Carbamate pesticides, a new type of broad-spectrum pesticides for controlling pests, mites, and weeds, are developed to address the shortcomings of organochlorine and organophosphorus pesticides. Their widespread use and slow degradation have led to environmental pollution, causing damage to ecosystems and human health. Managing pesticide residues is a pressing issue in the current environmental protection. This study aims to investigate the expression of SyEst870, a member of the SGNH/GDSL hydrolase family in Sphingobium yanoikuyae, in a prokaryotic system and evaluate the ability of the recombinant protein to degrade carbamate pesticides. The prokaryotic expression vector pET-32a-SyEst870 was constructed and transformed into the Escherichia coli BL21 for heterologous expression. The purified protein was studied in terms of enzyme activity and effects of temperature, pH, and metal ions on the enzyme activity, with p-nitrophenol acetate as the substrate and based on the standard curve of p-nitrophenol. LC-MS (liquid chromatography-mass spectrometry) was employed to examine the degradation effects of SyEst870 on carbaryl, metolcarb, and isoprocarb. GC-MS (gas chromatography-mass spectrometry) was employed to detect the degradation products of SyEst870 for the three pesticides. The soluble protein SyEst870 was successfully obtained through the heterologous expression in Escherichia coli, which yielded an enzyme with the activity of 677.5 U after affinity chromatography. SyEst870 exhibited degradation rates of 82.34%, 84.43%, and 92.87% for carbaryl, metolcarb, and isoprocarb, respectively, at an initial concentration of 100 mg/L within 24 h at 30 ℃ and pH 7.0. The primary degradation products of carbaryl were identified as α-naphthol and methyl isocyanate. Metolcarb was mainly degraded into m-cresol and methyl isocyanate, and isoprocarb was mainly degraded into 2-isopropylphenol and methyl isocyanate. Compared with the half-life of carbamate pesticides in the natural environment, which ranges from a few days to several weeks, the recombinant protein SyEst870 can rapidly eliminate the residues of carbamate pesticides. This study lays a foundation for addressing pesticide residues in the environment and in fruits and vegetables.
Escherichia coli/metabolism* ; Sphingomonadaceae/genetics* ; Recombinant Proteins/metabolism* ; Biodegradation, Environmental ; Esterases/metabolism* ; Pesticides/isolation & purification* ; Carbamates/isolation & purification*

Enantioselective hydrolysis of 2-carboxyethyl-3-cyano-5-methylhexanoic acid (CNDE) is the key step in chemoenzymatic synthesis of pregabalin. We purified an intracellular carboxyl esterase from Morganella morganii ZJB-09203, which exhibited high enantioselectivity and activity towards CNDE. The carboxyl esterase was purified to electrophoretic homogeneity by ammonium sulfate fraction precipitation, Phenyl Sepharose 6 FF hydrophobic interaction chromatography, anion exchange with DEAE Sephadex A-50 and Bio-Scale CHT column. The purified enzyme was a monomer with molecular mass of 68 kDa determined by SDS-PAGE and gel chromatography. Substrate specificity of the enzyme towards p-nitrophenyl esters suggested that the purified enzyme was an esterase. The optimal reaction pH for CNDE hydrolysis was 9.0, and optimal temperature was 45 degrees C. The esterase was stable between pH 7.0 and 9.0, and at 40 degrees C. The enzyme activity was enhanced by Ca2+, Cu2+ and Mn2+, whereas strongly inhibited by Co2+, Fe3+, Ni2+ and EDTA. Meanwhile, we investigated the kinetic parameters of the esterase towards p-nitrophenyl esters and effect of CNDE concentration on conversion. The present study reported the esterase capable of stereospecific hydrolysis of CNDE for the first time. Our research will provide foundations for industrial production of Pregabalin using the new biocatalyst.
Chromatography, Gel ; Electrophoresis, Polyacrylamide Gel ; Esterases ; metabolism ; Hydrogen-Ion Concentration ; Kinetics ; Molecular Weight ; Morganella morganii ; enzymology ; Substrate Specificity ; Temperature

The metabolic profile of pseudolaric acid B (PB) was investigated by using in vivo and in vitro tests. Pseudolaric acid C2 (PC2) was identified as the specific metabolite of PB in plasma, urine, bile and feces using HPLC and HPLC-ESI/MS(n) after both oral and intravenous administration to rats, and almost no prototype was detected in all kinds of samples. The metabolic behaviors of PB orally administered in rats treated with antibiotics to eliminate intestinal microflora were identical with those in untreated rats, demonstrating that the metabolism of PB is independent of intestinal microflora. PB was stable in 48 h respective incubation with artificial gastric juice and artificial intestinal juice, suggesting that neither pepsin nor trypsin is in charge of metabolism of PB, and also demonstrating that PB is stable in both pH environments of gastric tract and intestinal tract. In vitro research on metabolism of PB in rat liver microsomes incubation revealed that little PB was metabolized and that the proposed metabolites were the demethoxy and demethoxydecarboxy products of the prototype. The amount of metabolites was extremely low compared with the prototype, indicating that liver microsomes are not responsible for the metabolism of PB either. PB was gradually metabolized into PC2 during 1 h in whole blood incubation in vitro, and the metabolic process showed dynamically dependent manner with incubation time. Once absorbed into blood, PB was quickly metabolized into PC2, accordingly, little prototype was detected in all kinds of samples. The metabolism was attributed to the rapid hydrolysis of C-19 ester bond by plasma esterase. These results clarified the metabolic pathway of PB for the first time, which was of great significance to identify the in vivo active form and interpret acting mechanism of the active compounds of P. kaempferi.
Administration, Intravenous ; Administration, Oral ; Animals ; Bile ; metabolism ; Diterpenes ; blood ; metabolism ; urine ; Esterases ; metabolism ; Feces ; chemistry ; Hydrolysis ; Male ; Metabolic Networks and Pathways ; Microsomes, Liver ; metabolism ; Pinaceae ; chemistry ; Plant Bark ; chemistry ; Plants, Medicinal ; chemistry ; Rats ; Rats, Sprague-Dawley

Wild-type biocatalysts usually show high activity and selectivity towards their native substrates. Since non-native substrates are often used in synthetically useful biocatalytic transformations, it is necessary to engineer enzymes for improved activity, stability and selectivity (chemo-, regio- and stereoselectivity). Herein we give an overview of the recent advances in engineering the enantioselectivity of biocatalysts, with an aim to stimulate further development of this important field in China.
Animals ; Biocatalysis ; Epoxide Hydrolases ; genetics ; metabolism ; Esterases ; genetics ; metabolism ; Humans ; Lipase ; genetics ; metabolism ; Protein Engineering ; methods ; Stereoisomerism

OBJECTIVETo evaluate the molluscicidal activities of three Chinese plants N. indicum Mill, R stenoptera DC, and R. japonicum Houtt, and to clarify the molluscicidal mechanism.

METHODSN-butanol extracts and water extracts of the three plants were obtained. The reactions of EST isozyme, glycogen and total protein of snails to the plant extracts were studied.

RESULTSEST electrophoresis showed that EST was an important antidotal enzyme system and reacted strongly to environment. EST changed greatly during the whole exposure period so that it could be viewed as a pathological index of toxicity. Extracts decreased the glycogen content of the snails' soft tissues greatly, and also the protein content.

CONCLUSIONAll extracts show strong molluscicidal activity. The LD50 value of the water extract of N. indicum Mill is as low as 13.2 mg/L. EST can be viewed as a pathological index of toxicity. The energy metabolism abnormity is the key reason for the molluscicidal activities. The biochemical mechanism needs further research.

Animals ; Electrophoresis, Polyacrylamide Gel ; Esterases ; metabolism ; Glycogen ; metabolism ; Isoenzymes ; metabolism ; Juglandaceae ; chemistry ; toxicity ; Molluscacides ; toxicity ; Nerium ; chemistry ; toxicity ; Plant Extracts ; chemistry ; toxicity ; Rumex ; chemistry ; toxicity ; Snails ; drug effects

OBJECTIVEIn order to use facultative gut bacteria as an alternate to animals for the initial gastrointestinal toxicity screening of heavy metals, a comparative study on rat intestinal epithelial cells and resident gut bacteria was undertaken.

METHODSin vitro growth rate of four gut bacteria, dehydrogenase (DHA) and esterase (EA) activity test, intestinal epithelial and bacterial cell membrane enzymes and in situ effect of arsenite were analysed.

RESULTSGrowth profile of mixed resident population of gut bacteria and pure isolates of Escherichia coli, Pseudomonas sp., Lactobacillus sp., and Staphylococcus sp. revealed an arsenite (2-20 ppm) concentration-dependent inhibition. The viability pattern of epithelial cells also showed similar changes. DHA and EA tests revealed significant inhibition (40%-72%) with arsenite exposure of 5 and 10 ppm in isolated gut bacteria and epithelial cells. Decrease in membrane alkaline phosphatase and Ca2+ -Mg2+ -ATPase activities was in the range of 33%-55% in four bacteria at the arsenite exposure of 10 ppm, whereas it was 60%-65% in intestinal epithelial villus cells. in situ incubation of arsenite using intestinal loops also showed more or less similar changes in membrane enzymes of resident gut bacterial population and epithelial cells.

CONCLUSIONThe results indicate that facultative gut bacteria can be used as suitable in vitro model for the preliminary screening of arsenical gastrointestinal cytotoxic effects.

Animals ; Arsenites ; pharmacology ; Cell Membrane ; drug effects ; Culture Media ; Epithelial Cells ; drug effects ; enzymology ; microbiology ; Esterases ; metabolism ; Gram-Negative Bacteria ; drug effects ; enzymology ; growth & development ; Gram-Positive Bacteria ; drug effects ; enzymology ; growth & development ; Humans ; Intestines ; cytology ; drug effects ; microbiology ; Oxidoreductases ; metabolism ; Rats ; Teratogens ; pharmacology

OBJECTIVETo study the effects of intestinal microflora alteration on specific and nonspecific immune function and hematopoietic function of mice.

METHODSSixty BALB/C mice were divided at random into two groups, experimental group and control group, with 30 mice in each. The mice in the experimental group were given kanamycin 50 mg while those in the control group were given distilled water intragastrically everyday for consecutive 10 days. After the 10 day treatment all the mice were sacrificed, and the cecal contents were collected for quantitative analysis of the intestinal bacterial flora. Certain indexes of immune function, including phagocytosis rate of macrophages, number of T lymphocytes positively stained by esterase and serum interleukin 2 (IL-2) content, and the weight of the spleen, granulocyte-macrophage colony stimulating factor etc. as indexes of hematopoietic function were determined.

RESULTSIn the group, the quantity of Enterobacteriaceae, Enterococcus, Bifidobacterium and Lactobacillus were significantly lower than that in the control group (P < 0.01). The number of PFC (plaque forming cells), the phagocytosis rate of macrophage, the number of T lymphocytes with positive NANE staining, the level of IL-2 significantly decreased when compared with that in the control group (P < 0.01). The weight of the spleen in the experimental group decreased when compared with that in the control group (P < 0.01). Levels of IL-3, GM-CSF, the total number of WBC and the proportion of neutrophil remarkably decreased as compared to that in the control group (P < 0.01). Analysis of the correlations between normal microflora, immunologic and hematopoietic indexes showed that marked positive correlations between the quantity of Bifidobacteria and each immune index including the levels of IL-3 and GM-CSF. There was a positive correlation between IL-2 and IL-3, IL-2 and GM-CSF as well.

CONCLUSIONThe application of antibiotics may cause changes in the structure and quantity of intestinal microflora. The dysbacteriosis may decrease the immune function of organism. The dysbacteriosis may decrease the hemopoietic function. The dysbacteriosis, the decrease in immune and hematopoietic function may affect one another. The balance in microecosystem should be emphasized and antibiotics should be applied rationally to reduce the side effects such as dysbacteriosis.

Animals ; Anti-Bacterial Agents ; pharmacology ; Esterases ; biosynthesis ; Feces ; microbiology ; Granulocyte-Macrophage Colony-Stimulating Factor ; analysis ; Interleukin-2 ; blood ; Intestines ; drug effects ; microbiology ; Kanamycin ; pharmacology ; Macrophages ; drug effects ; physiology ; Mice ; Mice, Inbred BALB C ; Models, Animal ; Organ Size ; Phagocytosis ; drug effects ; Spleen ; drug effects ; pathology ; T-Lymphocytes ; drug effects ; metabolism