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PubMed1993

A comparison of the API Uriscreen with the Vitek Urine Identification-3 and the leukocyte esterase or nitrite strip as a screening test for bacteriuria.

The API Uriscreen is a rapid urine-screening test based on the detection of catalase activity present in somatic cells and in many of the bacteria commonly causing urinary tract infections. Of 487 routine, outpatient urine specimens processed by conventional quantitative culture, API Uriscreen, Vitek UID-3 panel, and a leukocyte esterase-nitrite strip, 142 had no growth. Of 336 urine specimens with > or = 10(3) colony-forming units (CFU)/ml, 79 were considered to be indicative of possible or probable urinary tract infection (Cumitech 2A). The sensitivity and specificity of the API Uriscreen for the detection of bacteriuria at > or = 10(5) CFU/ml were 62% and 85%, those of the leukocyte esterase-nitrite strip was 61% and 82%, those of the Vitek UID-3 panel were 91% and 66%. When bacteriurias were classified into possibly or probably indicative of urinary tract infection, the sensitivity and specificity of the API Uriscreen at > or = 10(5) CFU/ml were 87% and 78%, those of the leukocyte esterase-nitrite were 84% and 76%, those of the Vitek UID-3 were 93% and 55%. In this study, we consider the API Uriscreen did not have significant advantages over the leukocyte esterase-nitrite strip.

Bacteria↗
PubMed1984

Esterases of earthworms--III. Electrophoresis reveals that Eisenia fetida (Savigny) is two species.

Polyacrylamide gel electrophoresis of esterases in earthworms classified as Eisenia fetida (Savigny) revealed the existence of two species. The classification based upon esterase electromorphs coincided with classification into two colour variants formerly described as subspecies under the names E. fetida typica and E. fetida unicolor (André, 1963). Two main groups of esterases, acetylesterases and carboxylesterases were found. Because of less variability within species and higher stability the acetylesterases are regarded as the most useful in taxonomy. Sterile hybrids, with hybrid esterase patterns were produced at a low rate in the laboratory, but were never found with certainty in the wild populations. Eisenia fetida (Savigny) and Eisenia unicolor (André) are the names proposed for the two species.

Animals↗
PubMed1988

Polar fluorescein derivatives as improved substrate probes for flow cytoenzymological assay of cellular esterases.

Fluorescein esters are employed in assays of cell viability, membrane permeability and esterase activity. The ester most widely used, fluorescein diacetate (FDA), has the disadvantage of rapid cellular efflux of its hydrolysis product fluorescein. This is particularly problematic for flow cytoenzymology (FCE), where fluorescence is measured in individual cells allowing identification of subpopulations differing in esterase activity and/or membrane characteristics. We present a comparison of FDA with two potentially improved substrate probes for FCE, carboxyfluorescein diacetate (CFDA) and bis(carboxyethyl)-carboxyfluorescein-tetra acetoxy methyl ester (BCECF-AM). Substrates were characterized in terms of reaction and product efflux kinetics in EMT6 mouse mammary tumour cells, together with inhibition kinetics for the carbamoylating agent BCNU. Intact viable cells were analysed by FCE and spectrofluorimetry, and the latter was also used for cell sonicates and purified esterase. CFDA and BCECF-AM enter cells and are hydrolysed more slowly than FDA. CFDA and FDA hydrolyses obey Michaelis-Menten kinetics with Km values of around 19 and 2 microM, respectively, whereas BCECF-AM hydrolysis deviates from this classical behaviour. BCNU (5 X 10(-4) M) inhibits FDA and BCECF-AM hydrolyses by approximately 50%, compared to 30% for CFDA. CFDA may be partly hydrolysed by membrane-bound esterases. Efflux half-lives were 16 min, 94 min and greater than 2 h for products of FDA, CFDA and BCECF-AM, respectively. We conclude that BCECF-AM is the optimal substrate probe for FCE. This study emphasizes the need to optimize various parameters when selecting a substrate for flow cytoenzymological assay or when loading other reporter fluorochromes into cells via lipophilic esters.

Animals↗
PubMed1996

Changes in the methylation of amplified esterase DNA during loss and reselection of insecticide resistance in peach-potato aphids, Myzus persicae.

Insecticide resistance in peach-potato aphids, Myzus persicae, results from the amplification of genes encoding an esterase that hydrolyses and sequesters insecticides. Resistance is normally stable, but highly resistant aphid clones sometimes lose resistance when insecticidal selection pressure is removed. This loss of resistance, termed reversion, arises from a loss of elevated esterase enzyme through transcriptional control, i.e. without loss of the amplified esterase DNA sequences. We have shown that loss of the elevated enzyme occurred simultaneously with loss of methylation at CCGG sites in the amplified DNA sequences. During reselection of resistance in these revertant clones, enzyme levels increased, but there was no corresponding return of methylation to DNA sequences. Thus, although DNA methylation is closely correlated with expression of the amplified esterase genes during reversion, it may not be a factor in the reverse process.

Animals↗
PubMed2006

Chaperone-like activities of alpha-synuclein: alpha-synuclein assists enzyme activities of esterases.

Alpha-synuclein, a major constituent of Lewy bodies (LBs), has been implicated to play a critical role in the pathogenesis of Parkinson's disease (PD), although the physiological function of alpha-synuclein has not yet been known. Here we have shown that alpha-synuclein, which has no well-defined secondary or tertiary structure, can protect the enzyme activity of microbial esterases against stress conditions such as heat, pH, and organic solvents. In particular, the flexibility of alpha-synuclein and its C-terminal region seems to be important for complex formation, but the structural integrity of the C-terminal region may not be required for stabilization of enzyme activity. In addition, atomic force microscopy (AFM) and in vivo enzyme assays showed highly specific interactions of esterases with alpha-synuclein. Our results indicate that alpha-synuclein not only protects the enzyme activity of microbial esterases in vitro, but also can stabilize the active conformation of microbial esterases in vivo.

Enzyme Stability↗
PubMed2005

The esterase from Alicyclobacillus acidocaldarius as a reporter enzyme and affinity tag for protein biosynthesis.

Esterase from thermophilic bacteria Alicyclobacillus acidocaldarius can be produced up to 200 microg/ml by coupled in vitro transcription/translation system derived from Escherichia coli. The synthesized thermostable enzyme can be determined by photometrical and fluorescent assays at least up to 10(-8) M concentration or by activity staining in the polyacrylamide gels. Enhanced green fluorescence protein-esterase fusion protein was bound to a matrix with immobilized esterase inhibitor and purified by affinity chromatography. Thus, the esterase is suited as a reporter enzyme to monitor the expression of polypeptides coupled to its N-terminus and simultaneously, as a cleavable tag for polypeptide purification.

Affinity Labels↗
PubMed2006

Protease and esterase activity of staphylococci.

The aim of this work was to characterize protease and esterase activities of staphylococci in order to establish if they could contribute to the release of amino acids and short-chain fatty acids during ripening of fermented sausages. Eighteen Staphylococcus strains belonging to the species Staphylococcus xylosus (5), S. saprophyticus (3), S. equorum (4), S. carnosus (4) and S. simulans (2), previously isolated from different types of Southern Italian fermented sausages, were screened for proteinase, aminopeptidase and esterase activities. Most of the staphylococci strains lacked detectable levels of proteinase activity against casein-fluorescein isothiocynate. In the active strains, this activity was extracellular or cell-envelope associated. The studied staphylococci strains also showed low levels of aminopeptidase activities, which preferentially hydrolysed substrates containing L-methionine, L-leucine and L-phenylalanine as N-terminal residue. In contrast, all staphylococcal strains possessed significant activity against short-chain fatty acid esters. The maximum esterase activities were detected in whole-cell suspensions and cell-free extracts and to a lesser extent in the extracellular medium. The substrates preferentially hydrolysed were rho-nitrophenyl (rho-NP) butyrate and rho-NP caprylate and, secondly, rho-NP palmitate. The extracellular extracts of most of the strains were only active against rho-NP butyrate except for those of S. equorum (SI3, SI4) and S. simulans (Ssm12, Ssm21), which also hydrolysed rho-NP caprylate and rho-NP palmitate. The cell-free extracts and whole cells were mainly active against rho-NP butyrate and rho-NP caprylate, showing activity levels from 1760 U/mg of proteins to 54 U/mg of proteins and from 12,200 U/mg of proteins to 133 U/mg of proteins respectively. These activities were especially high in the strains that belonged to S. xylosus and S. equorum species. The diversity of the studied metabolic properties and, especially, the esterase activities in different staphylococcal species and even strains of the same species emphasize the relevance of these in vitro characterization studies for a rational selection of new starter cultures.

Aminopeptidases↗
PubMed2006

Functional expression and refolding of new alkaline esterase, EM2L8 from deep-sea sediment metagenome.

A metagenomics approach is an efficient method of isolating novel and useful genes from uncultured microorganisms in diverse environments. In this research, a gene encoding a new esterase (EM2L8) was cloned and characterized from the metagenomic DNA library of a deep-sea sediment. The gene consisted of 804bp encoding a polypeptide of 267 amino acids with a molecular mass of 28,952. The deduced amino acid sequence showed similarities with the BioH of Kurthia, the 3-oxoadipate enol-lactonase of Haloarcula and the acyltransferase of Thermoanaerobacter, which feature identities of 38%, 32%, and 33%, respectively. Residues essential for esterase activity, such as pentapeptide (GXSXG) and catalytic triad sequences, were uncovered. While the protein was overproduced mainly as inclusion body at 37 degrees C, it was mainly produced as a soluble active enzyme at 18 degrees C. A zymogram analysis revealed that purified EM2L8 taken from the soluble fraction could hydrolyze tributyrin substrate. Furthermore, the protein from the inclusion body fraction also showed strong activity on gel, thus indicating that the protein was refolded during SDS-gel electrophoresis and the ensuing incubation period. When the inclusion body was mixed with some anionic detergent solutions and diluted with a non-detergent buffer, the insoluble EM2L8 refolded rapidly and recovered its full esterase activity. Although EM2L8 had an optimum temperature of 50-55 degrees C, its activation energy in the range of 10-40 degrees C was 8.34kcal/mol, indicating that it is a cold-adapted enzyme. Moreover, it was found to have an optimum pH of 10-11, thus revealing that it is an alkaline enzyme. In this paper, the new esterase EM2L8 buried in a deep-sea sediment became known on the surface and was characterized biochemically.

Amino Acid Sequence↗
PubMed1985

Direct measurement of phagolysosomal esterase activity.

A new method of directly measuring esterase activity within phagolysosomes has been developed. Decanoyl fluorescein- binding microspheres were prepared and phagocytosed by human peripheral neutrophils. Within phagolysosomes lysosomal esterase hydrolyzed decanoyl fluorescein on the microspheres, causing the conversion of decanoyl fluorescein- binding microspheres (non-fluorescent) into fluorescein- binding microspheres (fluorescent). The activity of phagolysosomal esterase in intact neutrophils was assayed by the measurement of the fluorescence intensity without rupturing cells. By use of a flow cytometer, esterase activity within phagolysosomes in single cells was measured.

Esterases↗
PubMed1986

Proliferation, esterase activity, and propidium iodide exclusion in urologic tumor cells after in vitro exposure to chemotherapeutic agents.

After exposing urological tumor cells to anticancer agents in vitro, cellular esterase activity and the ability to exclude propidium iodide (PI) were examined as dual indicators of functionality or "viability." High esterase activity/PI exclusion was observed in assays in which anticancer agents failed to inhibit cellular proliferation, while low esterase activity/PI exclusion was often observed when proliferation had been significantly inhibited. In a number of instances, exposure to anticancer agents did produce significant inhibition of proliferation without lowering viability. In this setting, the recovery of proliferative capacity could be demonstrated with several transitional cell carcinoma cell lines, and this recovery was always associated with high esterase activity/PI exclusion. When the proliferation of primary urological tumor preparations was inhibited by drug exposure, estimates of elevated viability were obtained in 27 per cent of the determinations. Thus, viability estimates may be an indicator of the potential for tumor-cell recovery from exposure to anticancer agents. Moreover, the potential for recovery may explain differences between the results of chemosensitivity testing and actual clinical events by reconciling clinical failures with elevated viabilities indicative of this potential.

Antineoplastic Agents↗
PubMed1999

Relationship of human pancreatic cholesterol esterase gene structure with lipid phenotypes.

Pancreatic cholesterol esterase is one of the enzymes that plays a pivotal role in cholesterol absorption. Differences in the genotype of this enzyme could affect the susceptibility of individuals to dyslipidemia and/or cardiovascular disease. We undertook this study to investigate if any correlation exists between restriction fragment length polymorphism in the human pancreatic cholesterol esterase gene and serum lipid levels. DNA from 96 healthy adults was restricted with Stu I, Southern blotted, and probed with cDNA of human pancreatic cholesterol esterase. Results revealed six distinct patterns which were classified as A, B, C, D, E, and F which had a population frequency of 1%, 34.5%, 49%, 12.5%, 1% and 2% respectively. Correlation of the distribution of lipid and lipoprotein levels by pattern and sex revealed a significant interaction between pattern type and HDL (p=0.03) in the most common group (group C) for males. Male patients of pattern C tended to have a lower LDL cholesterol than non-pattern C males (p=0.07); in addition, 80% of all males in the study population with LDL cholesterol under 100 mg/dl were found in pattern C. Thus, the most common Stu I RFLP genotype is associated with a favorable lipid phenotype. This report shows an association between the human pancreatic cholesterol esterase genotype and serum lipid levels. Further analysis of a larger study group with Stu I and alternative polymorphic restriction enzymes is warranted, to confirm this biologically plausible result.

Adult↗
PubMed2003

Estrogenic and esterase-inhibiting potency in rainwater in relation to pesticide concentrations, sampling season and location.

In a year-round monitoring program (1998), pesticide composition and toxic potency of the mix of pollutants present in rainwater were measured. The goal of the study was to relate atmospheric deposition of toxic potency and pesticide composition to each other and to sampling period and local agricultural activity. Rainwater was collected in 26 consecutive periods of 14 days in a background location (BACK) and in two locations representative for different agricultural practices, i.e. intensive greenhouse horticulture (HORT) and flower bulb culture (BULB). Samples were chemically analyzed for carbamate (CARB), organophosphate (OP) and organochlorine (OC) pesticides and metabolites. Esterase inhibiting potency of rainwater extracts was measured in a specially developed bio-assay with honeybee esterases and was expressed as an equivalent concentration of the model inhibitor dichlorvos. Estrogenic potency of the extracts was measured in the ER-CALUX reporter gene assay and was expressed as an equivalent concentration of estradiol. Multivariate principal component analysis (PCA) techniques proved to be valuable tools to analyze the numerous pesticide concentrations in relation to toxic potency, sampling location, and sampling season. Pesticide composition in rainwater depended much more on sampling season than on sampling location, but differences between and were mainly attributed to local differences in agricultural practice. On average, the esterase inhibiting potency exceeded the maximum permissible concentration set for dichlorvos in The Netherlands, and was significantly higher in than in and . Esterase inhibition correlated significantly with OP and CARB concentrations, as expected given the working mechanism of these insecticides. The estrogenic potency incidentally exceeded NOEC levels reported for aquatic organisms and was highest in . Although estrogenic potency of rainwater correlated with OC concentrations, the ER-CALUX responses could not be attributed to any particular pesticides. Besides, the contribution of non-analyzed xeno-estrogens as alkylphenol(-ethoxylates) and bisphenol-A to the estrogenic potency of rainwater could not be excluded. Further research should focus on the chemical identification of estrogenic compounds in rainwater. In addition, more attention should be given to the ecological consequences of atmospheric deposition of individual pesticides and of total toxic potencies that regularly exceed environmental criteria for Dutch surface waters and/or toxic threshold values for aquatic organisms.

Agriculture↗
PubMed1975

[Purification and properties of an esterase excreted during sporulation of Bacillus subtilis].

After growth in Difco Nutrient Broth, several proteolytic enzymes are excreted by B. subtilis, Marburg strain, namely a metalloprotease and a seryl protease. We report here the purification and some biochemical properties of a third extracellular hydrolytic enzyme for which we propose the term of esterase. As the serylprotease, the esterase is a seryl enzyme endowed with both proteolytic and esterolytic activities. Nevertheless the esterase differs from serylprotease in many aspects. In particular, it is an acidic enzyme with a low proteolytic activity and a high esterolytic activity. Its specificity toward synthetic substrates is restricted. The enzyme is active only on esters of amino acids and particularly on those of tyrosine. With Bz Tyr O Et as substrate the esterase displays a maximum of activity between pH 7.2 and 8.1 with a Km of 1.3.10-minus 3 M at 30 degrees C. At the end of this work, two questions remain unanswered: 1) the origin of the multiplicity of the bands revealed by polyacrylamide electrophoresis in the purest fraction; 2) the nature of the physiological substrate of the enzyme.

2,2'-Dipyridyl↗
PubMed1977

1-naphthyl acetate esterases in fluids and tissues of jaw cysts.

The activity and electrophoretic mobility of 1-naphthyl acetate esterases in cystic fluids and cystic tissues of ameloblastomas, follicular and apical cysts were examined. The cystic fluids showed lower activities than sera but had very similar patterns on the electrophoretogram. The activity levels of the three kinds of cystic fluids were not statistically significantly different. The fluid esterases may have originated from serum but they were not produced by the cystic lining tissue. Ameloblastoma tissues showed the highest activity per wet weight and per mg protein of the three kinds of cyst lesions (P less than 0.05). On the electrophoretogram, the esterase-I activity constituted 41% of the total activity in ameloblastomas, whereas in follicular cysts and apical cysts the esterase-I activity constituted 32% and 24% of the total activity, respectively.

Ameloblastoma↗
PubMed1998

In situ evaluation of esterase stereoselectivity in two-dimensional electropherograms and tissue sections.

Staining with both enantiomers of an alpha-naphthyl ester plus a diazonium salt and comparing the color intensities given by the two enantiomers is a convenient method to evaluate the esterase stereoselectivity for that ester in two-dimensional electropherograms and tissue sections. Application of this method for rat liver has shown that (1) several esterases, e.g., one of pI 6.4 and Mr 118 kDa, are moderately stereoselective against alpha-naphthyl (R)-N-acetylalaninate and (R)-N-methoxycarbonylalaninate but strictly stereoselective against alpha-naphthyl (S)-N-methoxycarbonylvalinate, implying that esterase stereoselectivity may be inverted by changing the ester structure; and (2) these esterases are mainly contained in the hepatocytes around central veins.

Alanine↗
PubMed2003

The Serratia marcescens bioH gene encodes an esterase.

The 3.9 kb chromosomal DNA was cloned from Serratia marcescens Sr41, which confers on Escherichia coli cells a phenotype of clear halo formation on tributyrin agar plates. Three complete open reading frames (ORFs) were identified in the inserted DNA, and one ORF was demonstrated to encode a 28 kDa protein of 255 amino acids related to esterase activity. Interestingly, the ORF was 70% identical to a product of the E. coli bioH gene, which lies at a locus separated from the bioABFCD operon and acts in the early steps of the biotin synthetic pathway before pimeloyl-CoA synthesis. This gene complemented a bioH-deficient mutation of E. coli. From the sequence analysis, BioH is presumed to be a serine hydrolase, which belongs to the alpha/beta hydrolase-fold family comprising a wide variety of hydrolases including esterases. A catalytic triad composed of a nucleophilic residue (Ser80), an acidic residue (Asp206), and histidine (His234) was conserved in BioH, and the nucleophilic residue Ser, a catalytic center, was situated in the consensus sequence of G-X-S-X-G-G, a nucleophile elbow. Although the enzymatic function of BioH is not yet elucidated, the bioH gene products from S. marcescens and E. coli show esterase activity, which may imply the hydrolysis of a precursor leading to pimeloyl-CoA ester. The esterase activity of BioH and its CoA binding activity recently reported agree with a current hypothesis of pimeloyl-CoA ester synthesis from CoA and acylester derivatives including an acyl-carrier protein.

Amino Acid Sequence↗
PubMed1998

Typing of nosocomial strains of Serratia marcescens: comparison of pulsed-field gel electrophoresis of macrorestriction fragments with biotyping, esterase typing and ribotyping.

Fifty nosocomial isolates of Serratia marcescens, collected in six Belgian hospitals between 1986 and 1990, were characterized by using pulsed-field gel electrophoresis (PFGE) with XbaI. The results were compared with those previously obtained by three other methods: biotyping, esterase electrophoresis typing and ribotyping with EcoRI and HindIII. Macrorestriction analysis (42 PFGE groups) and esterase typing (42 zymotypes) proved to be the most discriminating, followed by ribotyping (28 ribotypes) and biotyping (10 biochemical profiles). Biotyping would serve as a screen to identify isolates, due to its accessibility. Esterase typing provided a reliable tool to make subdivisions within biotypes because of congruence between biochemical groups and esterase patterns. Additional discrimination was still achieved by ribotyping and PFGE. It is concluded that the combined results of these four markers were useful for distinguishing all epidemic and sporadic isolates.

Bacterial Typing Techniques↗
PubMed1999

The same amino acid substitution in orthologous esterases confers organophosphate resistance on the house fly and a blowfly.

Organophosphate (OP) insecticide resistance in certain strains of Musca domestica is associated with reduction in the carboxylesterase activity of a particular esterase isozyme. This has been attributed to a 'mutant ali-esterase hypothesis', which invokes a structural mutation to an ali-esterase resulting in the loss of its carboxylesterase activity but acquisition of OP hydrolase activity. It has been shown that the mutation in Lucilia cuprina is a Gly137-->Asp substitution in the active site of an esterase encoded by the Lc alpha E7 gene (Newcomb, R.D., Campbell, P.M., Ollis, D.L., Cheah, E., Russell, R.J., Oakeshott, J.G., 1997. A single amino acid substitution converts a carboxylesterase to an organophosphate hydrolase and confers insecticide resistance on a blowfly. Proc. Natl. Acad. Sci. USA 94, 7464-7468). We now report the cloning and characterisation of the orthologous M. domestica Md alpha E7 gene, including the sequencing of cDNAs from the OP resistant Rutgers and OP susceptible sbo and WHO strains. The Md alpha E7 gene has the same intron structure as Lc alpha E7 and encodes a protein with 76% amino acid identity to Lc alpha E7. Comparisons between susceptible and resistance alleles show resistance in M. domestica is associated with the same Gly137-->Asp mutation as in L. cuprina. Bacterial expression of the Rutgers allele shows its product has OP hydrolase activity. The data indicate identical catalytic mechanisms have evolved in orthologous Md alpha E7 and Lc alpha E7 molecules to endow diazinon-type resistance on the two species of higher Diptera.

Amino Acid Sequence↗