Characterization of Yersinia enterocolitica, Y. intermedia, Y. aldovae, Y. frederiksenii, Y. kristensenii and Y. pseudotuberculosis by electrophoretic polymorphism of acid phosphatase, esterases, and glutamate and malate dehydrogenases

Acid phosphatase, esterases, and glutamate and malate dehydrogenases of 192 strains of Yersinia enterocolitica, Y. intermedia, Y. aldovae, Y. frederiksenii, Y. kristensenii and Y. pseudotuberculosis were analysed by horizontal polyacrylamide agarose gel electrophoresis and by isoelectrofocusing in thin-layer polyacrylamide gels. The six species were clearly separated from each other by their distinct enzyme electrophoretic polymorphism. For Y. enterocolitica, the strains of biotype 5 were differentiated from the other biotypes by the mobility of glutamate dehydrogenase. For Y. frederiksenii, six zymotypes were delineated by pI and by the mobility of the enzymes. Variation in number or mobility of esterases within each species could represent a marker for epidemiological and ecological analyses. A linear relationship was obtained between the mean genetic diversity coefficient of enzymes and the mean percentage DNA-DNA relatedness of Y. intermedia, Y. aldovae, Y. enterocolitica and Y. frederiksenii.     

Enzyme activities of Lyme disease and relapsing fever borreliae

Activities of glycosidases ( n = 8), esterases ( n = 10), arylamidases ( n = 63), acid phosphatase, alkaline phosphatase and phosphoamidase were tested in 47 Borrelia strains. Forty-four were B. burgdorferi strains; 22 of which were isolated from human specimens (skin 13, cerebrospinal fluid six, and one each from blood, heart muscle and synovia), 13 were isolated from various organs of laboratory animals infected via tick bite or with human isolates, and nine from ticks. The remaining three were the relapsing fever strains B. coriaceae , B. hermsii , and B. turicatae. Strains were of low and high passage but the number of subcultures did not influence the enzyme patterns obtained by utilization of chromogenic substrates of constitutive enzymes. Glycosidase activity was absent in almost all strains tested. Esterase activity was high on molecules of chain length 9 carbons. 2-Naphthylamide derivatives were utilized by strains of human, rodent or tick origin in a range of 66.6 to 83.1%. Almost all strains utilized substrates for acid and alkaline phosphatase and phosphoamidase. Chymotrypsin activity was only found in three and two strains from specimens of human and rodent origin, respectively; and γ-glutamyltransferase activity only in three human skin isolates. No strain tested displayed trypsin activity. Overall, the specific activities of constitutive enzymes of the Borrelia strains tested are widely similar. Thus, the enzyme profiles did not discriminate between human, animal and tick isolates, or between human isolates of Borrelia whether cultivated from cerebrospinal fluid or from skin biopsy of Lyme borreliosis patients.     

Characterization of Enterobacter cloacae and E. sakazakii by electrophoretic polymorphism of acid phosphatase, esterases, and glutamate, lactate and malate dehydrogenases

Acid phosphatase, esterases, and glutamate, lactate and malate dehydrogenases of 34 strains of Enterobacter cloacae and 22 strains of Enterobacter sakazakii were analysed by horizontal polyacrylamide agarose gel electrophoresis and by isoelectrofocusing in thin-layer polyacrylamide gel. The two species could be separated on the basis of distinct electrophoretic patterns of all enzymes analysed. Glutamate dehydrogenase and acid phosphatase were detected exclusively in E. cloacae, whereas esterase bands were more intensively stained in E. sakazakii. For each species, two zymotypes could be distinguished, on the basis of electrophoretic mobilities of malate dehydrogenase and banding patterns of esterase for E. cloacae, and by both isoelectric point and electrophoretic mobilities of an esterase and of lactate and malate dehydrogenases for E. sakazakii. The high degree of enzyme polymorphism within the two species permitted precise identification of strains. The variations in electrophoretic patterns might therefore provide useful epidemiological markers.     

Native agarose-polyacrylamide gel electrophoresis allowing the detection of aminopeptidase, dehydrogenase, and esterase activities at the nanogram level: enzymatic patterns in some Frankia strains

Nanogram amounts of soluble aminopeptidases, dehydrogenases, and esterases were detected by nondenaturing ultralow gelling point agarose-polyacrylamide gel electrophoresis (ULGA-PAGE). Cytosolic fractions from Frankia sp. were electrophoresed at 4 degrees C in the presence of Co2+, Zn2+, or Mg2+ ions. Then, aminopeptidases and esterases were revealed by simultaneous capture staining by using fast garnet GBC diazonium salt as the chromogenic coupling compound. Dehydrogenases were revealed by using nitro blue tetrazolium salt as electron acceptor. A variety of aminopeptidases, dehydrogenases, and esterases could be identified by their migration in ULGA-PAGE and by their sensitivities to NaCl, CoSO4, ZnSO4, and MgCl2 when assayed "ingel." The presence of agarose was essential for the detection of the complex enzyme patterns. The patterns were remarkably similar for the five Frankia strains isolated from Allocasuarina and Casuarina host plants and differed from those of Frankia strains isolated from Comptonia and Hippopha? host plants. A nomenclature is proposed for aminopeptidases and other Frankia enzymes. The richness of the Frankia amino-peptidases and esterases zymograms makes them adequate marker enzymes for taxonomical, genetic, or biochemical studies. Dehydrogenases might also be useful, although a more restricted number of bands were found with L-lactic and L-malic acid as substrates.     

Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533, selected for the biotransformation of ferulic acid to vanillin, are also able to produce cell wall polysaccharide-degrading enzymes and feruloyl esterases

The filamentous fungal strains Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533, previously selected for the bioconversion of ferulic acid to vanillic acid and vanillin respectively, were grown on sugar beet pulp. A large spectrum of polysaccharide-degrading enzymes was produced by A. niger and very few levels of feruloyl esterases were found. In contrast, P. cinnabarinus culture filtrate contained low amount of polysaccharide-degrading enzymes and no feruloyl esterases. In order to enhance feruloyl esterases in A. niger cultures, feruloylated oligosaccharide-rich fractions were prepared from sugar beet pulp or cereal bran and used as carbon sources. Number of polysaccharide-degrading enzymes were induced. Feruloyl esterases were much higher in maize bran-based medium than in sugar beet pulp-based medium, demonstrating the ability of carbon sources originating from maize to induce the synthesis of feruloyl esterases. Thus, A. niger I-1472 could be interesting to release ferulic acid from sugar beet pulp or maize bran.     

Electrophoretic forms of glucose-6-phosphate dehydrogenase, acid phosphatase and esterase in Amoeba species amoebas

Glucose-6-phosphate dehydrogenase (G6PD), acid phosphatase and esterases in free-living amoebae of 7 Amoeba species were investigated with the use of disc-electrophoresis in polyacrylamide gel. The evidence provided is suggestive that the electrophoretic isoenzyme patterns of acid phosphatase and esterases (and G6PD in some cases), in addition to a few morphological characters, can serve as a taxonomic criterion for species identification within this genus, as well as for revealing erroneously classified species and strains. It is suggested that A. indica is an independent species whose preliminary diagnosis has been given in this paper. It is concluded that A. discoides and A. lescherae are strains of A. proteus, rather than two independent species. A and As-102 amoebian strains, kept in the collection of protozoan strains and species of the Institute of Cytology RAS and referred to as strains of A. proteus, belong in reality to another Amoeba species and even to another genus within the family Amoebidae. This conclusion has been documented by results of our analysis of electrophoretic patterns of acid phosphatase and esterases in these strains.     

Enzymatic systems involved in decomposition reflects the ecology and taxonomy of saprotrophic fungi

One hundred and eleven strains of Basidiomycota, 39 strains of Ascomycota and 2 strains of Mucoromycotina belonging to wood decomposers that cause white-rot (WR) or brown-rot (BR), other wood associated saprotrophs (WA), litter decomposing cord-forming Basidiomycota (LDF), and saprotrophic microfungi (SA), were screened for the production of hydrolytic enzymes and laccase. The presence of enzyme-encoding genes was also analysed in the published genomes of saprotrophic fungi. Several genes, including those for acidic phosphatase, β-glucosidase and N-acetylglucosaminidase, were common in the genomes with enzyme activity widely displayed by fungi, while other enzymes, such as certain hemicellulases or laccase, were produced less frequently. Enzyme production by saprotrophic fungi was shaped by the combination of their ecophysiology and taxonomy. Basidiomycota exhibited higher activities of all enzymes, except alkaline phosphatase, α-glucosidase, N-acetylglucosaminidase, α-mannosidase and α-fucosidase, than Ascomycota. The SA and BR fungi showed distinct enzyme production patterns, while the enzyme production by WR, LDF and WA was similar. Differences among species were typically reflected in the level of enzyme activity rather than in the absence of enzymes. Enzyme screening results showed that in several cases, fungi exhibited enzyme activity without the presence of the corresponding gene and vice versa. This indicates that the use of genome-derived information for the prediction of potential enzyme production has substantial limitations and cannot replace functional screening of fungal cultures.     

Histochemical investigation of the folliculogenesis in the ovary of the Mongolian gerbil (Meriones unguiculatus)

The ovaries of 70 mature Mongolian gerbils (Meriones unguiculatus) were investigated morphologically and enzyme histochemistry for the appearance of acid phosphatase, non-specific esterases, succinate dehydrogenase and thiamine pyrophosphatase. In the oocyte two particular enzyme active zones exist depending on the state of development. In young oocytes acid phosphatase, succinate dehydrogenase and thiamine pyrophosphatase can be found only in the perinuclear zone. From the late secondary follicle on, activity of acid phosphatase, succinate dehydrogenase and non-specific esterases can be detected only in a peripheral area of cytoplasm, whereas thiamine pyrophosphatase is present in the entire ooplasm. In the follicular epithelium a different pattern of enzyme distribution suggests a functional differentiation of the epithelial cells during folliculogenesis.     

Sequence‐ and activity‐based screening of microbial genomes for novel dehalogenases

Dehalogenases are environmentally important enzymes that detoxify organohalogens by cleaving their carbon-halogen bonds. Many microbial genomes harbour enzyme families containing dehalogenases, but a sequence-based identification of genuine dehalogenases with high confidence is challenging because of the low sequence conservation among these enzymes. Furthermore, these protein families harbour a rich diversity of other enzymes including esterases and phosphatases. Reliable sequence determinants are necessary to harness genome sequencing-efforts for accelerating the discovery of novel dehalogenases with improved or modified activities. In an attempt to extract dehalogenase sequence fingerprints, 103 uncharacterized potential dehalogenase candidates belonging to the α/β hydrolase (ABH) and haloacid dehalogenase-like hydrolase (HAD) superfamilies were screened for dehalogenase, esterase and phosphatase activity. In this first biochemical screen, 1 haloalkane dehalogenase, 1 fluoroacetate dehalogenase and 5 l -2-haloacid dehalogenases were found (success rate 7%), as well as 19 esterases and 31 phosphatases. Using this functional data, we refined the sequence-based dehalogenase selection criteria and applied them to a second functional screen, which identified novel dehalogenase activity in 13 out of only 24 proteins (54%), increasing the success rate eightfold. Four new l -2-haloacid dehalogenases from the HAD superfamily were found to hydrolyse fluoroacetate, an activity never previously ascribed to enzymes in this superfamily.     

Esterases in Folsomia candida (Collembola: Isotomidae). Characterization of enzymes among parthenogenic strains

Esterase enzymes from four strains of Folsomia candida were investigated using polyacrylamide gel electrophoresis. Up to 12 bands of enzymatic activity were present in each strain. Esterase bands were classified as choline esterases or as one of two groups of carboxyl esterases, based on mobility, on substrate specificity and on activity remaining after inhibition by class-specific chemicals. One strain-specific choline esterase was discovered which resisted the effects of many organophosphate inhibitors. Organophosphate inhibitor concentrations had to be 10 to 100 times greater to reduce the staining activity of this resistant choline esterase to the level of comparable esterases in other strains.     

Light and electron microscopic studies on the enzyme cytochemistry of leucocytes of eels, Anguilla species

The leucocytes of three anguillid eels were studied using enzyme cytochemistry. Leucocytes were stained for peroxidase, alkaline phosphatase, acid phosphatase, aryl sulphatase, β-glucuronidase, N-acetyl-β-glucosaminidase, β-galactosidase, lysozyme, a variety of non-specific esterases, chloroacetate esterase and two proteases. All cells were negative for aryl sulphatase, β-glucuronidase, N-acetyl-β-glucosaminidase, and β-galactosidase. Very few neutrophils, thought to be mature, and all eosinophils contained peroxidase-positive granules, and some monocytes showed very weak peroxidase staining. All leucocytes lacked alkaline phosphatase, but all cells except lymphocytes and thrombocytes of A. dieffenbachii contained acid phosphatase. Neutrophil acid phosphatase released into phagosomes was associated with Escherischia coli bacteriolysis. Neutrophils also secrete lysozyme and, with monocytes, produce and secrete a variety of esterases. The possible interaction of lysozyme, acid phosphatase and esterases in bacteriolysis is discussed.     

Microbial carbohydrate esterases in cold adapted environments

Psychrophiles produce cold-evolved enzymes that display a high catalytic efficiency, associated with a low thermal stability. In recent years, these enzymes have attracted the attention of scientists because of their peculiar properties that render them particularly useful in investigating the relationship existing between enzyme stability and flexibility on one hand, and enzyme activity on the other hand. Among these enzymes, the esterases, and particularly the feruloyl esterases, have potential uses over a broad range of applications in the agro-food industries. In recent years, the number of microbial feruloyl esterase activities has increased in the growing genome databases. Based on substrate utilization data and supported by primary sequence identity, four subclasses of esterase have been characterized so far. Up to the present, ten genomes from psychrophilic bacteria have been completely sequenced and additional fourteen genomes are under investigation. From the bacteria strains whose genome has been completely sequenced, we analyzed the presence of esterase genes, both the putative genes and the determined experimentally genes, and performed a ClustalW analysis for feruloyl esterases. Major details will be presented for the ORF PSHAa1385 from P. haloplanktis TAC125 that recently has been studied in our research group. In addition, the potential biotechnology applications of this class of enzymes will be discussed.     

Effects of freeze-preservation on some pollen enzymes: II. Freezing and freeze-drying stresses

The isozymes of lily and corn pollen esterases and acid phosphatase were studied in relation to freeze-drying and vacuum-drying. Fresh samples of Lilium longiflorum L. and Zea mays L. pollen were frozen at rates ranging between 200 and 100 °C/min and freeze-dried at temperatures from 0 to ?70 °C for approximately 48 to 70 hr. Freeze-dried samples were rehydrated slowly (10% relative humidity) and rapidly (90% relative humidity). Vacuum-drying was performed at room temperature (22 °C).Soluble pollen enzymes were analyzed by disc electrophoresis on polyacrylamide gels stained with substrate specific reagents. The stained gels were evaluated by densitometry for migration rate, isozyme pattern, and relative activity. The numerical data generated in this manner were then statistically analyzed.The following conclusions resulted from this study: (i) freeze-drying and freeze-thawing treatments were comparable except for corn esterases; (ii) freeze-drying induced alterations in enzyme activity except for corn acid phosphatase; (iii) the freezing rate, the final freezing temperature, and exposure to various relative humidities produced few changes in freeze-dried material; (iv) freeze-drying was less detrimental than vacuum-drying to the enzyme characteristics of corn; (v) freeze-drying yielded higher viabilities than vacuum-drying; and (vi) acid phosphatase alterations appeared to be related to pollen viability in most cases.     

Histochemical investigation of the folliculogenesis in the ovary of the Mongolian gerbil (Meriones unguiculatus)

Summary The ovaries of 70 mature Mongolian gerbils (Meriones unguiculatus) were investigated morphologically and enzyme histochemically for the appearance of acid phosphatase, non-specific esterases, succinate dehydrogenase and thiamine pyrophosphatase. In the oocyte two particular enzyme active zones exist depending on the state of development. In young oocytes acid phosphatase, succinate dehydrogenase and thiamine pyrophosphatase can be found only in the perinuclear zone. From the late secondary follicle on, activity of acid phosphatase, succinate dehydrogenase and non-specific esterases can be detected only in a peripheral area of cytoplasm, whereas thiamine pyrophosphatase is present in the entire ooplasm.In the follicular epithelium a different pattern of enzyme distribution suggests a functional differentiation of the epithelial cells during folliculogenesis.This investigation was supported by the Deutsche Forschungsgemeinschaft     

Comparative electrophoretic polymorphism of esterases and other enzymes in Escherichia coli

The electrophoretic polymorphism of esterases was compared with that of other enzymes in Escherichia coli populations by investigating allozyme distribution of four esterases (A, B, C and I) within both the subspecific groups I, II and III and the new groups A, B1, B2, C, D and E, which have been distinguished by electrophoretic analysis of 11 and 35 enzymes respectively in the 72 reference strains of the ECOR collection. Electrophoretic distribution of esterases was distinct for each of the three subspecific groups as indicated by distributions of allozymes and electrophoretic types (distinctive combination of allozyme for the four esterases). Esterase polymorphisms of the three subspecific groups appeared to have similar features to those of three previously studied natural populations of strains obtained from human and animal gastro-intestinal tracts and extra-intestinal infections in humans. Multiple correspondence analyses using data obtained from the four esterases and the 11 other enzymes also distinguished the groups A, B1, B2, C, D and E. All strains of group B2 showed the B2 electrophoretic pattern of esterase B, which appeared to be a marker of a distinct cluster of strains frequently implicated in extra-intestinal infections.     

Epidemiological typing of Acinetobacter strains by esterase electrophoresis

Fifty-three strains of Acinetobacter, belonging to the species A baumannii, A. haemolyticus and A. johnsonii, were differentiated by electrophoretic typing of their esterases, on the basis of both the enzyme specific activity profiles and their electrophoretic mobilities. Each esterase was defined by its spectrum of hydrolytic activity toward five synthetic substrates and its sensitivity to di-isopropyl fluorophosphate. Since each enzyme was not detected in all strains of a given species, several zymotypes could be defined by the patterns of combinations of esterases. Thus, 24 zymotypes were defined in the 32 A. baumannii strains, 4 were defined in the 10 A. haemolyticus strains and 6 were defined in the 11 A. johnsonii strains. When the electrophoretic mobilities of the various esterases were included, each of the 53 strains of Acinetobacter (with the exception of three A. haemolyticus strains) showed a distinct electrotype.     

Observations on morphology and hydrolytic enzyme histochemistry of excysted metacercariae of Himasthla rhigedana (Trematoda: Echinostomatidae)

The morphology of in vitro excysted metacercariae of Himasthia rhigedana was studied by light microscopy, enzyme histochemistry, and scanning electron microscopy (SEM). The body surface is covered with longitudinal rows of pointed spines which extend from the anterior end to just below the acetabulum. Histochemical methods for acid and alkaline phosphatases, β-glucoronidasc, leucine aminopeplidase, nonspecific esterase, acetylcholinesterase, butyrylcholincsterase, chymotrypsin-like protease, α-galactosidase and β-galactosidase were applied to the excysted metacercariae. Certain systems of the metacercaria containing these enzymes showed positive reactions to the appropriate methods and were selectively visualized. Reactions for alkaline phosphatase occur throughout most of the excretory system while acid phosphatase occurs in the gut, oral, and ventral suckers. Reactions for nonspecific esterases and cholinesterascs are found throughout the nervous system, in the gut, and in the oral and ventral suckers. The results of including specific inhibitors or activators in the incubation medium after preineubation m 10^?5m diethyl p-nitrophenyl phosphate (E-600) suggested that A-and C-typc esterases are present in the gut. Inclusion of 10^?4m eserine in the incubation medium abolished cholinesterase activity in the nervous system.     

Histological and histochemical studies on testis, epididymis and ductus defrens of the rooster (Gallus domesticus)]

Testis, epididymis and ductus deferens of the adult domestic fowl and male gonads of juvenile roosters have been studied by means of histochemical and histological methods. Testicular interstitial cells: According to enzyme-histochemical results oxidative energy production seems to be of minor importance. An extraordinarily high activity of lysosomal enzymes (acid phosphatase and esterases in the adult, esterases only in the immature) is observed. Positive reactions of 3beta-steroid-dehydrogenase and enzymes of the pentose phosphate cycle indicate steroid hormone production; the pathways of the steroid synthesis, however, are probably different in adult and immature testes. A remarkable LAP content of juvenile Leydig cells is a parameter of an increased protein metabolism. Areas of reserve cells: Focal accumulation of these cell types are observed in testis and epididymis of the immature and in the epididymis of the adult fowl. Reserve cells reveal distinct activities of LAP (prospective growth ability) and 3beta-HstDH (reserve capacity for steriod synthesis). All other enzymes studied react weakly, thus pointing to a generally low metabolic activity. Seminiferous tubules: The strong reaction of alkaline phosphatase in the peritubular cells may play a part in energy disposition for contractions. Sertoli cells of adult animals are rich in lysosomal enzymes and enzymes of the glycolytic chain but oxidoreductases react weaker than in mammalian Sertoli cells. This indicates that nutritive interactions between germ and Sertoli cells in birds are different from those in mammals: The basally orientated germ cells of birds contain strong activities of diaphorases, LDH, SDH, Cyto-Ox and seem to be metabolically rather indipendent from Sertoli cells.     

Enzyme-coupled assay of acetylxylan esterases on monoacetylated 4-nitrophenyl beta-D-xylopyranosides

Three different monoacetates of 4-nitrophenyl beta-D-xylopyranoside were tested as substrates for beta-xylosidase and for microbial carbohydrate esterases and a series of non-hemicellulolytic esterases. The acetyl group in 2-O-acetyl, 3-O-acetyl, and 4-O-acetyl 4-nitrophenyl beta-D-xylopyranoside makes the glycoside resistant to the action of beta-xylosidase (EC 3.2.1.37). This fact was explored to introduce a new enzyme-coupled assay of acetylxylan esterases (EC 3.1.1.72) and other carbohydrate-deacetylating enzymes. The deacetylation converts the monoacetates into the substrate of beta-xylosidase, the auxiliary enzyme. The effect of the acetyl group migration along the xylopyranoid ring in aqueous media can be avoided by shortening the assay duration. The assay enables an easy examination of the positional specificity of the enzymes, which is important for classification of acetylxylan esterases and for elucidation of the structure-function relationship among carbohydrate esterases in general. Non-hemicellulolytic esterases showed different positional specificity of deacetylation than did acetylxylan esterases.     

The electrophoretic polymorphism of bacterial esterases

Abstract: The specific activities of esterases and certain other molecular properties including immunospecificity indicate that the electrophoretic variations of these enzymes in bacterial populations are the result of allelic variations at specific gene loci. The esterase polymorphism of Enterobacteriaceae and some other species isolated from man or animals demonstrates that esterases can distinguish between bacteria at the species or subspecies level, both by their biochemical properties and by their electrophoretic differences. The esterase data complement DNA hybridization studies and agree with ribosomal DNA polymorphism, especially for delineating a phylogenetically distinct group of highly pathogenic strains in Escherichia coli . A two-dimensional electrophoretic profile obtained by establishing a direct correspondence between homologous esterase bands resolved by independent runs of isoelectric focusing and standard electrophoresis considerably improves the detection of allelic variations, whereas protein titration curves (electrophoresis in pH gradient) can be used to demonstrate the real electrophoretic homogeneity of allozymes or evalue their molecular relationship in terms of apparent amino acid substitutions. This overview establishes that esterases, by their significant electrophoretic polymorphism, are reliable molecular markers for systematics and epidemiology, and are suitable enzyme systems for studying population genetics and phylogeny.