Objective: To explore the relationship between extracellular enzymes activity and virulence of Candida glabrata clinical isolates based on the infection model of Galleria mellonella larvae. Methods: Using experimental research methods, 71 strains of non-repetitive Candida glabrata were collected from Qinghai Provincial People's Hospital from June 2021 to January 2022. Bovine serum protein agar medium, egg yolk agar medium, sheep blood agar medium, Tween-80 agar medium and triglyceride agar medium were used to detect the aspartyl protease activity, phospholipase activity, hemolysis activity, esterase activity and lipase activity of Candida glabrata. Median lethal concentration (LC₅₀) was calculated by using 1.25×10⁸ CFU/ml,2.50×10⁸ CFU/ml,3.75×10⁸ CFU/ml,5.00×10⁸ CFU/ml suspension of Candida glabrata ATCC2001 to infect Galleria mellonella larvae. Histopathological and etiological analysis was performed to determine whether the infection model was successfully established. The clinical isolates of Candida glabrata were configured to infect Galleria mellonella larvae with LC₅₀ concentration to detect the pathogenicity of Galleria mellonella larvae.Spearman test or Pearson test were used to analyze the correlation between the extracellular enzyme activity of Candida glabrata clinical isolates and the pathogenicity of Galleria mellonella larvae. Results: 71 strains of Candida glabrata isolated clinically were detected to have low hemolytic activity after 2 days of culture. Aspartyl protease was detected after 4 days of culture, among which 7 strains (9.86%), 19 strains (26.76%) and 45 strains (63.38%) showed low, medium and high aspartyl protease activity. After 7 days of culture, 71 strains did not detect phospholipase, esterase and lipase activities. Candida glabrata on Galleria mellonella larvae of LC₅₀=2.5×10⁸ CFU/ml Fungal spore were found in the intestinal tissue pathological section of Galleria mellonella larvae in the experimental group, and Candida glabrata was identified by the microbial Mass Spectrometry after culture, while no fungi were found in the pathological section and culture of the control group. Spearman test shows that, there was a linear positive correlation between aspartyl protease activity and the survival rate of Galleria mellonella larvae (r = 0.73, P<0.01), the difference was statistically significant.Pearson test shows that, there was no significant linear relationship between hemolytic activity and survival rate of Galleria mellonella larvae (r = 0.16, P = 0.34), the difference was not statistically significant. Conclusion: The clinical isolates of Candida glabrata in this study had aspartyl protease activity and low hemolytic activity, but no phospholipase, esterase and lipase activity. The activity of aspartyl aspartyl protease of Candida glabrata was positively correlated with the pathogenicity of Galleria mellonella larvae.
Animals ; Sheep ; Larva/microbiology* ; Virulence ; Candida glabrata ; Agar ; Moths/microbiology* ; Esterases ; Aspartic Acid Proteases ; Lipase

Whole-genome sequencing of Flammulina ononidis, a wood-rotting basidiomycete, was performed to identify genes associated with carbohydrate-active enzymes (CAZymes). A total of 12,586 gene structures with an average length of 2009 bp were predicted by the AUGUSTUS tool from a total 35,524,258 bp length of de novo genome assembly (49.76% GC). Orthologous analysis with other fungal species revealed that 7051 groups contained at least one F. ononidis gene. In addition, 11,252 (89.5%) of 12,586 genes for F. ononidis proteins had orthologs among the Dikarya, and F. ononidis contained 8 species-specific genes, of which 5 genes were paralogous. CAZyme prediction revealed 524 CAZyme genes, including 228 for glycoside hydrolases, 21 for polysaccharide lyases, 87 for glycosyltransferases, 61 for carbohydrate esterases, 87 with auxiliary activities, and 40 for carbohydrate-binding modules in the F. ononidis genome. This genome information including CAZyme repertoire will be useful to understand lignocellulolytic machinery of this white rot fungus F. ononidis.
Basidiomycota ; Esterases ; Flammulina* ; Fungi ; Genome ; Glycoside Hydrolases ; Glycosyltransferases ; Polysaccharide-Lyases

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 explore genetic relationships of the 39 materials in six species of Curcuma.

METHODThe peroxidase isozyme (POD) and esterase isozyme (EST) were studied using vertical slab polyacrylamide gel electrophoresis (PAGE) technique, and the zymograms were analyzed using the software of NTSYSpc2. 1.

RESULTThe interspecific zymogramatic differences were obvious. Each species possessed its own specific zymogram distinguishing form the others. In the analysis of EST isozyme, C. phaeocaulis, C. wenyujin, C. kwangsiensis and C. chuanhuangjiang had their own specific zymogram. In the analysis of POD isozyme, just C. phaeocaulis and C. kwangsiensis had their specific zymogram.

CONCLUSIONThe genetic relationships are not associated with the geographical distributions and the genetic relationship between C. longa and C. sichuanensis are very close.

Cluster Analysis ; Curcuma ; classification ; enzymology ; genetics ; Electrophoresis, Polyacrylamide Gel ; Esterases ; analysis ; genetics ; Isoenzymes ; analysis ; genetics ; Peroxidase ; analysis ; genetics ; Phylogeny ; Species Specificity

Animals ; Apolipoproteins ; blood ; Cattle ; Colostrum ; Esterases ; blood ; Female ; Insulin-Like Growth Factor I ; Lipids ; blood ; Nephrotic Syndrome ; blood ; Pregnancy ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the genotypes , allele frequencies and dynamic distribution on resistance associated esterase genes of Culex pipiens complex in Hangzhou.

METHODSThe PCR-restriction fragment length polymorphism (PCR-RFLP) assay was applied to type the resistance associated esterase genes, and dynamic surveillance on frequencies of the resistance associated esterase gene of natural population of Culex pipiens complex in Hangzhou during 2003-2005, and phenotype of the resistance associated esterase genes were detected by esterase starch gel electrophoresis technique.

RESULTSThe PCR-RFLP assay of esterase allele genes for three consecutive years disclosed four esterase genotypes, namely, the world-wide highly active homozygous Est beta 1(1) (50%-54%), homozygous Est beta 2 (29%-34%), heterozygous Est beta 1(1)/beta 2 (5%-10%) and Est beta N (3.13%) of a new homozygous genotype. The research of the resistance associated esterase genes phenotype in natural population of Culex pipiens complex in Hangzhou in 2005 with esterase starch gel electrophoresis technique revealed four major types, namely, Est beta 1(1) (61%), Est alpha 2/beta 2 (12%), Est alpha 8/beta 8 (7%) and sensitive phenotype (29%).

CONCLUSIONThere should be various resistance associated esterase genotypes in natural population of Culex pipiens complex in Hangzhou. During the period of 2003-2005, Est beta 1(1) was the major type; Est alpha 2/beta 2 was the second. Est beta N was a new esterase genotype detected in 2005 only with a mere percentage of 3.13%. As for its resistance to the new insecticide, a follow-up study should be needed. The molecular typing of the amplified esterase gene should be consistent with the resistance associated esterase genes phenotype.

Alleles ; Animals ; China ; Culex ; genetics ; physiology ; Esterases ; analysis ; genetics ; Gene Frequency ; Genotype ; Insecticide Resistance ; genetics ; Phenotype

OBJECTIVETo investigate the species resources of Changium smyrnioides.

METHODNine habitat populations of C. smyrnioides, included with population environment, time limit, morphological characters and so on were investigated. The phylogenetic relationship of different habitat populations of C. smyrnioides was studied by isoenzyme electrophoretic analysis of the EST.

RESULTThe results showed that wild resources of C. smyrnioides were endangered. Seed germinating rate of cultivated plant was significantly higher than that of wild seed, but polysaccharide content in the root of wild species was significantly higher than that of cultivated plant. There existed a geographic phylogenetic relationship among different habitat populations.

CONCLUSIONThe wild C. smyrnioides resources are endangered. The wild C. smyrnioides around Nanjing city are better than the others, it could be used for the further selecting of species resources.

Apiaceae ; chemistry ; classification ; growth & development ; Breeding ; China ; Conservation of Natural Resources ; Ecosystem ; Electrophoresis ; methods ; Esterases ; analysis ; Germination ; Isoenzymes ; analysis ; Pharmacognosy ; Phylogeny ; Plant Proteins ; analysis ; Plant Roots ; chemistry ; growth & development ; Polysaccharides ; analysis ; Seeds ; chemistry ; growth & development

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