Different Concentrations Of Enzyme Research Articles

Saliva with different concentrations of enzyme with esterolytic activity, and its effect on the immediate and long-term bond strength of adhesive systems to dentin

The present study evaluated whether different concentrations of albumin would influence the immediate or long-term bond strength of adhesive systems to dentin. Human third molars (N = 90) were sectioned at the dentin-enamel junction to obtain middle dentin occlusal surfaces. After sectioning into discs, the molars were restored with resin composite (Filtek Z250, 3 M/ESPE) by using an etch-and-rinse (Adper Scotchbond Multi-Purpose, 3 M/ESPE) or a self-etch (Clearfil SE Bond, Kuraray) adhesive system. The specimens were then sectioned into beams and stored in artificial saliva containing albumin in regular (100 μg/mL; SA100) or elevated (700 μg/mL; SA700) concentration—both concentrations simulated the range in which this enzyme may be present clinically, depending on the patient's periodontal condition. Beams from the control group (CO) were kept in artificial saliva with no addition of albumin. Half of the beams from each group were stored for 24 h, and the remainder, for 12 months. Then, the microtensile bond strength test was performed, and the failure mode was analyzed (adhesive, cohesive, or mixed). Bond strength data were submitted to the generalized linear model, followed by the Tukey tests, and failure modes were compared using chi-square tests (α = 5%). There were no significant interactions, but there was a significant effect between the albumin concentration (p = 0.015) and the time factor (p = 0.016). The beams stored in SA700 had significantly lower bond strength in comparison with the CO beams, regardless of the adhesive system. At 12 months, the bond strength decreased significantly for all groups. SA700 caused a significant increase in the adhesive failures after 12 months (p = 0.005). It was concluded that the bond strength of both adhesive systems was impaired by the albumin concentration aging.

Films Sensor Containing Extracted Natural Dyes for Monitoring Freeze-Thawing of Frozen Chicken

Films sensors include natural sensitive dyes and enzymes were used to create freezing-thawing sensitive films. Gliadin/glycerol, as well as anthocyanin, chlorophyll, and beta-carotene, were used to make the films. This type of biosensor is an efficient way to deal with the problem of freeze-thawing meat and provides a low-cost, colorimetric indicator for raising consumer awareness about freeze-thawed chicken. As a result of response enzymes, Glucose oxidase, and alpha-amylase have an impact on intensifying pigments films and reacting to freezing-thawing meat, the naked eye is capable of distinguishing between the colors of films, which changed from red, yellow, and green to faint colors. Sensing films’ color stability was tested, and the results demonstrated that the films were extremely stable and could be kept for longer than three weeks. Finally, real samples could be used to monitor misuse of utilizing storage temperature for chicken meat by using freeze-thawing sensitive colorimetric films. The best concentrations of GOX enzyme are 0.9%(activity270U/0.09g), 0.9%(117U/0.09g) of α-Amylase, and 5% of natural extracted pigments for preparing freezing-thawing sensitive films were of enzymes and, while the best concentration1:0.1 of Gliadin/glycerol composition films. The results of the colorimetric enzymatic system produced with different concentrations of enzymes and exposed to 8 and 35°C revealed that the same concentration of enzymes results in varying stability times when exposed to different temperature conditions, pH levels, and moisture.

Effect of Co-digestion Ratio and Enzyme Treatment on Biogas Production from Grass Silage and Chicken Litter

Biomethane production potential was evaluated by anaerobic co-digestion of grass silage (GS) and chicken litter (CL) in batch experiments. The aim of this study was to identify optimum enzyme treatment and co-digestion ratio for these substrates. The study is divided into trial A and trial B for enzyme treatment and co-digestion respectively. Mixed substrates were utilised in both trials which were maintained for a period of 30 days in 125 mL serum bottles at 37 °C. Preliminary batch assays in trial A for biomethane potential determination were performed using a co-digestion ratio of 2:1 (GS:CL) treated with five different concentrations of enzyme (w/v)—0.05, 0.1, 0.25, 0.5 and 1%. The highest specific methane yield (SMY) of 61 L CH4/kg was observed for 0.1% (w/v) enzyme treatment. Trial B assays were conducted for nine different GS:CL ratios—0:1, 1:0, 1:1, 1:2, 1:3, 2:3, 3:2, 3:1, and 2:1 respectively. Highest SMY were recorded for the ratios 1:0, 1:1 and 1:3 of GS:CL which correspond to 124 L CH4/kg, 137 L CH4/kg and 135 L CH4/kg respectively.

The Development of Leucine Dehydrogenase and Formate Dehydrogenase Bifunctional Enzyme Cascade Improves the Biosynthsis of L-tert-Leucine

Leucine dehydrogenase (LDH) and formate dehydrogenase (FDH) were assembled together based on a high-affinity interaction between two different cohesins in a miniscaffoldin and corresponding dockerins in LDH and FDH. The miniscaffoldin with two enzymes was further absorbed by regenerated amorphous cellulose (RAC) to form a bifunctional enzyme complex (miniscaffoldin with LDH and FDH adsorbed by RAC, RSLF) in vitro. The enzymatic characteristics of the bifunctional enzyme complex and free enzymes mixture were systematically compared. The synthesis of L-tert-leucine by the RSLF and free enzyme mixture were compared under different concentrations of enzymes, coenzyme, and substrates. The initial L-tert-leucine production rate by RSLF was enhanced by 2-fold compared with that of the free enzyme mixture. Ninety-one grams per liter of L-tert-leucine with an enantiomeric purity of 99% e.e. was obtained by RSLF multienzyme catalysis. The results indicated that the bifuntional enzyme complex based on cohesin-dockerin interaction has great potential in the synthesis of L-tert-leucine.

A comparative study of enzyme initiators in a hydrogel cell encapsulation system

Event Abstract Back to Event A comparative study of enzyme initiators in a hydrogel cell encapsulation system Justine J. Roberts1* and Penny Martens1* 1 UNSW Australia, Graduate School of Biomedical Engineering, Australia Introduction: Phenol-modified hydrogels for cell encapsulation are a growing area of research, although an appropriate initiator needs to be selected. A variety of chemical and enzymatic initiators have been used, with a range of results. Of note, radicals produced by chemical initiators have been shown to be cytotoxic[1]. Therefore, this work explores the use of enzymes to catalyse the crosslinking process. Many previous studies using enzyme crosslinkers use biological-based hydrogels. However many biological polymers can act as antioxidants, obscuring the effects of the initiating system on cells[1]. Therefore we chose poly(vinyl alcohol) (PVA) modified with tyramine (PVA-Tyr) groups to act as a “blank slate” material that does not interfere with the initiating system. The aim was to highlight both the advantages and challenges of four enzyme initiators. Specifically, the commonly used HRP/H2O2 was evaluated along with hematin/H2O2, laccase, and tyrosinase. These enzymes were evaluated for gel formation and cell response to encapsulation. Materials and Methods: PVA-TYR was synthesised as described previously[2]. The initiators were added to the PVA-TYR solution (5% w/w) under gentle mixing. Initiators used were HRP (0-0.5 U/mL) with H2O2 (0-12 mM), hematin (0-0.08% w/w) with H2O2 (0-12 mM), laccase (0-25 U/mL) and tyrosinase (0-2000 U/mL). The samples were gelled at 37ºC and 5% CO2 for 4 hours. The gelation kinetics were measured on a spectrophotometer for up to 6 hrs at 37°C. The wet and dry mass of the hydrogels was measured at 0 and 48 hrs to obtain the mass loss and swelling (q) of the hydrogels. L929 murine dermal fibroblasts were added to the macromer solution prior to crosslinking at 1x106 cells/ml. Cell viability within gels was determined over one week of culture via a live/dead assay and the MTS assay. Results: All of the enzymes evaluated can lead to the formation of highly crosslinked hydrogel networks. UV-Vis confirmed complete gelation for all enzymes within 4 hrs. A range of concentrations was tested for each enzyme to determine the concentration which produced the optimal gel, i.e., the lowest sol fraction. In all cases, the sol fraction was less than 30%, except for hematin which had 50% sol fraction, even at the highest concentrations. The optimal concentrations of enzymes were then used for the cell encapsulation studies. It is recognised that this method resulted in different concentrations of enzymes per system, but the goal was to keep the hydrogel network approximately the same, i.e., similar swelling and mesh size. The live/dead assay demonstrated that cells were alive in all systems initially; however there were stark contrasts in the viability and proliferation after 7 days, with tyrosinase and laccase outperforming the other two. Cell number was determined over 7 days by the MTS assay (See Figure 1), and further proved this finding. Conclusions: These results demonstrate that a thorough evaluation of enzyme initiators should be performed, as the initiator will impact the quality of the hydrogel formed and cellular proliferation, which is critical for biomedical applications. The Whitaker foundation for a grant to JJ Roberts.

Synthesis of a novel cyclic prodrug of S-allyl-glutathione able to attenuate LPS-induced ROS production through the inhibition of MAPK pathways in U937 cells.

A novel cyclic prodrug of S-allyl-glutathione (CP11), obtained by using an acyloxy-alkoxy linker, was estimated for its pharmacokinetic and biological properties. The stability of CP11 was evaluated at pH 1.2, 7.4, in simulated fluids with different concentrations of enzymes, and in human plasma. The anti-inflammatory ability of CP11 was assessed in U937 cells, an immortalized human monocyte cell line. Results showed that CP11 is stable at acidic pH showing a possible advantage for oral delivery due to the longer permanence in the stomach. Having a permeability coefficient of 2.49 × 10(-6) cm s(-1), it was classified as discrete BBB-permeable compound. Biological studies revealed that CP11 is able to modulate inflammation mediated by LPS in U937 cells preventing the increase of ROS intracellular levels through interaction with the MAPK pathway.

Enzymatic transformations of cellulose assessed by quantitative high‐throughput fourier transform infrared spectroscopy (QHT‐FTIR)

Enzymatic hydrolysis of bacterial microcrystalline cellulose was performed with the thermophile enzyme system of Thermobifida fusca Cel5A (a classical endocellulase), Cel6B (a classical exocellulase), Cel9A (a processive endoglucanase), and a synergistic mixture of endo- and exocellulases. Different concentrations of enzymes were used to vary the extent of hydrolysis. Following standardization, the concentration of cellulose was directly correlated to the absorbance of the cellulose signals. Crystallinity indexes (Lateral Order Index (LOI), Total Crystallinity Index, Hydrogen Bonding Index), allomorphic composition, conversion of specific atomic bonds (including the β-glucosidic bonds) were extracted from the spectral data obtained by QHT-FTIR. By quantifying the disruption of the H-bonding in complement to the sugar production, a more dynamic and complex picture of the role of cellulases in the hydrolysis of cellulose was demonstrated. The disruption of the H-bonding within the cellulose matrix appears as a quantifiable activity of the enzymes which was not correlated with the production of sugars in solution. The results also demonstrate that Cel9A activities from the cellulose transformation standpoint were partially similar to the activities of the synergistic mixture. In addition, Cel9A preferentially degraded the I(α) fraction of the crystalline cellulose while the Cel5A and Cel6B synergistic mixture preferentially degraded the I(β) fraction.

Pre-treatment and ethanol fermentation potential of olive pulp at different dry matter concentrations

Renewable energy sources have received increased interest from the international community with biomass being one of the oldest and the most promising ones. In the concept of exploitation of agro-industrial residues, the present study investigates the pre-treatment and ethanol fermentation potential of the olive pulp, which is the semi solid residue generated from the two-phase processing of the olives for olive oil production. Wet oxidation and enzymatic hydrolysis have been applied aiming at the enhancement of carbohydrates' bioavailability. Different concentrations of enzymes and enzymatic durations have been tested. Both wet oxidation and enzymic treatment were evaluated based on the ethanol obtained in a subsequent fermentation step by Saccharomyces cerevisiae and Thermoanaerobacter mathranii. It was found that a four-day hydrolysis time was adequate for a satisfactory release of glucose and xylose. The combination of wet oxidation and enzymatic hydrolysis resulted in the glucose and xylose concentration increase of 138 and 444%, respectively, compared to 33 and 15% with only enzymes added. However, the highest ethanol production was obtained when only enzymic pre-treatment was applied, implying that wet oxidation is not a recommended pre-treatment process for olive pulp at the conditions tested. It was also showed that increased dry matter concentration did not have a negative effect on the release of sugars, indicating that the cellulose and xylan content of the olive pulp is relatively easily available. The results of the experiments in batch processes clearly emphasize that the simultaneous saccharification and fermentation (SSF) mode is advantageous in comparison with the separate hydrolysis and fermentation (SHF) mode concerning process contamination.

Influence of Type of Substrate and Enzyme Concentration on Formation of Galacto-oligosaccharides

Different substrates and different concentrations of enzyme (Maxilact LX 5000) for galacto-oligosaccharides synthesis were tested. Lactose in phosphate buffer (138 mmol/l), ultrafiltration permeate (115 mmol/l), recombined whey (136 mmol/l) were used as substrates. Concentrations of used enzyme were from 0.15 to 15 U/ml for lactose in buffer, from 0.12 U/ml to 1.5 U/ml for ultrafiltration permeate and 1.5 U/ml for recombined whey. Reaction products were analysed by HPLC. There was obtained 6.4 ± 0.4 mmol/l of galacto-oligosaccharides (GOS) for lactose in buffer, it means that 0.0633 ± 0.0025 g/g of lactose was converted to GOS. The conversions of lactose to GOS for recombined whey and ultrafiltration permeate were 0.0669 ± 0.0079 and 0.0920 ± 0.0010 g/g. There was obtained 7.3 ± 0.1 mmol/l of GOS for ultrafiltration permeate and for recombined whey 5.9 ± 0.1 mmol/l of GOS.

Further studies of the effect of magnetic fields on the oscillating peroxidase–oxidase reaction

We have performed an experimental study of the effect of DC magnetic fields on the oscillating peroxidase–oxidase reaction to obtain additional information about the mechanism of the action of the magnetic field. The changes in oscillation amplitude and frequency were measured at low (5.1) and high (6.3) pH, respectively. Furthermore, the effect of a magnetic field with a field strength of 1000 G on the oscillation amplitude was measured at different concentrations of enzyme and the modifiers methylene blue and 4-hydroxybenzoic acid. Changes in pH and the concentration of methylene blue seemed to have little or no effect on the reaction's magnetosensitivity. However, increases in the enzyme concentration or the concentration of 4-hydroxybenzoic acid were accompanied by increases in magnetosensitivity. These results lend support to a previously proposed mechanism in which the magnetosensitive steps involve the transfer of electrons to two enzyme intermediates, compounds I and II.

Characteristics of enzymatically produced agar-starch sponges

Sponges with a cellular structure were created by subjecting agar-starch gels to α-amylase activity prior to freeze-dehydration. Various starch concentrations (0.5–1.5%), enzyme concentrations (1000–1500 ppm) and times of exposure to the enzyme were selected to change the structure and mechanical properties of the hydrocolloid cellular solid. The sponges were compressed to 80% deformation between parallel lubricated plates, yielding the sigmoid-shape curve typical of compressed cellular solids. The influence of starch concentration and exposure time to different concentrations of enzyme on the mechanical properties of the dry sponges was studied. Once enough substrate (starch) was embedded within the sponge matrix, structure and mechanical properties were solely dependent on enzymatic activity. α-Amylase activity was confirmed by ion chromatography.

A simple model for predicting the response of chicks to dietary enzyme supplementation.

Two experiments with Leghorn chicks and data from five publications were analyzed to determine whether a simple general equation could be used to predict the relationship between the amount of a crude enzyme added to a diet and chick performance. The maximum improvements in weight gain and feed: gain ratio in Leghorn chicks fed rye diets containing different concentrations of enzymes were as high as 61 and 42%, respectively. Regression analyses demonstrated that there was usually a high linear correlation (r2 > 0.9, P < 0.05) between the concentration of the enzyme when transformed into its logarithmic value and weight gain or the feed:gain ratio. The general prediction equation was Y = A + B(logX), where Y is the performance value (i.e., weight gain, grams), A is the intercept (y-axis), B is the slope of the line (change in performance per units of enzyme in the diet), and X is the amount of enzyme in the diet. The slope of the line provides an index of the overall efficacy of the enzyme treatment. The log-linear model shows that for every ninefold increase in amount of enzyme in the diet (i.e., when the amount was increased to 10 times the starting amount), there was only a doubling of improvement in chick performance. High correlations (r2 values) were also obtained when data from the literature were analyzed. The equation was applicable to different classes and ages of poultry fed diets containing rye, wheat, barley, and lupins. These studies demonstrate that there is a linear relationship between the amount of enzyme added to the diet, when expressed as a logarithmic value, and the corresponding performance of chickens.

Activation of purified cytoplasmic 17β-hydroxysteroid dehydrogenase from human placenta by human albumin and globulin

The cytoplasmic 17β-hydroxysteroid dehydrogenase of human placenta, purified more than 2500-fold, was activated by small amounts of human albumin and globulin. This activation was dependent on substrate concentration. At 20 μM estradiol (10 × K M ) and two different concentrations of enzyme (0.01 and 2 μg/ml), the activation was greatest at albumin or globulin concentrations between 0 and 30 μg/ml. At “low” concentrations of estradiol (20 nM = 10 −2 × K M ) and enzyme (0.01 μg/ml), maximal activity occurred at approximately 10 μg/ml. Higher concentrations of albumin and globulin led to a decline in activity.

Model discrimination and nonlinear parameter estimation in the analysis of the mechanism of action of β-hydroxybutyrate dehydrogenase from Rhodopseudomon as spheroides

1. 1. β-Hydroxybutyrate dehydrogenase ( d-3-hydroxybutyrate:NAD + oxide-reductase, EC 1.1.1.30) from Rhodopseudomonas spheroides was prepared and initial velocity studies in the presence and absence of product at zero time were performed. 2. 2. The product inhibition pattern was found to be consistent with an ordered sequential mechanism. 3. 3. Nonlinear regression analyses were applied to selected combinations of the velocity data to provide estimation of the parameters, their standard deviations, and confidence limits. 4. 4. Analysis of variance provided a basis for determination of the acceptability of the model and for discrimination of points that showed extreme lack of fit. 5. 5. A data-blocking procedure has been introduced that permits the estimation of parameters using combinations of data sets that were obtained with different concentrations of enzyme. 6. 6. The lack of one-to-one correspondence between the set of kinetic constants and the set of rate constants imposed difficulties in the initial attempt to apply the nonlinear regression analysis to the full rate equation in rate constant form. 7. 7. A reformulation of the kinetic constants based on the requirement for a non-zero Jacobian for the transformation has been developed that provides a one-to-one correspondence with the set of rate constants. 8. 8. A method for the evaluation of the approximate 95% confidence regions of the parameters and for the assessment of the experimental design by means of a determination of the eigenvalues of the solution matrix ( X′ X) is introduced.

Examination of the Conditions for Protoplast Isolation from Tobacco Cells Cultured in vitro

Protoplasts were isolated from cultured tobacco cells by removing the cell wall enzymatically. We examined the time courses of treatment with some different concentrations of enzymes (cellulase and macerozyme) and with some different kinds and concentrations of sugars (sucrose, sorbitol and mannitol) which produce good conditions for protoplast isolation. The best conditions for protoplast preparation from tobacco cells cultured in vitro were: use of actively growing cells and isolation of protoplasts with 5% cellulase and 0.1% macerozyme in 0.5% sorbitol.

Uricase, amino acid oxidase, and xanthine oxidase

The object of this paper is the comparative study of three oxidizing enzymes—uricase, amino acid oxidase, and xanthine oxidase. We shall describe first the main properties of uricase and amino acid oxidase, laying special stress on characters which have not been sufficiently investigated by previous workers. This will include the study of the effect of various factors on the activity of these enzymes, the reaction between these enzymes and their substrates, the activation of the substrate molecules, their reaction with the molecular oxygen, and the reduction of the latter to hydrogen peroxide. We shall then examine briefly the main characters of xanthine oxidase, and this will enable us to compare the properties of these three enzymes and to determine some of the characters they have in common. II−Uricase or Urico-oxidase 1− Previous Work That uricase or urico-oxidase catalyses the oxidation of uric acid to allantoin has been known since the work of Schittenhelm (1905), Wiechowski and Wiener (1909), and others, but its main properties have been established only by Battelli and Stern (1909, 1912) in their important investigation on this subject. According to these authors, for the oxidation of a molecule of uric acid to allantoin one atom of oxygen and one molecule of water are taken up while one molecule of CO 2 is given off. The reaction consists, therefore, in oxidation, hydration, and decarboxylation, and the R. Q. of the reaction is usually equal to 2. It varies slightly, however, according to the age of the enzyme preparation. The relationship which these authors have established between the amount of uric acid disappearing, the oxygen taken up, and the CO 2 given off, has made possible the study of the reaction by the estimation of either oxygen or CO 2 . The velocity of oxidation of uric acid was found to depend on the oxygen tension, being, for instance, at least twice as great in pure oxygen as in air. The main results obtained by Battelli and Stern have been recently confirmed by other workers, who have, however, paid special attention to the study of the kinetics of this reaction (Felix, Scheel, and Schuler, 1929; Schuler, 1932; Rô, 1931; Grynberg, 1931). The velocity of the reaction was measured by these authors in terms of the amount of uric acid oxidized, of oxygen absorbed, and of CO 2 liberated; and these reactions were studied at various hydrogen ion concentrations, at different tensions of oxygen, and at different concentrations of enzyme and substrate. The oxidation of uric acid catalysed by the enzyme was also compared with that obtained by permanganate and by hydrogen peroxide. One of the important conclusions which resulted from this work was that the enzyme does not catalyse directly the oxidation of uric acid to allantoin, but that the reaction takes place in two steps: (1) the catalytic oxidation and hydration of uric acid by uricase to an oxyacetylene-diurein carboxylic acid, and (2) the decarboxylation of this unstable compound to allantoin, which is independent of the enzyme (Biltz and Schauder, 1923; Felix and his co-workers, 1929; Grynberg, 1931; and Schuler, 1932).