Academic literature on the topic 'Fluorogenic substrates. eng'

New fluorogenic ceramidase substrates derived from the N-acyl modification of our previously reported probes (RBM14) are reported. While none of the new probes were superior to the known RBM14C12 as acid ceramidase substrates, the corresponding nervonic acid amide (RBM14C24:1) is an efficient and selective substrate for the recombinant human neutral ceramidase, both in cell lysates and in intact cells. A second generation of substrates, incorporating the natural 2-(N-acylamino)-1,3-diol-4-ene framework (compounds RBM15) is also reported. Among them, the corresponding fatty acyl amides with an unsaturated N-acyl chain can be used as substrates to determine alkaline ceramidase (ACER)1 and ACER2 activities. In particular, compound RBM15C18:1 has emerged as the best fluorogenic probe reported so far to measure ACER1 and ACER2 activities in a 96-well plate format.

Abstract Zinc metallopeptidases of bacterial pathogens are widely distributed virulence factors and represent promising pharmacological targets. In this work, we have characterized Zmp1, a zinc metallopeptidase identified as a virulence factor of Mycobacterium tuberculosis and belonging to the neprilysin (NEP; M13) family, whose X-ray structure has been recently solved. Interestingly, this enzyme shows an optimum activity toward a fluorogenic substrate at moderately acidic pH values (i.e., 6.3), which corresponds to those reported for the Mtb phagosome where this enzyme should exert its pathological activity. Substrate specificity of Zmp1 was investigated by screening a peptide library. Several sequences derived from biologically relevant proteins were identified as possible substrates, including the neuropeptides bradykinin, neurotensin, and neuropeptide FF. Further, subsequences of other small bioactive peptides were found among most frequently cleaved sites, e.g., apelin-13 and substance P. We determined the specific cleavage site within neuropeptides by mass spectrometry, observing that hydrophobic amino acids, mainly phenylalanine and isoleucine, are overrepresented at position P1′. In addition, the enzymatic mechanism of Zmp1 toward these neuropeptides has been characterized, displaying some differences with respect to the synthetic fluorogenic substrate and indicating that the enzyme adapts its enzymatic action to different substrates.

Different aminopeptidase and endopeptidase substrates were assessed for the detection of enzymatic activity of microorganisms collected from the surface of aerobically cold-stored pork and beef. The most sensitive substrates were fluorogenic Ala-7-amino-4-methylcoumarin (Ala-AMC) or Leu-AMC and colorogenic Ala-p-nitroanilide (Ala-pNA). Activity on natural oligopeptides, e.g., bradykinin or αs1 casein fragment 1 to 23, was very low. The correlation coefficient (r) between log surface counts of 66 meat samples and log fluorescence or absorbance after incubation of surface microbial cells for 2 h with Ala-AMC, Leu-AMC, and Ala-pNA was 0.89, 0.83, and 0.82, respectively. A distinct yellow color was obtained with Ala-pNA when the surface count was ∼106 CFU/cm2. Although correlation and sensitivity was better, no clear advantage is obtained with the use of the fluorogenic Ala-AMC or Leu-AMC instead of Ala-pNA, a substrate proposed by Alvarado et al. (J. Food Sci. 57:1330, 1992) for rapidly assessing the microbial quality of refrigerated meat. The correlation coefficient (r) between time of cold storage and surface count was 0.69.

Abstract Background: Antithrombin III (ATIII) is a stoichiometric inhibitor of factor Xa and thrombin and a major physiological regulator of coagulation. Reduction of ATIII levels, for example in patients with congenital ATIII deficiency, causes a rise in active thrombin which is associated with risk of venous and arterial thrombosis (1, 2). The calibrated automated thrombogram (CAT) assay is widely used to measure thrombin generation in human plasma. Therewith, the fluorogenic substrate Z-G-G-R-AMC is cleaved by the thrombin emerging over time in the plasma sample. Thus, an increase in fluorescence signal reflects the amount of thrombin provided that fluorogenic substrate is in excess over thrombin. To account for substrate consumption, plasma color, and 'inner filter' effect, the fluorescence signal is compared to alpha-2-macroglobulin thrombin complex, the thrombin calibrator. In this complex, thrombin is protected from its physiological inhibitors, but retains its ability to cleave small substrates (3). Aim: We assessed the technical feasibility of measuring the effect of ATIII reduction on thrombin generation in normal and FVIII-inhibited human plasma in the tissue factor (TF) triggered CAT assay. Methods: CAT was performed in ATIII deficient plasma with 1 pM of TF to trigger thrombin generation. The plasma was supplemented with 0.125 - 2.5 µM ATIII to achieve ATIII plasma levels of 5 - 100% of normal. The ATIII titration was also done in ATIII deficient plasma inhibited with a goat polyclonal anti-human FVIII antibody to simulate hemophilia A conditions. Raw data analysis focused on the velocity of fluorescence signal increase, which is usually automatically converted to nM of thrombin by the Thrombinoscope software. Results: Reduced ATIII levels resulted in an apparent concentration dependent increase in thrombin generation in both normal and FVIII inhibited human plasma. For example, a reduction from 2.5 to 1.25 µM ATIII resulted in an apparent increase of 45 - 55% in thrombin peak and ETP values. Most importantly, analysis of the raw data showed that the fluorescence signal reached a plateau after 30 min (normal plasma) and 50 min (FVIII-inhibited plasma) in samples with <1.25 µM ATIII. The plateau (at ~1700 FU) is due to complete depletion of fluorescent substrate. In comparison, samples with ³1.25 µM ATIII did not reach a plateau and show substrate conversion until the end of the assay. In summary, a plasma level of at least 1.25 µM ATIII (50% of normal) was required to obtain thrombin generation profiles which were not compromised by fluorogenic substrate depletion. Below 1.25 µM ATIII, it is not possible to distinguish between inhibition of thrombin and excessive consumption of fluorescence substrate, and therefore, data may be misinterpreted. Conclusion: Thrombin generation at low ATIII plasma levels is difficult to interpret as the physiological inhibition of the generated thrombin is reduced. Consequently, the thrombin substrate is consumed, resulting in substrate depletion. Therefore, we conclude that the CAT assay is technically not suitable to assess the effect of ATIII levels < 1.25 µM ATIII corresponding to 50% of normal plasma. Disclosures Scheiflinger: Baxalta Innovations GmbH: Employment.

Control and manipulation of bacterial populations requires an understanding of the factors that govern growth, division, and antibiotic action. Fluorescent and chemically reactive small molecule probes of cell envelope components can visualize these processes and advance our knowledge of cell envelope biosynthesis (e.g., peptidoglycan production). Still, fundamental gaps remain in our understanding of the spatial and temporal dynamics of cell envelope assembly. Previously described reporters require steps that limit their use to static imaging. Probes that can be used for real-time imaging would advance our understanding of cell envelope construction. To this end, we synthesized a fluorogenic probe that enables continuous live cell imaging in mycobacteria and related genera. This probe reports on the mycolyltransferases that assemble the mycolic acid membrane. This peptidoglycan-anchored bilayer-like assembly functions to protect these cells from antibiotics and host defenses. Our probe, quencher-trehalose-fluorophore (QTF), is an analog of the natural mycolyltransferase substrate. Mycolyltransferases process QTF by diverting their normal transesterification activity to hydrolysis, a process that unleashes fluorescence. QTF enables high contrast continuous imaging and the visualization of mycolyltransferase activity in cells. QTF revealed that mycolyltransferase activity is augmented before cell division and localized to the septa and cell poles, especially at the old pole. This observed localization suggests that mycolyltransferases are components of extracellular cell envelope assemblies, in analogy to the intracellular divisomes and polar elongation complexes. We anticipate QTF can be exploited to detect and monitor mycobacteria in physiologically relevant environments.

Salivary gland secretions of three species of the medicinal leech differ in the level of lysozyme peptidoglycan-lysing activity. Using the synthetic fluorogenic substrate, 4-methyl-umbelliferyl tetra N-acetyl-β-chitotetraosid, the glycosidase activity (as one of peptidoglycan-lysing activities) of salivary gland secretion of three species of the medicinal leech was quantitatively evaluated in comparison with egg lysozyme. It is supposed, that lysozyme activity of the leech secretions is determined not only by 5 isoforms of destabilase-lysozyme, but by some other enzymes which can utilize this substrate. These may be lysozymes other than i- (invertebrate) lysozymes (such as destabilase-lysozyme, or related enzymes).

Reaction conditions for the reducing-end-specific derivatization of cellulose substrates with the fluorogenic compound, anthranilic acid, have been established. Hydrolysis of fluorescence-labelled celluloses by cellobiohydrolase Cel7A from Trichoderma reesei was consistent with the active-site titration kinetics (burst kinetics), which allowed the quantification of the processivity of the enzyme. The processivity values of 88±10, 42±10 and 34±2.0 cellobiose units were found for Cel7A acting on labelled bacterial cellulose, bacterial microcrystalline cellulose and endoglucanase-pretreated bacterial cellulose respectively. The anthranilic acid derivatization also provides an alternative means for estimating the average degree of polymerization of cellulose and, furthermore, allows the quantitative monitoring of the production of reducing end groups on solid cellulose on hydrolysis by cellulases. Hydrolysis of bacterial cellulose by cellulases from T. reesei revealed that, by contrast with endoglucanase Cel5A, neither cellobiohydrolases Cel7A nor Cel6A produced detectable amounts of new reducing end groups on residual cellulose.

Abstract Glucuronidase is present in most strains of Escherichia coli but absent in most other enteric microorganisms; therefore, an assay for this enzyme is useful for determining the presence of the organism. The substrate 4-methylumbelliferyl beta-D-glucuronide (MUG) is incorporated into either lauryl tryptose (LT) broth or EC medium; the inoculated tubes are then incubated under specified conditions and examined under longwave UV light for the presence of a fluorogenic glucuronidase end product. When compared with the 10-day most probable number (MPN) procedure of AOAC, the LT-MUG and the EC-MUG tests required 24 and 96 h, respectively, and gave comparable mean log MPN values for samples of crabmeat, sunflower kernels, and walnut pieces. However, false-positive and falsenegative reactions were observed with foods tested by both of these rapid methods. Overall, method sensitivity was not compromised by using the LT-MUG rather than the EC-MUG method. Incorporation of 25 αg MUG/mL into LT broth resulted in diminished fluorescence of positive reactions, whereas MUG concentrations of 50 and 100 αg/mL provided decisive fluorogenic reactions.

Abstract An assay for the enzyme glucuronidase was used to determine the presence of Escherichia coli in selected, naturally contaminated high moisture foods. Raw pork sausage, ground turkey, and ground beef were inoculated into tubes containing the substrate 4-methylumbelliferyl beta-D-glucuronide (MUG) in lauryl tryptose (LT) medium. After incubation at 35°C for 24 h, the inoculated LT-MUG tubes were examined under longwave ultraviolet light for the presence of a fluorogenic glucuronidase end product. A fluorescing tube indicated the presumptive presence of E. coli. The 10 day most probable number method of the AOAC and the LT-MUG procedure gave comparable recoveries of E. coli.

Abstract A collaborative study was conducted to compare a proposed LSTMUG method with the AOAC official method for Escherichia coli detection. E. coli produces an enzyme, β-glucuronidase, which cleaves the substrate, 4-methyl-umbelliferyl-β-D-glucuronide (MUG), to yield a fluorescent end product. Incorporation of the MUG substrate into lauryl tryptose broth (LST) enables a rapid quantitative method for screening E. coli, which is detected by fluorescence of the medium under longwave UV light. In this collaborative study, 5 food samples, 2 frozen (entree sauce/gravy and dairy topping) and 3 chilled (hamburger, pork sausage, and cheese), were tested for E. coli detection by 17 collaborating laboratories. Results indicate that the LST-MUG method is equal to or better than the current AOAC method for detecting E. coli. The LST-MUG method has been adopted official first action

Orientador: Eleni Gomes
Banca: Luiz Juliano Neto
Banca: João Ruggiero Neto
Resumo: Fungos termofílicos têm despertado grande interesse acadêmico e industrial por produzirem uma variedade de enzimas termoestáveis com potenciais aplicações em processos biotecnológicos como biocatálise nas indústrias de couro, farmacêutica, têxtil e alimentícia, e na preparação de produtos de limpeza e cosméticos. Particularmente, as proteases, além de participarem de inúmeros processos fisiológicos vitais como vias metabólicas, hemostasia e sinalização celular, também representam hoje cerca de 60% do mercado mundial de enzimas. Neste trabalho, descrevemos a produção, purificação e caracterização bioquímica de uma serino-protease produzida por um fungo termofílico do gênero Myceliophthora. As taxas de atividade proteolítica foram avaliadas através de fermentação em meio sólido (FES) e submerso (FSM) e observou-se um rendimento na atividade proteolítica 4,5 vezes maior para o meio sólido. A enzima bruta obtida por ambos os procedimentos (FES e FSM) exibiu a mesma temperatura ótima de 50 ºC, porém em relação ao pH ótimo houve um deslocamento de 7 (FSM) para 9 (FES) sugerindo que o perfil enzimático do fungo difere de acordo com suas condições de fermentação. Baseado nesses resultados prosseguiu-se os estudos com o extrato bruto obtido por FES. A imobilização da enzima bruta em esferas de alginato de cálcio resultou no aumento da temperatura ótima e na estabilidade térmica quando comparado com a enzima livre. O extrato bruto obtido por FES foi, então, fracionado por métodos cromatográficos como exclusão molecular e troca-iônica que resultaram na protease pura com peso molecular de 28,2 kDa determinado por espectrometria de massa. A protease pura demonstrou pH ótimo de 9,0 e temperatura ótima de 45 °C que corroboram... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Thermophilic fungi have attracted great academic and industrial interest because they produce a variety of thermostable enzymes with potential applications in biotechnological processes such as biocatalysis in the industries of leather, pharmaceutical, textile and food, and the preparation of detergents and cosmetics. In particular, proteases not only participate in many vital physiological processes such as metabolic pathways, cell signaling and homeostasis, but also currently represent about 60% of the world market of enzymes. In this work, we describe the production, purification and biochemical characterization of a serine protease produced by a thermophilic fungus of the genus Myceliophthora. The levels of proteolytic activity were evaluated either by solid fermentation (SSF) and submerged (SmF). The crude enzyme obtained by both procedures (SSF and SmF) exhibited similar optimum temperature of around 50 ºC, but in relation to the optimum pH was shifted of 7 (SmF) to 9 (SSF), suggesting that the enzymatic profile of the fungus differs from according to its fermentation conditions. Based on these results, the studies were followed with crude extract obtained by SSF. The immobilized enzyme on beads of calcium alginate resulted in increased optimum temperature and thermal stability when compared to the free enzyme. The crude extract obtained by SSF was then fractionated by chromatographic methods including molecular exclusion and ion-exchange that resulted in the pure protease with molecular weight of 28.2 kDa as determined by mass spectrometry. The pure protease showed optimum pH of 9.0 and optimum temperature of 45 °C that corroborate to the preliminary characterization of the crude extract. Inhibition tests resulted in complete inhibition by PMSF, a canonical... (Complete abstract click electronic access below)
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