Valine Aminopeptidase Research Articles

Extracellular Hydrolytic Enzyme Activities of the Heterotrophic Microbial Communities of the Rouge River: An Approach to Evaluate Ecosystem Response to Urbanization

The potential effects of urbanization on the bioavailability of dissolved organic carbon (DOC) were tested by determining the extracellular enzyme activities of the heterotrophic microbial communities of the Rouge River. The activities of 19 enzymes were monitored across two water samples (river water and groundwater) at different spatial and temporal scales. High phosphatase, esterase, and aminopeptidase activities was observed in site 9 (site most exposed to anthropogenic sources) showed higher concentrations of DOC compared to sites 1 and 8 (sites exposed to less anthropogenic sources), where moderate activities of diverse range of enzymes were observed. High relative contributions of phosphatase, esterase, and aminopeptidase activities to the overall enzyme activity as observed in site 9 stressed the increased importance of peptides as C source for heterotrophic communities and high in-stream carbon processing, which account for high nonspecific extracellular enzyme activities. In contrast, high contribution of glycosyl hydrolases occurred consistently across all sites, which highlights the significance of microbial detrital and plant biomass as carbon sources. Majority of the enzymes showed evidence of activity at various extents during spring and summer. However, higher activities of leucine aminopeptidase, valine aminopeptidase, β-glucosidase, and α-mannosidase were observed in the summer; and alkaline phosphatase and α-glucosidase in the spring. The results presented here suggest a shift in organic carbon bioavailability across all sites of contrasting urbanization, despite similarities in DOC concentrations. Hence, API ZYM technique can be used as an effective indicator of river water and groundwater system health across an urban gradient.

Biochemical composition and digestive enzyme activity during naupliar development of Artemia spp from three solar saltworks in Greece

Three parthenogenetic brine shrimp Artemia populations from saltworks in Greece (Messolongi, Milos and Polychnitos) were studied with regard to their biochemical composition and the activity of digestive enzymes in four developmental stages from decapsulated cysts to nauplii instar III. The content of protein and RΝΑ was highest in nauplii instar II (65.10 and 6.23% dry weight, respectively), while that of total lipid was highest in nauplii instar I (11.40% dw). The carbohydrate content was highest in decapsulated cysts (10.30% dw) and about one half that value in nauplii instar I. The DNA content was ten-fold higher in nauplii instar I than in decapsulated cysts with the highest levels in nauplii instar III (3.67% dw). The activity of alkaline phosphatase, leucine aminopeptidase, valine aminopeptidase, cystine aminopeptidase, β-galactosidase, β-glucosidase, esterase lipase (C8), esterase (C4), N-acetyl-β-glucosaminidase, α-fucosidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase increased significantly from decapsulated cysts to nauplii instar II and remained constant or decreased in nauplii instar III. In nauplii instar II, leucine aminopeptidase, β-galactosidase, alkaline phosphatase and esterase (C4) demonstrated higher activity levels (140.90, 133.62, 66.01 and 45.47 nmoles/100 µg dw within 5 min, respectively) of the aforementioned enzymes. The Artemia population of Messolongi had significantly higher levels of DNA, protein and alkaline phosphatase activity per individual, as well as body weight, compared to the Artemia populations of Milos and Polychnitos. The latter population showed the lowest level of lipid and the highest esterase activity (C4) per individual. There was a reverse relationship between the protein content and leucine aminopeptidase activity, lipid content and esterase (C4) activity, carbohydrate content and β-galactosidase activity among the three Artemia populations. The variations observed in the biochemical composition and the activity of some specific enzymes should be taken into consideration in selecting these Artemia populations from Greece for application in aquaculture.

English

  A mixture of polymeric substrates (simulating a complex wastewater) was transformed under sewer conditions and aerobiosis by Aspergillus niger in a tanks-in-series reactor at a hydraulic retention time of 14 h. Starch was totally removed after 7 h of incubation. Removal of the protein portion with a molecular weight larger than 2 kDa followed the starch removal and the total proteins were the latest to be removed. Alkaline phosphatase, leucine aminopeptidase, valine aminopeptidase, a-bglucosidase and a-mannosidase were abundantly secreted in the growth medium. This research is the first report on mixed polymeric substrate biodegradation under sewer condition by A. niger, and could be considered as an open window on fungal biomass valorisation in wastewater treatment.   Keywords: Polysaccharide, wastewater, starch, bovine serum albumin, macromolecules, tank-in-series reactor, enzyme.

Characterization of lactic acid bacteria isolated from a Greek dry-fermented sausage in respect of their technological and probiotic properties

A total of 147 lactic acid bacteria was isolated from two types of naturally fermented dry sausages at four different stages of the ripening process studied in order to select the most suitable strains according to their technological characteristics including probiotic properties and antimicrobial activity against food-borne pathogens. Identification of the isolates revealed that 90% were lactobacilli, 4% enterococci, 3% Pediococcus sp. and sporadic isolates of Weissella viridescens, Leuconostoc pseudomesenteroides, and Leuconostoc sp. The isolated strains of Lactobacillus sakei (49 isolates), Lactobacillus curvatus (24 isolates) and Lactobacillus plantarum (7 isolates) were further characterized. All strains could grow at 15 °C, whereas the majority of the strains was able to grow in the presence of 6.5% NaCl and on acetate agar. The enzymatic potential of the strains was evaluated using the API ZYM system. During in vitro investigations all strains exhibited high leucine and valine aminopeptidase activities and moderate acid phosphatase and phosphohydrolase activities. Some strains showed very weak lipolytic activity. The enzyme profiling is an important factor for selection of strains as starter cultures. A large majority of the strains tolerated 0.1% bile salts whereas 58% of Lactobacillus curvatus strains and all Lactobacillus plantarum strains were resistant to 0.3% bile salts. All Lactobacillus sakei strains and the majority of Lactobacillus curvatus and Lactobacillus plantarum strains exhibited an anti-listerial activity against three Listeria monocytogenes strains. A percentage of 75, 50 and 29% of Lactobacillus sakei, L. curvatus and L. plantarum strains, respectively, could inhibit two Staphylococcus aureus strains. The contribution of the selected strains to a possible inhibition of Listeria monocytogenes and S. aureus in situ on fermented meats would be of considerable interest to enhance the hygienic quality of these products.

Microbial Population Dynamics and Enzyme Activities During Composting

The changes in population size of different microbial groups (total aerobic heterotrophs, actinomycetes, fungi, fecal coliforms, ammonium- and nitrite-oxidizing bacteria, and denitrifying bacteria) and the activities of 19 different enzymes (three phosphatases, three esterases, two proteases, three amino-peptidases, and eight glycosyl-hydrolases) were examined during cocomposting of poultry litter (a mixture of poultry manure, waste feed, feathers, and wood shavings) and yard trimmings (a mixture of grass clippings, leaves, and wood barks). Three piles with forced aeration were established by mixing 2:1 (v/v) ratio of poultry litter and yard trimmings. During composting, samples were taken at three different locations (top, middle, and bottom) of the forced aeration piles for microbial and enzyme analyses. Results demonstrated that population size of different microbial groups was not a limiting factor in this composting process as the microorganisms in the poultry litter + yard trimmings compost are in great abundance. Although the numbers of these microbial groups were reduced by high temperature, their populations multiplied rapidly as composting progressed. Fecal coliforms were eliminated by day 49, suggesting that the poultry litter + yard trimmings compost showed an overall increase in diversity and relative abundance of extracellular enzymes present as composting progressed. The population of fungi and actinomycetes (microorganisms active in degradation of cellulose, hemicellulose, and lignin) were positively correlated with esterase, valine amino-peptidase, α-galactosidase, β-glucosidase, and lipase. Of all 19 enzymes examined, ß-galactosidase (enzyme involved in the hydrolysis of lactose) had the most significant positive correlation with microbial populations, such as total aerobic heterotrophs, ammonium- and nitrite-oxidizing bacteria, denitrifying bacteria, and fecal coliforms. Cystine amino peptidase, chymotrypsin, and trypsin showed no evidence of activity during the entire period of composting. This composting process represented a combined activity of a wide succession of environments in the compost pile as one microbial group/enzyme overlapped the other and each emerged gradually as a result of the continual change in temperature as well as moisture content, O2 and CO2 level, and progressive breakdown of complex compounds to simpler ones.

A comparative survey of the hydrolytic enzymes of ectoparasitic and free-living mites

Extracts of ectoparasitic mites of birds ( Dermanyssus gallinae), sheep ( Psoroptes ovis) and plants ( Tetranychus urticae) and of free-living mites ( Acarus siro) contained acid and alkaline phosphatase, C4 and C8 esterases, lipase, leucine and valine aminopeptidases and a range of glycosidase activities. Dermanyssus gallinae and P. ovis, species highly adapted to an animal parasitic lifestyle, had very similar profiles and contained low activities of glycosidases. In contrast, the polyphagous species A. siro contained moderate to high activities of every glycosidase examined, whereas the phytophagous species, T. urticae, displayed high activities of only β-galactosidase and β-glucuronidase. All extracts hydrolysed haemoglobin with optima below pH 6, and this hydrolysis was associated with an aspartic proteinase and variable cysteine proteinase activity dependent on species. Inhibitor-labelling with biotinyl-Phe-Ala-FMK revealed the presence of cysteine proteinases with molecular masses of 25–33.5 kDa. Each mite species contains the enzymes necessary to complete digestion of the diet in the intracellular lysosomal compartment. The absolute and relative activities of each enzyme varied, and are discussed according to phylogeny and dietary habit.

Immunological control of scab mites: digestive enzymes as candidate compounds

Phosphatases, C4 and C8 esterases, leucine and valine aminopeptidases, n-acetyl-β-glucosaminidase, β-glucosidase, β-galactosidase and β-glucuronidase were active in extracts of scab mites ( Psoroptes spp.) raised on sheep or rabbits. Trypsin and chymotrypsin activities were not detected. Haemoglobin was hydrolysed by a detergent-soluble fraction of the mite extracts in a pH-dependent fashion with an optimum of pH 3–5. Acid proteinase activity was greater in mites raised on rabbits than in those raised on sheep. Inhibitors of cysteine, serine and metallo-proteinases failed to inhibit the hydrolysis of H–Pro–Thr–Glu–Phe–Phe(NO 2)Arg–Leu–OH while pepstatin A, a specific inhibitor of aspartic proteinases, totally inhibited its hydrolysis at a concentration of 1 nM.

Hydrolytic enzymes of Psoroptes cuniculi (Delafond)

Phosphatases, C4 and C8 esterases, leucine and valine aminopeptidases, N-acetyl-β-glucosaminidase, β-glucosidase, β-galactosidase and β-glucuronidase were detected in extracts of the parasitic mite Psoroptes cuniculi. Lipase, trypsin-like and chymotrypsin-like activities were not present. Haemoglobin was hydrolysed by a detergent-soluble fraction of the mite extracts with a maximum hydrolysis between pH 3 and 5. Acid proteinase activity was greater against haemoglobin than bovine serum albumin. Inhibitors of cysteine, serine and metallo-proteinases failed to inhibit the hydrolysis of H–Pro–Thr–Glu–Phe–Phe(NO 2)–Arg–Leu–OH while pepstatin A inhibited its hydrolysis in a dose-dependent manner (IC 50 8.02×10 −11M (±0.30×10 −11). Thermal inactivation of the proteolytic activity followed an exponential decay pattern. Typical K m and V max values were 7.2×10 −5 (±0.7×10 −5) M −1 and 1.13×10 −3 (±0.05×10 −3) OD unit −1 min −1 respectively. Acid proteinase activity eluted from a size exclusion column in a single, major peak representing a molecular weight range of 21–24.5 kDa. The major endoproteinase of P. cuniculi therefore appears to be a cathepsin D-like aspartic proteinase.

Identification of Pasteurella haemolytica A1 isolates from market-stressed feeder calves by use of enzyme and antimicrobial susceptibility profiles

SUMMARY An epidemiologic study of Pasteurella haemolytica serovar 1 (Ph1) in market-stressed feeder calves from 7 farms in eastern Tennessee was conducted. The nasal mucus of each calf was cultured sequentially at the farm of origin (day 0), at an auction market (day 133), and at a feedyard in Texas (days 141, 148, 155, and 169). Of the 103 calves tested, 77 were culture-positive, including 1 on day 0, 1 on day 133, 20 on day 141, 57 on day 148, 50 on day 155, and 14 on day 169. From the 143 Ph1 isolates, 20 enzyme profiles were determined by use of a commercial enzyme system that detects 19 enzymatic reactions; 4 antimicrobial susceptibility profiles were obtained, using the disk-diffusion method, which evaluated susceptibility to 11 antibacterial drugs. All isolates were positive for acid phosphatase and alkaline phosphatase, but were negative for α-galactosidase, α-mannosidase, β-glucosidase, β-glucuronidase, cystine aminopeptidase, N-acetyl-β-glucosaminidase, and trypsin. Other positive enzyme reactions included: leucine aminopeptidase, 140 Ph1 isolates; phosphohydrolase, 90 isolates; α-fucosidase, 63 isolates; esterase (C4), 59 isolates; valine aminopeptidase, 30 isolates; esterase lipase (C8), 24 isolates; β-galactosidase, 2 isolates; and α-glucosidase, chymotrypsin and lipase (C14), 1 isolate each. Thirty-four Ph1 profiles were identified, using combined enzyme and antimicrobial susceptibility profiles. The data indicate that the strains isolated during the feedyard period may have been determined more by farm of origin (P ≤ 0.001) than by habitation with calves from other farms while in the feedyard. The combined enzyme and antimicrobial susceptibility profile method is a rapid and simple epidemiologic technique for tracking Ph1 strains in market-stressed feeder calves.

Biochemical Activities of Bacillus Species Isolated from Flat Sour Evaporated Milk

Forty strains of bacilli were isolated from flat sour evaporated milk and were characterized as Bacillus stearothermophilus (5 strains), Bacillus licheniformis (10 strains), Bacillus coagulans (15 strains), Bacillus macerans (5 strains), and Bacillus subtilis (5 strains). The hydrolases of these strains were examined with the API ZYM system, and the ability of these strains to produce acid in milk incubated at different temperatures was also examined. Esterase, esterase lipase, lipase, valine aminopeptidase, phosphoamidase, β-glucuronidase, and β-glucosidase were the activities found in all strains tested; they were strain-dependent and ranged between 1 and 5 by the API ZYM test. The same strains produced various concentrations of acid in milk. These organisms may be responsible for the flat sour spoilage in evaporated milk.

Pathogenic factors of Pseudomonas cepacia isolates from patients with cystic fibrosis

One hundred and nineteen isolates of Pseudomonas cepacia, 98 of which were from cystic fibrosis (CF) patients and 21 from environmental and other human sources, were examined for biochemical and exo-enzymatic properties that may contribute to the pathogenicity of this bacterium. The following characteristics were demonstrated significantly more frequently in isolates from CF patients than in control isolates: production of catalase, ornithine decarboxylase, valine aminopeptidase, C14 lipase, alginase and trypsin; reduction of nitrate to nitrite; hydrolysis of urea and xanthine; complete haemolysis on bovine red blood cells; cold-sensitive haemolysis on human red blood cells; greening of horse and rabbit red blood cells. The role of these factors in the pulmonary disease associated with cystic fibrosis is not clear. However, several factors which have been reported previously as being associated with pathogenic processes with other bacteria have now been described in P. cepacia. Additional factors not previously reported as "pathogenicity factors" are also described.

Evaluation of the API-ZYM system for identification of Listeria

The API-ZYM system (Analytab Products) was evaluated for potential use for confirming the identity of Listeria isolates. A total of 68 Listeria strains from various sources were assayed using standard procedures. All isolates were positive for esterase (C4), esterase lipase (C8), phosphohydrolase (one exception), β-glucosidase, acid phosphatase and lipase (one exception). Negative responses were observed consistently for valine aminopeptidase, trypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-fucosidase and cystine aminopeptidase (one exception). Variable responses were noted for alkaline phosphatase, leucine aminopeptidase, chymotrypsin, α-glulucosidase and N-acetyl glucosaminidase. Comparison of these results with those of other Gram positive species indicated that the API-ZYM system could be used for rapid confirmation of Listeria to the subgenus level.

Biochemical Activities of Pediococcus pentosaceus isolates of Dairy Origin

Eighty-three strains of pediococci were isolated from raw goat milk and Feta and Kaseri cheese and were characterized as Pediococcus pentosaceus (75 strains) and P. pentosaceus subspecies intermedins (8 strains). The hydrolases of 49 P. pentosaceus strains were examined with the API ZYM system. Leucine aminopeptidase and valine aminopeptidase were the strongest activities found (means 4.98 and 4.92, respectively, on a five-point color intensity scale) and were found in all strains. Activities of β-galactosidase and N-acetyl-β-glucosamidase were found in 96 and 92% of the strains, respectively, and were slightly weaker (mean activities of 4.61 and 4.17, respectively). Considerably lower were the activities of β-glucosidase (mean 2.99) and phosphoamidase (mean 1.55) and were detected in 98 and 55% of the strains, respectively. Very weak (<1) esterase, esterase:lipase, lipase, phosphoamidase, cystine aminopeptidase, or acid phosphatase activities were found in various numbers of strains. These results give information about the biochemical activities of P. pentosaceus species found in milk and cheese.

ヒト混合唾液中の酵素活性 歯口清掃中止による変化

The purpose of this study was to elucidate 19 enzymatic activities in whole mixed saliva and their changes in men without oral hygiene.Ten healthy, unmedicated male students with excellent oral hygiene and clinically normal gingiva, between the ages of 22 and 26 years, were the experimental subjects. At the start of the experimental period, whole mixed saliva and parotid saliva were collected and the clinical parameters were recorded for each of them. Then, the experimental no-cleansing period of 7 days was started, and whole mixed saliva was collected and the clinical parameters were recorded every day. Enzymatic activities were determined using API ZYM systems (MONTALIEU VERCIEU, France).Alkaline phosphatase, butyrate esterase, caprylate esterase-lipase, myristate lipase, leucine aminopeptidase, valine aminopeptidase, and trypsin activities in whole mixed saliva supernatant were significantly higher at the end of the no-cleansing period than at the start of experiment. Myristate lipase, valine aminopeptidase, and trypsin were detected in whole mixed saliva sediment, but not in parotid saliva. This suggests that these enzyme activities in whole mixed saliva are useful in estimating the oral hygiene status.

Dipeptidyl-aminopeptidases and aminopeptidases in Dictyostelium discoideum

Extracts prepared from culminating cells of Dictyostelium discoideum have been found to contain dipeptidyl-aminopeptidases I (EC 3.4.14.1), II (EC 3.4.14.2), III (EC 3.4.14.4), arginine aminopeptidase (EC 3.4.11.6) and valine aminopeptidase. Dipeptidyl-aminopeptidase III was the most active of the dipeptidyl-aminopeptidases; its molecular weight was 158,000, with a pH optimum of 10.2 and gave a single peak of activity on gel-filtration or when fractionated by chromatofocusing. The specific activities of dipeptidyl-aminopeptidases I and III increased during development being highest during the culmination stage before decreasing during sorocarp formation; dipeptidyl-aminopeptidase II and arginine aminopeptidase decreased progressively throughout development. The presence of these dipeptidyl-aminopeptidases suggests the possibility that processing of peptides may be necessary during the development of Dictyostelium.

Aminopeptidase activity of Capnocytophaga.

Studies have shown that Capnocytophaga species are unique among the periodonlal microorganisms so far tested in that they produce marked leucine aminopeptidase, valine aminopeptidase, and cystine aminopeptidase activities. In this paper, we used the expanded API ZYMAP (API‐System, La Balme les Grottes, France) system to survey 18 Capnocytophaga strains for their ability to hydrolyze 57 synthetic substrates of β‐naphthylamide derivalives of amino acids, dipeplides, tripeptides, tetrapeptides, and pentapeptides. In general, the study strains exhibited marked hydroiylic activity against all test β‐naphthylamides bearing a free N‐terminal α‐amino group, The hydrolytic activity varied little among the strains. Since aminopeptidases can be involved in bradykinin formation and degradation of collagen fragments and other events ofthe inflammatory process, the Capnocytophaga aminopeptidases may be important virulence factors in Capnocytophaga‐associated periodonial diseases.

Hydrolytic enzymes of Moraxella bovis.

Certain strains of Moraxella bovis produce tissue-damaging enzymes which may initiate or potentiate infectious bovine keratoconjunctivitis. Thirteen reference strains of this species were characterized physiologically and screened for production of various enzymes by some conventional biochemical tests and the API ZYM system (Analytab Products, Plainview, N.Y.). All 13 strains were hemolytic. All hydrolyzed Tween 80 and Tween 85 and displayed C4 esterase, C8 esterase-lipase, and C14 lipase activities. All produced phosphoamidase and phosphatase. All were able to hydrolyze casein and gelatin. All produced leucine and valine aminopeptidases and fibrinolysin. Twelve produced hyaluronidase or were agarolytic. Three hydrolyzed chondroitin sulfate. Nine strains autoagglutinated. Five produced catalase, and two produced cystine aminopeptidase.