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In this paper a device, based on urease-loaded microspheres, is presented. The first task of this work was the optimization of a procedure for the alginate microspheres realization, having a radius as close as possible to the optimal one necessary to achieve the maximum enzyme exploitation. This optimal radius was calculated theoretically through a mathematical model which describes the concentration of substrate (urea) inside the microspheres on the assumption of a diffusion-reaction mechanism. The enzyme-loaded microspheres were successfully tested in a prototypal device aimed at the depletion of urea from a circulating fluid simulating blood flow: the results showed that urea concentration in the circulating fluid drops down to less than 25% of the initial value after 5 h. [ABSTRACT FROM AUTHOR]

The immobilization of a multi-enzyme extract is an excellent method for multi-step biotransformations. This paper describes how a multi-enzyme extract from Agrobacterium radiobacter , rich in d -hydantoinase and N-carbamyl- d -amino acid amidohydrolase was immobilized on chitin for its application on the synthesis of p-hydroxyphenylglycine. The adsorption derivative showed a higher activity than the covalent one. Compared to the soluble multi-enzymatic extract, the adsorption derivative showed greater pH-stability in the pH range under study. Its optimum pH ranged from 7-8. Furthermore, it showed high activity at low temperature. [ABSTRACT FROM AUTHOR]

Peptidoglycan (PG), a heteropolysaccharide component of the mycobacterial cell wall can be shed during tuberculosis infection with immunomodulatory consequences. As such, changes in PG structure are expected to have important implications on disease progression and host responses during infection with Mycobacterium tuberculosis. Mycobacterial amidases have important roles in remodeling of PG during cell division and are implicated in susceptibility to antibiotics. However, their role in modulating host immunity remains unknown. We assessed the bacterial burden and host immune responses to M. tuberculosis mutants defective for either one of two PG N-acetylmuramyl-L-alanine amidases, Ami1 and Ami4, in bone marrow-derived macrophages (BMDM) and C57BL/6 mice. In infected BMDM, the single deletion of both genes resulted in increased proinflammatory cytokine responses. In mice, infection with the Δami1 mutant led to differential induction of pro-inflammatory cytokines and chemokines, decreased cellular recruitment and reduced lung pathology during the acute phase of the infection. While increased proinflammatory cytokines production was observed in BMDM infected with the Δami4 mutant, these effects did not prevail in mice. Infection using the Δami1 and Δami4 Mtb mutants showed that these genes are dispensable for intracellular mycobacterial growth in macrophages and mycobacterial burden in mice. These findings suggest that both Ami1 and Ami4 in M. tuberculosis are not essential for mycobacterial growth within the host. In summary, we show that amidases are important for modulating host immunity during Mtb infection in murine macrophages and mice. [ABSTRACT FROM AUTHOR]

In the present study it is shown that a preparation of highly purified plasma kallikrein (specific activity 81 S-2302 U/mg) still contained small amounts of an IgG fraction. Amidase assays of the fresh enzyme with four peptide substrates (S-2302, Bz-Pro-Phe-Arg-pNA, S-2366, S-2222) did not reveal any inhomogeneity, and immunoblot experiments with antibodies against prekallikrein yielded only an 85 kD double band. After a storage period (2 years at -70 degrees C), S-2366 and S-2222 amidase activities not reflecting the initial kallikrein appeared. Immunoblot studies with Fc-specific antibodies against IgG showed a 170 kD band, and immunoblots with a monoclonal antibody against prekallikrein demonstrated that, in addition to the 85 kD band, a band with a mol weight of about 152 kD could also be detected. Immunoblots showed that only 85 kD kallikrein was recovered in the eluate from Protein G columns, and amidase assays based on S-2302 and Bz-Pro-Phe-Arg-pNA showed a recovery of about 70%. The other part of the kallikrein (30%) was removed along with the additional S-2366 and S-2222 activities and the IgG3 fraction present. The theory is advanced that the additional activity reflects a kallikrein fraction with a mol weight of about 152 kD, and is present in the fresh enzyme preparation in inactive complex with IgG. Storage of the kallikrein preparation led to some weakening of this complex and the appearance of functional activities of the kallikrein fraction involved. [ABSTRACT FROM AUTHOR]

This study investigated the relationship between a recently proposed alkaline hydrolysis method for estimating the chemical index of nitrogen (N) mineralization potential of soils and the activities of arylamidase and four amidohydrolases involved in hydrolysis of organic N (ON) in soils. Nitrogen mineralization was studied in 13 soils from uncultivated fields in Iowa, USA, by direct steam distillation of 1 g field-most soil treated with 1 M KOH or 1 M NaOH. The distillate was collected in boric acids, which was changed every 5 min for a total of 40 min. The NH4+-N in the distillate was determined by titration with 0.005 M H2SO4. The cumulative amounts of N hydrolyzed were fitted to the first-order exponential equation to determine the "potentially hydrolyzable N (Nmax)" for the soils. The activities of arylamidase, L-asparaginase, L-glutaminase, amidase, and L-aspartase were assayed at their optimal pH values. Results showed that estimated Nmax values were strongly correlated with the activities of arylamidase and amidohydrolases. The activities of arylamidase and the amidohydrolases were significantly correlated, indicating that the activities of the two groups of enzymes are coupled in mineralization of ON in soils. Based on the specificity of enzyme reactions and the strong relationship between estimated Nmax values and the activities of arylamidase and amidohydrolases, we concluded that similar amide-N bonds were susceptible to enzymatic and alkaline hydrolysis, and that alkaline hydrolyzable ON can be used as an index of N mineralization in soils. [ABSTRACT FROM AUTHOR]

In this study, an amidohydrolase activity of amidase in whole cells ofRhodococcussp. MTB5 has been used for the biotransformation of aromatic, monoheterocyclic and diheterocyclic amides to corresponding carboxylic acids. Benzoic acid, nicotinic acid and pyrazinoic acid are carboxylic acids which have wide industrial applications. The amidase of this strain is found to be inducible in nature. The biocatalytic conditions for amidase present in the whole cells of MTB5 were optimized against benzamide. The enzyme exhibited optimum activity in 50 mM potassium phosphate buffer pH 7.0. The optimum temperature and substrate concentrations for this enzyme were 50 °C and 50 mM, respectively. The enzyme was quite stable for more than 6 h at 30 °C. It showed substrate specificity against different amides, including aliphatic, aromatic and heterocyclic amides. Under optimized reaction conditions, the amidase is capable of converting 50 mM each of benzamide, nicotinamide and pyrazinamide to corresponding acids within 100, 160 and 120 min, respectively, using 5 mg dry cell mass (DCM) per mL of reaction mixture. The respective percent conversion of these amides was 95.02%, 98.00% and 98.44% achieved by whole cells. The amidase in whole cells can withstand as high as 383 mM concentration of product in a reaction mixture and above which it undergoes product feedback inhibition. The results of this study suggest thatRhodococcussp. MTB5 amidase has the potential for large-scale production of carboxylic acids of industrial value. [ABSTRACT FROM PUBLISHER]

Objective: To analyze methylation profiles of known preeclampsia/eclampsia (PE) candidate genes in normal (NL) and preeclamptic (PE) women at delivery.Methods: A matched case-control study comparing methylation in 79 CpG sites/33 genes from an independent gene set in maternal leukocyte DNA in PE and NL (n = 14 each) on an Illumina BeadChip platform. Replication performed on second cohort (PE = 12; NL = 32).Results: PE demonstrates differential methylation in POMC, AGT, CALCA, and DDAH1 compared with NL.Conclusion: Differential methylation in four genes associated with PE may represent a potential biomarker or an epigenetic pathophysiologic mechanism altering gene transcription. [ABSTRACT FROM AUTHOR]

Purpose: To describe clinical and CT features of L-asparaginase-associated pancreatitis (L-AP) and to correlate CT grades with clinical parameters. Methods: A total of 16 children (M:F = 9:7; mean age, 8.1 years) who developed L-AP after L-asparaginase (L-asp) treatment and underwent abdominal CT scan were included. We retrospectively reviewed clinical data (age, sex, signs, and symptoms related to pancreatic toxicity and its complications, the number of L-asp doses receiving before L-AP); laboratory test results (serum amylase, lipase, C-reactive protein (CRP), calcium, blood urea nitrogen (BUN), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), glucose, and serum albumin); and clinical course (the number of days of hospitalization, number of NPO days, use of nasogastric tube, intravenous (IV) narcotics, total parenteral nutrition (TPN) or any surgical intervention). We also reviewed CT images and modified CT severity index (MCSI) for grading the severity of AP and classified to three groups (mild, moderate, and severe) or two groups (low and high score) according to MCSI. Results: L-AP typically occurred early in the course of therapy. Use of IV narcotics ( P = .014) and peak amylase ( P = .009) showed a significant difference between mild and severe L-AP groups according to MCSI. Between the low and high score groups, Use of IV narcotics ( P = .046), BUN ( P = .039), and peak amylase level ( P = .013) was significantly different. However, the L-asp dose, hospital day, and other clinical date associated with prognosis did not show any significant difference. Conclusion: In L-AP with pediatric ALL patients, MCSI may correlate with usage of IV narcotics, BUN, and peak amylase levels. [ABSTRACT FROM AUTHOR]

A biosensor for urea has been developed based on the observation that urea is a powerful active-site inhibitor of amidase, which catalyzes the hydrolysis of amides such as acetamide to produce ammonia and the corresponding organic acid. Cell-free extract from Pseudomonas aeruginosa was the source of amidase (acylamide hydrolase, EC 3.5.1.4) which was immobilized on a polyethersulfone membrane in the presence of glutaraldehyde; an ion-selective electrode for ammonium ions was used for biosensor development. Analysis of variance was used for optimization of the biosensor response and showed that 30 μμL of cell-free extract containing 7.47 mg protein mL−−1, 2 μμL of glutaraldehyde (5%, v/v) and 10 μμL of gelatin (15%, w/v) exhibited the highest response. Optimization of other parameters showed that pH 7.2 and 30 min incubation time were optimum for incubation of membranes in urea. The biosensor exhibited a linear response in the range of 4.0--10.0 μμM urea, a detection limit of 2.0 μμM for urea, a response time of 20 s, a sensitivity of 58.245 % per μμM urea and a storage stability of over 4 months. It was successfully used for quantification of urea in samples such as wine and milk; recovery experiments were carried out which revealed an average substrate recovery of 94.9%. The urea analogs hydroxyurea, methylurea and thiourea inhibited amidase activity by about 90%, 10% and 0%, respectively, compared with urea inhibition. [ABSTRACT FROM AUTHOR]

The article discusses the use of comparative modeling to predict the three-dimensional (3D) structure of cysteine, histidine-dependent amidohydrolase and peptidase domain of the LysK endolysin (CHAP k protein) which is derived from bacteriophage K. It notes the presence of highly conserved amino acid residues in CHAP proteins which include an invariant cystine (Cys), and histidine (His). The author also cites the use of web-based servers, and visualization programs in the comparative analysis.

In the last decades the idea to use enzymes for environmental bioremediation has been more and more proposed and, in the light of this, new solutions have been suggested and detailed studies on some classes of enzymes have been performed. In particular, our attention in the last few years has been focused on the enzymes belonging to the amidohydrolase superfamily. Several members of this superfamily are endowed with promiscuous activities. The term 'catalytic promiscuity' describes the capability of an enzyme to catalyse different chemical reactions, called secondary activities, at the active site responsible for the main activity. Recently, a new family of microbial lactonases with promiscuous phosphotriesterase activity, dubbed PTE-Like Lactonase (PLL), has been ascribed to the amidohydrolase superfamily. Among members of this family are enzymes found in the archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius, which show high thermophilicity and thermal resistance. Enzymes showing phosphotriesterase activity are attractive from a biotechnological point of view because they are capable of hydrolysing the organophosphate phosphotriesters (OPs), a class of synthetic compounds employed worldwide both as insecticides and chemical warfare agents. Furthermore, from a basic point of view, studies of catalytic promiscuity offer clues to understand natural evolution of enzymes and to translate this into in vitro adaptation of enzymes to specific human needs. Thermostable enzymes able to hydrolyse OPs are considered good candidates for the set-up of efficient detoxification tools. [ABSTRACT FROM AUTHOR]

The inhibition of jack bean urease by 2,3-dichloro-1,4-naphthoquinone (DCNQ) was studied at ambient temperature in 20 mM phosphate buffer, pH 7.8. The process was investigated by incubation procedure in the absence of substrate. It was found that DCNQ acted as a time- and concentration-dependent inactivator of urease. The time course of the reaction displayed a biphasic mode. Each phase followed a pseudo-first-order kinetics, however the inactivation rate at the first phase was significantly faster than at the next one. The biphasity indicated the complex mechanism of DCNQ action on urease. Quinones action on proteins has been elucidated as at least two processes: direct arylation of essential protein thiols and/or indirect oxidation of essential thiols by reactive oxygen species (ROS) realising during quinone reduction to semiquinones. The next evidence of the studied mechanism was provided by the reactivation experiment that showed the participation of reversible and irreversible processes in the inactivation. The application of dithiothreitol (DTT) into DCNQ blocked-urease solution resulted in an effective enzyme activity regain which quickly returned to 70 ± 10%. The irreversible inactivation of urease was attributed to DCNQ arylation of thiol residues in the protein. On the other hand, it was assumed that the reversible inactivation was a result of the action of ROS such as H2O2. Presence of H2O2 in the incubation system was proved by an experiment with the use of catalase. The enzyme by the elimination of H2O2 decreased DCNQ inactivating influence on urease. The comparison of participation of the fast and slow phase in the inactivation with the percentage of the process reversibility was assumed that the fast period was a result of the arylation mechanism while the slow phase was related to the oxidative influence of H2O2. [ABSTRACT FROM AUTHOR]

The interaction of a variety of substrates with Pseudomonas aeruginosa native amidase (E.C. 3.5.1.4), overproduced in an Escherichia coli strain, was investigated using difference FTIR spectroscopy. The amides used as substrates showed an increase in hydrogen bonding upon association in multimers, which was not seen with esters. Evidence for an overall reduction or weakening of hydrogen bonding while amide and ester substrates are interacting with the enzyme is presented. The results describe a spectroscopic approach for analysis of substrate--amidase interaction and in situ monitoring of the hydrolysis and transferase reaction when amides or esters are used as substrates. [ABSTRACT FROM AUTHOR]

Pig trypsin was chemically modified with the bifunctional compound ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester) to yield EG-trypsin. EG-trypsin showed greater thermal stability (100% active beyond 100 min at 55°°C; native only 53% active at 100 min) together with slightly increased tolerance toward some organic solvents. Arg/Lys hydrolysis ratio changed little. Esterase/amidase activity ratio of EG-trypsin in buffer was 11-fold greater than that of native pig trypsin, but 5-fold less in 30% v/v acetonitrile. In buffer, EG-trypsin synthesized the dipeptide benzoyl-Arg-Leu-NH2 at a 3-fold higher rate than native trypsin, but native trypsin outperformed EG-trypsin in 30% v/v acetonitrile. [ABSTRACT FROM AUTHOR]

The aim of this work was to develop a biosensor for toxic amides using whole cells of Pseudomonas. aeruginosa containing amidase activity, which catalyses the hydrolysis of amides such as acrylamide producing ammonia and the corresponding organic acid. Whole cells immobilized in several types of membrane in the presence of glutaraldehyde and an ammonium ion-selective electrode, were used for biosensor development. This biosensor exhibited a linear response in the range of 0.1-4.0×10-3 M of acrylamide, a detection limit of 4.48×10-5 M acrylamide, a response time of 55 s, a sensitivity of 58.99 mV mM-1 of acrylamide and a maximum t1/2 of 54 days. The selectivity of this biosensor towards other amides was investigated, which revealed that it cross-reacted with acetamide and formamide, but no activity was detected with phenylacetamide, p-nitrophenylacetamide and acetanilide. It was successfully used for quantification of acrylamide in real industrial effluents and recovery experiments were carried out which revealed an average substrate recovery of 93.3%. The biosensor is cheap since whole cells of P. aeruginosa can be used as source of amidase activity. [ABSTRACT FROM AUTHOR]

Twelve fungi were screened for the potential of their extracellular enzymes to increase the hydrophilicity of polyamide (PA) materials. The most pronounced increase in hydrophilicity was found for enzymes from Beauveria brongniartii and B. bassiana. The 55 kDa polyamidase from B. brongniartii was purified using ultrafiltration, anion exchange chromatography and size exclusion chromatography. This polyamidase was able to hydrolyse adipic acid bishexylamide and various typical amidase substrates, but did not show protease activity. In contrast, the 27 kDa protease from B. brongniartii did not show activity on PA. The improvement of hydrophilicity due to hydrolysis with the 55 kDa polyamidase from B. brongniartii based on rising height was 11 cm for PA 6 Perlon fibres and 5 cm for PA 6.6 Nylon. The drop dissipation measurement corroborated the improvement of the hydrophilicity giving 7 s and less than 1 s for the two enzyme treated materials, respectively. The surface tension s of Perlon increased from 46.1 to 67.4 mNm after enzyme treatment. [ABSTRACT FROM AUTHOR]

The identification of arachidonylethanolamide (anandamide) as an endogenous cannabinoid is one of the most important developments in cannabinoid research. Anandamide and all known analogs exhibit significant selectivity for the first cannabinoid receptor subtype (CB1) and modest to very low affinity for the second cannabinoid receptor subtype (CB2). Elucidating how structurally different anandamide analogs bind to the CB1 receptor is an important goal. The experimental binding data of 26 anandamide analogs from Sheskin et al. [J. Med. Chem. 40, 659 (1997)] were chosen. These workers used the centrifugation method for their binding assay study and found that the addition of an amidase inhibitor, PMSF, does not lower the binding affinity Ki. A predictive model was developed based on data calculated by the Hartree-Fock method with the 6-31G** basis set. This model is based on linear combinations of calculated molecular descriptors that have been fit to the experimental binding activities. This model contains six molecular descriptors and has a standard deviation of 0.2436 for the 26 anandamide analogs. The predictive power of the model was tested for four other anandamide anlogs that were not included in the original basis set and found to be good. [ABSTRACT FROM AUTHOR]

A new flavonoid datisdirin (1), along with eight known compounds tectochrysine (2), cearoin (3), sideroxyline (4), ursolic acid (5), corosolic acid (6), arjunolic acid (7), erythrodiol (8) and oleanolic acid (9), were isolated from the ethyl acetate fraction of D. cannabina Linn. The structure of compound 1 was deduced on the basis of its spectral data. Datisdirin showed activity against the ureases enzyme. [ABSTRACT FROM AUTHOR]

Influences of temperature and moisture content on diffusional transport property and kinetics of both urease inactivation and color change during soybean treatment with superheated steam in the temperatures of 120–150°C were investigated. The experimental results have shown that the effective diffusivity and apparent rates of such reactions are related to temperature in a way that the rates of water transport, urease inactivation, and brown pigment formation are higher with higher temperature. In addition, these rates are also enhanced by increasing moisture content, except for the browning reaction that is inhibited by this factor due to the dilution effect. The reduction in urease activity is reasonably described by modified first-order reaction and the color change of soybeans is characterized by three Hunter parameters in which the changes of L-, a- and b-values are followed according to zero-order, modified Monod and first-order reactions, respectively. The kinetics of inactivation and color change, along with the experimental data of protein solubility and lysine content, have been suggested that the soybean should be treated with superheated steam at the temperature ranging from 120 to 135°C. The superheated-steam fluidized-bed technique can simultaneously be applied for both drying and inactivating such components in soybeans by a single operation when the initial moisture content falls within a suitable range, approximately lower than 20%d.b. At elevated initial moisture contents, two-stage drying technique is recommended in order to avoid excessive cooked soybeans. [ABSTRACT FROM AUTHOR]

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide by human tissue kallikrein (hK1) was studied in the absence and in the presence of increasing concentrations of the following chloride salts: sodium, potassium, calcium, magnesium and aluminium. The data indicate that the inhibition of hK1 by sodium, potassium, calcium and magnesium is linear competitive and that divalent cations are more potent inhibitors of hK1 than univalent cations. However the inhibition of hK1 by aluminium cation is linear mixed, with the cation being able to bind to both the free enzyme and the ES complex. This cation was the best hK1 inhibitor. Aluminium is not a physiological cation, but is a known neurotoxicant for animals and humans. The neurotoxic actions of aluminium may relate to neuro-degenerative diseases. [ABSTRACT FROM AUTHOR]

Arachidonoyl-serotonin inhibits in a mixed-type manner the metabolism of the endocannabinoid anandamide by the enzyme fatty acid amidohydrolase. In the present study, compounds related to arachidonoyl-serotonin have been synthesised and investigated for their ability to inhibit anandamide hydrolysis by this enzyme in rat brain homogenates. Removal of the 5-hydroxy from the serotonin head group of arachidonoyl-serotonin produced a compound ( N -arachidonoyltryptamine) that was a 2.3-fold weaker inhibitor of anandamide hydrolysis, but which also produced its inhibition by a mixed-type manner ( K i(slope) 1.3 μM; K i(intercept) 44 μM). Replacement of the amide linkage in this compound by an ester group further reduced the potency. In contrast, replacement of the arachidonoyl side chain by a linolenoyl side chain did not affect the observed potency. N -(Fur-3-ylmethyl)arachidonamide (UCM707), N -(fur-3-ylmethyl)linolenamide and N -(fur-3-ylmethyl)oleamide inhibited anandamide hydrolysis with pI 50 values of 4.53, 5.36 and 5.25, respectively. The linolenamide derivative was also found to be a mixed-type inhibitor. It is concluded that the 5-hydroxy group of arachidonoyl-serotonin contributes to, but is not essential for, inhibitory potency at fatty acid amidohydrolase. [ABSTRACT FROM AUTHOR]