A novel esterase gene, pytH, encoding a pyrethroid-hydrolyzing carboxylesterase was cloned from Sphingobium sp. strain JZ-1. The gene contained an open reading frame of 840 bp. Sequence identity searches revealed that the deduced enzyme shared the highest similarity with many ␣/␤-hydrolase fold proteins (20 to 24% identities). PytH was expressed in Escherichia coli BL21 and purified using Ni-nitrilotriacetic acid affinity chromatography. It was a monomeric structure with a molecular mass of approximately 31 kDa and a pI of 4.85. PytH was able to transform p-nitrophenyl esters of short-chain fatty acids and a wide range of pyrethroid pesticides, and isomer selectivity was not observed. No cofactors were required for enzyme activity.Pyrethroid pesticides are now the major class of insecticides used for insect control in agriculture and households as a replacement for more toxic organophosphorus pesticides, and their usage is continuing to grow (10). Although pyrethroid pesticides generally have lower acute oral mammalian toxicity than organophosphate insecticides, exposure to very high levels of pyrethroid pesticides might cause endocrine disruption, lymph node and spleen damage, and carcinogenesis (6, 12). In addition, most pyrethroid pesticides possess acute toxicity to some nontarget organisms, such as bees, fish, and aquatic invertebrates, often at concentrations of less than 0.5 g/kg (19,22). Great concerns have been raised about the persistence and degradation of pyrethroid pesticides in the environment.In general, pyrethroid pesticides are degraded by both abiotic and biotic pathways, including photooxidation, chemical oxidation, and biodegradation. Microorganisms play the most important role in degradation of pyrethroids in soils and sediments. Many pyrethroid-degrading microorganisms have been isolated from soils (13,16,24,27).The major routes of pyrethroid metabolism in pyrethroidresistant insects and pyrethroid-degrading microorganisms include oxidation by cytochrome P450s and ester hydrolysis by carboxylesterases (9). Carboxylesterases are a family of enzymes that are important in the hydrolysis of a large number of endogenous and xenobiotic ester-containing compounds, such as carbamates, organophosphorus pesticides, and pyrethroids. Carboxylesterases from Bacillus cereus SM3 (17), Aspergillus niger ZD11 (13), Nephotettix cincticeps (2), and mouse liver microsomes (23) hydrolyzing the carboxyl ester linkage of the pyrethroids were purified to homogeneity and characterized. Genes encoding the pyrethroid-hydrolyzing carboxylesterases from mouse liver microsomes and Klebsiella sp. strain ZD112 were cloned and functionally expressed (23,27).Pyrethroids differ from many other pesticides in that they contain one to three chiral centers; the chirality may arise from the acid moiety, the alcohol moiety, or both (Fig. 1). A pyrethroid compound therefore consists of two to eight isomers. Isomers of a chiral compound often differ from each other in biological properties. Isomer selectivity has been widely observe...

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Cyhalofop-butyl (CyB) is a widely used aryloxyphenoxy propanoate (AOPP) herbicide for control of grasses in rice fields. Five CyB-degrading strains were isolated from rice field soil and identified as Agromyces sp., Stenotrophomonas sp., Aquamicrobium sp., Microbacterium sp., and Pseudomonas azotoformans; the results revealed high biodiversity of CyB-degrading bacteria in rice soil. One strain, P. azotoformans QDZ-1, degraded 84.5% of 100 mg L(-1) CyB in 5 days of incubation in a flask and utilized CyB as carbon source for growth. Strain QDZ-1 could also degrade a wide range of other AOPP herbicides. An esterase gene, chbH, which hydrolyzes CyB to cyhalofop acid (CyA), was cloned from strain QDZ-1 and functionally expressed. A chbH-disrupted mutant dchbH was constructed by insertion mutation. Mutant dchbH could not degrade and utilize CyB, suggesting that chbH was the only esterase gene responsible for CyB degradation in strain QDZ-1. ChbH hydrolyzed all AOPP herbicides tested as well as permethrin. The catalytic efficiency of ChbH toward different AOPP herbicides followed the order quizalofop-P-ethyl ≈ fenoxaprop-P-ethyl > CyB ≈ 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, whereas the substitutions in the aromatic ring had only a slight influence.

De-esterification is an important degradation or detoxification mechanism of sulfonylurea herbicide in microbes and plants. However, the biochemical and molecular mechanisms of sulfonylurea herbicide de-esterification are still unknown. In this study, a novel esterase gene, sulE, responsible for sulfonylurea herbicide de-esterification, was cloned from Hansschlegelia zhihuaiae S113. The gene contained an open reading frame of 1,194 bp, and a putative signal peptide at the N terminal was identified with a predicted cleavage site between Ala37 and Glu38, resulting in a 361-residue mature protein. SulE minus the signal peptide was synthesized in Escherichia coli BL21 and purified to homogeneity. SulE catalyzed the de-esterification of a variety of sulfonylurea herbicides that gave rise to the corresponding herbicidally inactive parent acid and exhibited the highest catalytic efficiency toward thifensulfuron-methyl. SulE was a dimer without the requirement of a cofactor. The activity of the enzyme was completely inhibited by Ag ؉ , Cd 2؉ , Zn 2؉ , methamidophos, and sodium dodecyl sulfate. A sulE-disrupted mutant strain, ⌬sulE, was constructed by insertion mutation. ⌬sulE lost the de-esterification ability and was more sensitive to the herbicides than the wild type of strain S113, suggesting that sulE played a vital role in the sulfonylurea herbicide resistance of the strain. The transfer of sulE into Saccharomyces cerevisiae BY4741 conferred on it the ability to de-esterify sulfonylurea herbicides and increased its resistance to the herbicides. This study has provided an excellent candidate for the mechanistic study of sulfonylurea herbicide metabolism and detoxification through de-esterification, construction of sulfonylurea herbicide-resistant transgenic crops, and bioremediation of sulfonylurea herbicide-contaminated environments. S ulfonylurea herbicides are an important class of herbicides used worldwide for controlling weeds in all major agronomic crops. The herbicides inhibit acetohydroxy acid synthase (AHAS), a key enzyme in the biosynthesis pathway of branched-chain amino acids valine, leucine, and isoleucine in bacteria, fungi, and plants (3,4,8,13). The use of sulfonylurea herbicides has developed rapidly because of their high efficacies at low dosages and multicrop selectivities. The sulfonylurea products are now the second most common kind of herbicides after the glyphosates, and more than 30 products have been commercialized.Most sulfonylurea herbicides are weak acids, vulnerable to acid hydrolysis under acidic conditions. However, in neutral to alkaline soils, some of the herbicides, such as metsulfuron-methyl, chlorsulfuron, and ethametsulfuron-methyl, are degraded at a very slow rate and persist from several months to more than 1 year (15,20,24,30). The residues of herbicides in the soil seriously damage subsequent rotation of sulfonylurea-sensitive crops, like legumes and oilseeds, which can result in serious agricultural loss. Thus, great concern and interest have been raised regarding the enviro...

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...

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Colla corii asini (CCA) is a protein-based traditional Chinese medicine made from donkey skins. Because it has the ability to nourish blood, its demand is increasing rapidly. The shortage of donkey skins increases the risk of the adulteration of CCA products with other animal skins. To ensure the drug efficacy and safety of CCA products, a proteomics technique was applied to reveal proteins in the skins of donkey, horse, cattle, and pig. Species-specific peptides for each animal species were predicted using bioinformatics, and their presence in the skins and gelatin samples was examined by nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS). One unique marker peptide for each animal species was selected to develop an LC-MS/MS multiple reaction monitoring method. The capability of this method to identify donkey, horse, cattle, and pig materials was demonstrated by analyzing in-house-made donkey gelatins containing different amounts of other animal skins and commercial CCA products. The adulteration of non-donkey species could be sensitively detected at a low level of 0.5%. Hybrid animals, such as mules and hinnies, were also differentiated from donkeys. We provide a practical tool for the quality control of CCA products. The strategy can also be used to study other important traditional Chinese medicines which contain animal proteins.

S-Adenosyl-L-methionine (SAM) plays important roles in trans-methylation, trans-sulfuration, and polyamine synthesis in all living cells, and it is also an effective cure for liver disease, depressive syndromes, and osteoarthritis. The increased demands of SAM in pharmaceuticals industry have aroused lots of attempts to improve its production. In this study, a multiple-copy integrative plasmid pYMIKP-SAM2 was introduced into the chromosome of wild-type Saccharomyces cerevisiae strain ZJU001 to construct the recombined strain R1-ZJU001. Further studies showed that the recombinant yeast exhibited higher enzymatic activity of methionine adenosyltransferase and improved its SAM biosynthesis. With a three-phase fed-batch strategy in 15-liter bench-top fermentor, 8.81 g/L SAM was achieved after 52 h cultivation of R1-ZJU001, about 27.1 % increase over its parent strain ZJU001, whereas the SAM content was also improved from 64.6 mg/g DCW to 91.0 mg/g DCW. Our results shall provide insights into the metabolic engineering of SAM pathway in yeast for improved productivity of SAM and subsequent industrial applications.