with an isoelectric point of 5.4. AmpA displays maximum enzymatic activity at 40°C and a pH of between 7.5 and 8.0, and it is very stable at pHs ranging from 5.5 to 10.0 and at temperatures up to 50°C. AmpA efficiently hydrolyzes a variety of secondary amine compounds such as propanil, 4-acetaminophenol, propham, chlorpropham, dimethoate, and omethoate. The most suitable substrate is propanil, with K m and k cat values of 29.5 M and 49.2 s ؊1 , respectively. The benzoylurea insecticides (diflubenzuron and hexaflumuron) are also hydrolyzed but at low efficiencies. No cofactor is needed for the hydrolysis activity. AmpA shares low identities with reported arylamidases (less than 23%), forms a distinct lineage from closely related arylamidases in the phylogenetic tree, and has different biochemical characteristics and catalytic kinetics with related arylamidases. The results in the present study suggest that AmpA is a good candidate for the study of the mechanism for amide pesticide hydrolysis, genetic engineering of amide herbicide-resistant crops, and bioremediation of amide pesticide-contaminated environments.A variety of amide compounds are used as pesticides to control insects, pathogens, and weeds in agriculture. These compounds include benzoylurea insecticides (hexaflumuron, diflubenzuron, etc.), organophosphate insecticides with an amide group (dimethoate, omethoate, etc.), carbamate insecticides or herbicides (carbofuran, carbaryl, propham, chlorpropham, etc.), benzimidazole fungicides (carbendazim, benomyl, etc.), acetanilide herbicides (acetochlor, butachlor, propanil, etc.), substituted phenylurea herbicides (diuron, linuron, etc.), and sulfonylurea herbicides (chlorsulfuron, metsulfuron-methyl, etc.) (33). However, the widespread use of amide pesticides has resulted in the contamination of the environment and agricultural products. Moreover, many amide pesticides are hazardous to human health and may damage crops when used improperly (7,8,33). Thus, the removal of amide pesticides from the environment and agricultural products is of paramount importance.Microorganisms play a significant role in the degradation or detoxification of amide pesticides in the environment. Bacterial strains that are able to degrade amide pesticides (carbofuran, carbaryl, acetochlor, carbendazim, dimethoate, etc.) have been isolated, and their microbial metabolic pathways have also been elucidated (12,22,25,32,43). To date, the sequence information for the following amide pesticide-hydrolyzing enzymes is...

Biodegradation of propyzamide by Comamonas testosteroni W1 and cloning of the propyzamide hydrolase gene camH

“…The presence of serine hydrolase PMSF and the surfactants (Tween 80, Triton X-100 and SDS) resulted in approximately 2.7-22.4% residue of the enzymatic activity. While EDTA showed only slight inhibitory effect suggesting that CamH is a metal ion-independent hydrolase (Wang et al, 2014;Zhang et al, 2012). …”

“…RIT357 (GenBank accession number EZP62832, 37% identity). CamH has a highly conserved serine-and glycinerich sequence (GGSSGGAAVAVA, sites 149-160), which is a characteristic of the amidase family (Zhang et al, 2012). A highly conserved catalytic triad in amidase family, Ser-Ser-Lys (Ser151, Ser175, and Lys75), was also identified (Fig.…”

Biotechnology: Herbicide-Resistant Crops

“…There are numerous papers on potential genes for HR crops, such as that of Zhang et al (2012), which describes the microbial gene for an arylamidase that can degrade a number of herbicides with amide groups (e.g., propanil). A few such genes have been used to transform plants in order to successfully impart herbicide resistance.…”

Mineralization of the herbicide swep by a two-strain consortium and characterization of a new amidase for hydrolyzing swep

“…The shotgun method was used to clone the hydrolase gene and performed as described previously [21]. The transformants were plated onto LB agar containing 100 mg/L ampicillin and 0.5 mM 4-acetaminophenol (a structural analogue of swep) and then incubated at 37 °C for 12 h. Brown colonies potentially harboring the target amidase were selected for exhibiting the capacity to transform 4-acetaminophenol into the brown product 4-aminophenol [22]. Positive clones were further tested for their ability to hydrolyze swep by HPLC, and then sequenced immediately.…”