Enzymatic Parameters Research Articles

A Novel Combined Microstrip Resonator/Nanospray Ionization Source for Microwave-Assisted Trypsin Digestion of Proteins.

Enzymatic digestion of proteins is a critical step in bottom-up and middle-down proteomics. Here, we demonstrate a method for decreasing the time required for proteolytic digestion of proteins from multiple hours to minutes by using an in-line microstrip cavity for programmed microwave heating. When a nanospray emitter tip, containing a digestion sample, is exposed to a region of highly focused microwave field, the rate of proteolytic digestion is enhanced and the time required for digestion greatly decreased. The design is advantageous for mass spectrometry because the solution-based digestion can then be directly sprayed from a nanoelectrospray tip emitter, decreasing sample transfer loss and allowing the system to be used in a flow-through proteolytic workflow. Microwave-assisted digestion using this method is evaluated against standard overnight digestion protocols using a variety of proteins, evaluating sequence coverage and observed peptide location, digestion rate, and overall efficacy. The influence of applied microwave power is investigated, and enzymatic kinetic parameters are evaluated to estimate temperature within the microreactor. Finally, the modulation of the proteolytic digestion of proteins based upon the modulation of applied microwave power is demonstrated on a time scale of seconds in a flow-through system.

Cd- and Cu-induced phytotoxicity on 2–3 leaf stage of Cyamopsis tetragonoloba and its regulation by nitrate reductase and ROS quenching enzyme

The present study focused on Cd- and Cu-induced nitrate reductase activity and its relation to ROS scavenging enzyme in C. tetragonoloba. Hydroponically adapted seedling treated with different metal concentration (Cd and Cu) in the series from 10 to 100 µM. Seedlings were harvested after 96 h of metal treatment and analyze cellular homeostasis and metal tolerance mechanism by determining growth, stress and enzymatic parameters. The nitrate reductase activity was observed to be highest at 70 µM Cd and 50 µM Cu in cyamopsis roots, i.e., 2.31 and 2.59 times higher than control. Exogenous supplementation of SNP (100 µM) enhanced the NR activity in roots of C. tetragonoloba. Polymorphism between different metal treated leaves and roots were also observed. Higher polymorphism was observed in metal-treated roots in comparison to metal-treated leaves. The activity of other antioxidants except catalase (ascorbate peroxidase, guaiacol peroxidase and nitrate reductase was also recorded to be highest at 70 µM Cd and 50 µM Cu. Increased activity of Nitrate reductase and other antioxidants were directly associated with reduction in MDA and H2O2 level at 70 µM Cd and 50 µM Cu. The present investigation is highlighted the defensive role of Nitrate reductase in mitigating Cd and Cu induced toxicity by regulation of antioxidants and ROS metabolism. The study is important as this is the first report exemplifying the protective role of Nitrate reductase in detoxifying ROS in C. tetragonoloba under Cd and Cu stress.

The possible role of extra magnesium and nitrogen supply to alleviate stress caused by high irradiation and temperature in lemon trees

Citrus trees are subjected to variation of environmental conditions throughout the annual cycle, especially during the flowering and fruit set. Heat waves have affected the environmental conditions required for optimum crop growth and have reduced fruit yield and quality worldwide. This study aimed to evaluate an extra supply of magnesium (Mg) and/or nitrogen (N) to increase citrus tolerance to excessive increased irradiation and consequent air temperature. Young lemon trees were grown in a greenhouse with extra Mg, N and Mg + N supply for five months and then exposed to heat for 12 days. After imposing stress condition, photosynthetic and enzymatic antioxidant system parameters were assessed to evaluate the impact of the extra nutrient supply in alleviating stress induced by combination of elevated irradiance and air temperature. Extra nutrient supply increased plant tolerance to the environmental stress, allowing maintenance of high photosynthetic and transpiration rates and low ratio of apparent electron transport rate per photosynthetic carbon assimilated. Enhanced Mg supply also increased activity of the antioxidant enzyme system and decreased the oxidative stress of plants. Trees treated with extra N exhibited increased chlorophyll concentrations and ability to harvest light energy. Citrus trees under stressed condition, such as elevated air temperature and radiation, associated to heat waves, exhibited less damage in the photosynthetic mechanism and high activity of the antioxidant enzymatic system when supplied with extra nutrient. It was concluded that extra Mg and N supply are important tools to prevent damages and enhance lemon trees biochemical and physiological responses to alleviate the harmful effects of unfavorable environmental condition.

A comparative study of enzymatic and immunological parameters in Planorbarius corneus and Biomphalaria glabrata exposed to the organophosphate chlorpyrifos

This study aimed to investigate the acute effects of chlorpyrifos on biomarkers related to neurotoxicity and immunotoxicity in two allopatric freshwater gastropod species belonging to the family Planorbidae. For this purpose, Planorbarius corneus and Biomphalaria glabrata were exposed to chlorpyrifos (active ingredient or commercial formulation) for 48 h at environmentally realistic concentrations (1 and 7.5 μg L−1). Basal acetylcholinesterase activity in soft tissues and hemolymph was almost one order of magnitude higher in P. corneus than in B. glabrata. However, upon chlorpyrifos exposure, statistically significant inhibition of enzymatic activity was registered in both species. Acetylcholinesterase was more sensitive to inhibition in soft tissues than in hemolymph. The highest inhibition was observed in the B. glabrata soft tissues exposed to the commercial formulation (88 % at 1 μg L-1 and 93 % at 7.5 μg L−1). Hemocyte number and lysosomal membrane stability did not show significant changes with respect to controls in any of the exposed groups. Superoxide anion generation was diminished (21–46 %) in P. corneus hemocytes exposed to the active ingredient and in B. glabrata hemocytes exposed to the active ingredient or the formulation. In contrast, hemocyte phagocytic activity increased in all exposed groups. Phagocytosis was most stimulated (89 %) in hemocytes sampled from B. glabrata treated with 7.5 μg L−1 chlorpyrifos. Altogether the results suggest that the freshwater gastropods P. corneus and B. glabrata are suitable model animals for environmental monitoring studies in the Northern Hemisphere and Latin America, respectively. Furthermore, these results add information on the relevance of testing pesticide formulations and on the usefulness of acetylcholinesterase inhibition and immunological parameters as biomarkers of the acute effects of chlorpyrifos in these species.

IDENTIFICATION OF POTENTIAL ANTIOXIDANT AND HEPATOPROTECTIVE CONSTITUENTS OF VITEX DONIANA BY UHPLC/+ESI-QQTOF-MS/MS ANALYSIS

Objective: This study evaluated the antioxidant and hepatoprotective activities of the dichloromethane (DCM-F) and n-butanol (BUT) fractions of Vitex doniana and identified the bioactive constituents using liquid chromatography-mass spectrometry (LC-MS) analysis.
 Methods: The methanol extract of the leaves was subjected to liquid-liquid fractionation and the BUT soluble separated by vacuum LC (VLC) using binary mixtures of DCM in methanol (1 L) sequentially in the ratios of 9:1 BF1–1:9 BF5. The antioxidant activity was investigated in vitro using 1, 1-diphenyl-2-picrylhydrazyl (DPPH) scavenging, phosphomolybdenum total antioxidant capacity, and ferric ion reducing antioxidant power assay methods. The in vivo antioxidant potential was performed by measuring the concentrations of both enzymatic and non-enzymatic parameters in carbon tetrachloride (CCl4)-induced hepatic injury.
 Results: The extract, DCM-F, and the VLC sub-fractions showed good antioxidant activity which is comparable with ascorbic acid (53.6%). DCM-F showed the highest antioxidant activity (71.6%), while the least activity was shown by BF5 (15.0%). Peak dereplication of LC-MS chromatograms identified several putative compounds in the BUT soluble. DCM-F caused a significant increase (p<0.05) in superoxide dismutase, catalase, glutathione peroxidase, and Vitamins C and E in CCl4-induced rats compared to standard. The levels of serum alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase, and malondialdehyde that were elevated in CCl4-induced rats were also significantly reduced (p<0.05) by the DCM-F compared to standard.
 Conclusion: The ability of V. doniana leaves to mop up free radicals and reverse the CCl4-induced hepatic injury in rats suggests antioxidant potentials. The bioactive compounds identified in BUT fraction could support its ethnomedicinal uses in the treatment of illnesses associated with radical scavenging activity.

The enzymology of the human prostanoid pathway.

Prostanoids are short-lived autocrine and paracrine signaling molecules involved in a wide range of biological functions. They have been shown to be intimately involved in many different disease states when their regulation becomes dysfunctional. In order to fully understand the progression of any disease state or the biological functions of the well state, a complete evaluation of the genomics, proteomics, and metabolomics of the system is necessary. This review is focused on the enzymology for the enzymes involved in the synthesis of the prostanoids (prostaglandins, prostacyclins and thromboxanes). In particular, the isolation and purification of the enzymes, their enzymatic parameters and catalytic mechanisms are presented.

Determining hematological, biochemical and immunological reference values in healthy adults with high-risk for HIV acquisition in Mozambique.

IntroductionIn many African countries, laboratory reference values are not established for the local healthy adult population. In Mozambique, reference values are known for young adults (18-24yo) but not yet established for a wider age range. Our study aimed to establish hematological, biochemical and immunological reference values for vaccine trials in Mozambican healthy adults with high-risk for HIV acquisition.MethodsA longitudinal cohort and site development study in Mozambique between November 2013 and 2014 enrolled 505 participants between 18 to 35 years old. Samples from these healthy participants, were analyzed to determine reference values. All volunteers included in the analysis were clinically healthy and human immunodeficiency virus (HIV), hepatitis B and C virus, and syphilis negative. Median and reference ranges were calculated for the hematological, biochemical and immunological parameters. Ranges were compared with other African countries, the USA and the US National Institute of Health (NIH) Division of AIDS (DAIDS) toxicity tables.ResultsA total of 505 participant samples were analyzed. Of these, 419 participants were HIV, hepatitis B and C virus and syphilis negative including 203 (48.5%) females and 216 (51.5%) males, with a mean age of 21 years. In the hematological parameters, we found significant differences between sex for erythrocytes, hemoglobin, hematocrit, MCV, MCH and MCHC as well as white blood cells, neutrophils and platelets: males had higher values than females. There were also significant differences in CD4+T cell values, 803 cells/μL in men versus 926 cells/μL in women. In biochemical parameters, men presented higher values than women for the metabolic, enzymatic and renal parameters: total and direct bilirubin, ALT and creatinine.ConclusionThis study has established reference values for healthy adults with high-risk for HIV acquisition in Mozambique. These data are helpful in the context of future clinical research and patient care and treatment for the general adult population in the Mozambique and underline the importance of region-specific clinical reference ranges.

Evaluating Pfeiffer Chromatography for Its Validation as an Indicator of Soil Quality

Soil monitoring is fundamental to promote sustainability agroecosystems. It is necessary to consider indicators that bring together biological, physical, chemical and inter-relational attributes. Pfeiffer chromatography (PC) represents an important method for soil diagnosis, so the present study aims to analyze it in order to contribute to its validation. The soil samples of 12 production systems were evaluated in quintuplicate. The chromas were elaborated using Whatman no 4 filter paper, and the soil extract was performed by sodium hydroxide solution 1%. The results were obtained using revealing solution (AgNO3 0.5%) ascension by capillarity, and the chrome zones: central, internal, medium and external were correlated to soil penetration, chemical, biological and enzymatic parameters, respectively. Variance analysis was applied to the results that presented normal distribution and the means were compared by Scott-Knott test. T-test for Spearman correlations and principal component analysis were used to evaluate the correlations. There was negative correlation between the central zone and the average penetration resistance within the range 0-40 cm in depth. The internal, medium and external zones presented positive correlation with organic matter, carbon microbial biomass, and enzymatic activities, respectively. Quality standard features such as coloration, size and proportion of zones, presence of enzymatic clouds, peaks and radial lines were also confirmed. Thus, the standardization developed by this study contributed to validation of PC. Since PC is a low-cost and easy-to-perform method, it proves to be a useful tool allowing farmers autonomy to monitoring different agricultural systems, contributing to their production sustainable.

Temperature and food availability alters the physiology and aerobic capacity of tambaqui (Colossoma macropomum)

Fish of the Amazon experience both daily and seasonal variation in temperature and food availability. In the present work, we investigated the influence of nutrient status changes resulting from feeding Colossoma macropomum five flooded forest fruits on aerobic and swimming performance. To assess the effects of diet, three groups of fish were provided different types of food. One group of was provided with commercial feed, another was given fruit feed, and the last group was not fed. The effects of temperature were tested by keeping half of each group of animals at either 26 °C or 33°C for 30 days. After this period, the animals were transferred to either a respiratory chamber or swimming tunnel. Then, tissues were collected for relevant analyses. Results showed that C. macropomum depresses aerobic metabolism when food is withheld (27% reduction in ṀO2), and observed effects were intensified at the highest temperature (40% reduction in ṀO2 at 33 °C). Further, increased temperature negatively influenced hematological (Ht, Hb, VCM), biochemical (glucose, triglycerides, cholesterol), hepatossomatic index and enzymatic (MDH and CS in white muscle) parameters. The animals feeding commercial and fruits enriched diets, have a physiological and biological advantage when compared to the unfed animals at both temperatures. Metabolic suppression is an important strategy for maintaining swimming capacity in C. macropomum. It is suggested that even after a long period of fasting, carbohydrates are important energetic substrates.

An Allosteric Site Residue Critical for Transmission of the Allosteric Signal of the Vibrio cholerae SpeG Enzyme

SpeG is a polyamine N‐acetyltransferase enzyme that belongs to the superfamily of Gcn5‐related N‐acetyltransferases (GNATs). We previously showed this enzyme from Vibrio cholerae (VcSpeG) acetylates spermine and spermidine. SpeG is thought to contribute to biofilm formation in several pathogenic bacteria and several studies have shown that it also plays a role in bacterial polyamine homeostasis under stressful conditions. This GNAT is unique because it forms a dodecameric structure and has an allosteric site in each monomer. Additionally, it binds polyamines at both allosteric and active sites. Previous X‐ray crystallographic studies have shown that the E41 residue in the allosteric site of VcSpeG forms a hydrogen bond with N10 of spermine and N6 of spermidine. We hypothesized that mutating this residue would affect the allosteric network of the enzyme. Therefore, we created a series of E41 point mutations and investigated the enzymatic activity and kinetic parameters toward spermine and spermidine. Changing the identity of the residue at this location either increased the catalytic efficiency and altered the sigmoidicity of the substrate saturation curve or decreased the catalytic efficiency and affected cooperativity. This implies that the identity of the residue at this position affects the allosteric signal that is transmitted. Further investigation into other residues in this site is warranted.Support or Funding InformationResearch reported in this work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM133506 (to MLK).

In vitro hepatic aflatoxicol production is related to a higher resistance to aflatoxin B1 in poultry

A study was conducted to determine the cytosolic in vitro hepatic enzymatic kinetic parameters Vmax, KM, and intrinsic clearance (CLint) for aflatoxin B1 (AFB1) reductase [aflatoxicol (AFL) production] and AFL dehydrogenase (AFB1 production) in four commercial poultry species (chicken, quail, turkey and duck). Large differences were found in AFB1 reductase activity, being the chicken the most efficient producer of AFL (highest CLint value). Oxidation of AFL to AFB1 showed only slight differences among the different poultry species. On average all species produced AFB1 from AFL at a similar rate, except for the turkey which produced AFB1 from AFL at a significantly lower rate than chickens and quail, but not ducks. Although the turkey and duck showed differences in AFL oxidation Vmax and KM parameters, their CLint values did not differ significantly. The ratio AFB1 reductase/AFL dehydrogenase enzyme activity was inversely related to the known in vivo sensitivity to AFB1 being highest for the chicken, lowest for the duck and intermediate for turkeys and quail. Since there is no evidence that AFL is a toxic metabolite of AFB1, these results suggest that AFL production is a detoxication reaction in poultry. Conversion of AFB1 to AFL prevents the formation of the AFB1-8,9-exo-epoxide which, upon conversion to AFB1-dihydrodiol, is considered to be the metabolite responsible for the acute toxic effects of AFB1.

Composting versus mechanical–biological treatment: Does it really make a difference in the final product parameters and maturity

One of crucial waste management problems is the management of organic waste. This activity employs the composting. In case of green waste, its application seems reasonable, whereas the use of selected mixed waste raises problems related to the compost quality. Across countries, the non-sterile organic fraction of municipal solid waste is being separated through the mechanical-biological treatment. The technology is a solution of waste treatment and meets objectives set out in the Landfill Directive. There are many problems associated with the use of output products. The use of compost as a fertilizer requires determination of its impact on the environment. Compost quality can be assessed using analytical methods and phytotoxicity tests. Therefore, the aim of this study was to describe changes in physico-chemical, enzymatic, phytotoxicity and vegetation parameters occurring in composts from two systems - a prismatic installation for green waste, and a mechanical-biological treatment installation. The compost from green waste exhibited greater stability. Values of dehydrogenase activity were lower if compared with the mechanically and biologically treated compost, which indicates lower compost maturity. The biomass production of Brassica napus L. and Fetuca rubra L. was higher in the variant with the application of green compost. The influence on Hordeum vulgare L., Cannabis sativa L., and Sinapis alba L. depended on the plant type and the compost used. Nevertheless, the compost from green waste was less toxic. The evidence from this study suggests that the mechanical-biological treatment had problems associated with the maturation and quality of the final product.

Altered Structural Expression and Enzymatic Activity Parameters in Quiescent Ulcerative Colitis: Are These Potential Normalization Criteria?

Mucosal healing determined by endoscopy is currently the remission standard for ulcerative colitis (UC). However, new criteria for remission are emerging, such as histologic normalization, which appears to correlate better to the risk of relapse. Here, we study mucosal healing on a molecular and functional level in quiescent UC. We obtained endoscopic biopsies from 33 quiescent UC patients and from 17 controls. Histology was assessed using Geboes score. Protein and mRNA levels were evaluated for the tight junction proteins claudin-2, claudin-4, occludin, and tricellulin, as well as Cl−/HCO3− exchanger DRA, and cyclo-oxygenase enzymes (COX-1, COX-2). The mucosal activity of COX-1 and COX-2 enzymes was assessed in modified Ussing chambers, measuring electrogenic ion transport (short-circuit current, SCC). Chronic inflammation was present in most UC patients. The protein level of claudin-4 was reduced, while mRNA-levels of claudin-2 and claudin-4 were upregulated in UC patients. Surprisingly, the mRNA level of COX-1 was downregulated, but was unaltered for COX-2. Basal ion transport was not affected, while COX-2 inhibition induced a two-fold larger decrease in SCC in UC patients. Despite being in clinical and endoscopic remission, quiescent UC patients demonstrated abnormal mucosal barrier properties at the molecular and functional level. Further exploration of mucosal molecular signature for revision of current remission standards should be considered.

A simple and affordable kinetic assay of nucleic acids with SYBR Gold gel staining.

Labeling substrates or products are paramount in determining enzymatic kinetic parameters. Several options are available; many laboratories use either radioactive or fluorescent labeling because of their high sensitivity. However, those methods have their own drawbacks such as half-life decay, expensive and hazardous. Here, we propose a novel, simple, economical and fast alternative to substrate labeling for studying the kinetics of nucleic acids: post-migration gel staining with SYBR Gold. Cleavage rates similar to the ones reported in the literature for the I-R3 DNA-cleaving DNA enzyme in the presence of zinc chloride are an indication of the quality of the new method. Moreover, the activity of the hammerhead ribozyme was also monitored by our method to illustrate its versatility. This labeling-free method has several advantages such as its ease of use as well as cost effective and versatility with both non-structured and structured RNAs or DNAs.

CtpB is a plasma membrane copper (I) transporting P-type ATPase of Mycobacterium tuberculosis

BackgroundThe intracellular concentration of heavy-metal cations, such as copper, nickel, and zinc is pivotal for the mycobacterial response to the hostile environment inside macrophages. To date, copper transport mediated by P-type ATPases across the mycobacterial plasma membrane has not been sufficiently explored.ResultsIn this work, the ATPase activity of the putative Mycobacterium tuberculosis P1B-type ATPase CtpB was associated with copper (I) transport from mycobacterial cells. Although CtpB heterologously expressed in M. smegmatis induced tolerance to toxic concentrations of Cu2+ and a metal preference for Cu+, the disruption of ctpB in M. tuberculosis cells did not promote impaired cell growth or heavy-metal accumulation in whole mutant cells in cultures under high doses of copper. In addition, the Cu+ ATPase activity of CtpB embedded in the plasma membrane showed features of high affinity/slow turnover ATPases, with enzymatic parameters KM 0.19 ± 0.04 µM and Vmax 2.29 ± 0.10 nmol/mg min. In contrast, the ctpB gene transcription was activated in cells under culture conditions that mimicked the hostile intraphagosomal environment, such as hypoxia, nitrosative and oxidative stress, but not under high doses of copper.ConclusionsThe overall results suggest that M. tuberculosis CtpB is associated with Cu+ transport from mycobacterial cells possibly playing a role different from copper detoxification.

Oenological tannins to prevent Botrytis cinerea damage in grapes and musts: Kinetics and electrophoresis characterization of laccase

Enzymatic parameters (KM and Vmax), residual activity, effect of bentonite and electrophoresis characterization of laccase in the presence of different oenological tannins (OT) were investigated in relation to B. cinerea negative effects in grapes and musts. Five OT were tested (gallotannin, ellagitannin, quebracho, grape-skin and grape-seed) in comparison with ascorbic acid (AA), sulfur dioxide (SO2) and bentonite. We added OT, AA, SO2 and bentonite to botrytized must obtained by inoculation of grapes with B. cinerea strain 213. Laccase activity was measured by the syringaldazine method at different concentrations of substrate. Enzymatic parameters were determined using Michaelis-Menten and Lineweaver-Burk plots. The B. cinerea strain was also grown in a liquid medium for laccase production. Molecular weight of laccases and effect of OT upon these laccases were studied by SDS-PAGE. Results confirm that bentonite, contrary to OT, did not permit to reduce laccase activity. Regardless the tannin considered, Vmax, KM and laccase activity were reduced and gallotannin, grape-skin and grape-seed tannin presented the greatest ability. Efficiency of grape-seed tannin addition in order to reduce the laccase activity, was comparable to that of AA or SO2 at the typical doses employed in oenology for each one. Oenological tannins appear to be excellent processing aids to prevent laccase effects and contribute to reduce the use of SO2 in grapes and musts.

Effect of astaxanthin nanoparticles in protecting the post-thawing quality of rooster sperm challenged by cadmium administration

The protective role of astaxanthin nanoparticles (Ast NPs, 25 mg/kg p.o) against cadmium (Cd, 1 mg/100 g b.w. SC), a known inductor of lipid peroxidation and changes in the antioxidant defense system in the Ross 308 breeder roosters sperm, was examined. Sperm motility (computer-assisted sperm motility analysis), membrane integrity (hypoosmotic swelling test), viability, total abnormality, and enzymatic parameters were assessed after thawing. The testis/body weight (mg/kg) ratio and HE staining results of testis were also performed. The obtained results showed that Cd induced detrimental effects on testis and sperm, while Cd treated by Ast NPs (Cd Ast) diminished this change compared to the Cd group. Cd-treated group resulted in significantly (P < 0.05) lowest total (37.29 ± 2.46) and progressive (5.84 ± 0.47) motility and decreased antioxidant enzyme activity (CAT, TAC, and GPx), as well as producing a significant (P < 0.05) decrease in testis weight (mg) compared to the control group. Treatment with Ast NPs (Ast NPs + Cd) had reversed Cd-induced changes in the antioxidant defense system and significantly prevented Cd-induced testis damage. In conclusion, the results of our work suggest that Ast NPs at 25 mg/kg act as a potent antioxidant in protecting rooster testes against oxidative stress induced by Cd.

ATP sulfurylase activity of sulfate-reducing bacteria from various ecotopes.

Sulfate-reducing bacteria (SRB) are widespread in various ecotopes despite their growth and enzymatic features not compared. In this study, the enzymatic parameters of ATP sulfurylase in cell-free extracts of sulfate-reducing bacteria isolated from various ecotopes such as soils, corrosion products and human large intestine were determined. Comparative analysis of both enzyme characteristics and growth parameters were carried out and similar research has not been reported yet. The initial and maximum rates of enzymatic reaction catalyzed by ATP sulfurylase were significantly different (p < 0.05) in the bacterial strains isolated from various environmental ecotopes. The specific activity of this enzyme in sulfate-reducing bacteria was determined for corrosive and intestinal strains 0.98-1.56 and 0.98-2.26 U × mg-1 protein, respectively. The Michaelis constants were 1.55-2.29mM for corrosive and 2.93-3.13mM for intestinal strains and the affinity range were demonstrated. Based on cluster analysis, the parameters of physiological and biochemical characteristics of sulfate-reducing bacteria from different ecotopes are divided into 3 clusters corresponding to the location of their isolation (soils, heating systems and human intestine). Understanding the enzymatic parameters of the initial stages of sulfate consumption in the process of dissimilatory sulfate reduction will allow the development of effective methods for controlling the production of toxic metabolites, including hydrogen sulfide.

Faba bean protein in reduced fat/cholesterol mayonnaise: extraction and physico-chemical modification process.

Effect of faba bean enzymatic protein extraction process parameters were investigated at different ultrasound powers (200, 300 and 400W), sonication times (15, 25 and 35min), enzyme dosages (0.15, 0.3 and 0.45%) and enzyme treatment times (15, 25 and 35min). Physico-functional characterization of the protein samples through solubility, zeta potential, color, fat adsorption, emulsifying and foaming capacity measurements suggested faba protein as a promising substituent to egg yolk powder in reduced fat mayonnaise formulation. Reduced-fat mayonnaise formulation containing different faba protein/egg yolk powder ratios were prepared and characterized through rheological analyses as well as emulsion stability, texture, color and microstructural evaluations. The formulations containing equal compositions of faba bean protein and egg yolk powder (0.375%) and also the one with 0.5% faba bean protein and 0.25% egg yolk powder were finally suggested to substitute the conventional formulation.

Automatic construction of metabolic models with enzyme constraints

BackgroundIn order to improve the accuracy of constraint-based metabolic models, several approaches have been developed which intend to integrate additional biological information. Two of these methods, MOMENT and GECKO, incorporate enzymatic (kcat) parameters and enzyme mass constraints to further constrain the space of feasible metabolic flux distributions. While both methods have been proven to deliver useful extensions of metabolic models, they may considerably increase size and complexity of the models and there is currently no tool available to fully automate generation and calibration of such enzyme-constrained models from given stoichiometric models.ResultsIn this work we present three major developments. We first conceived short MOMENT (sMOMENT), a simplified version of the MOMENT approach, which yields the same predictions as MOMENT but requires significantly fewer variables and enables direct inclusion of the relevant enzyme constraints in the standard representation of a constraint-based model. When measurements of enzyme concentrations are available, these can be included as well leading in the extreme case, where all enzyme concentrations are known, to a model representation that is analogous to the GECKO approach. Second, we developed the AutoPACMEN toolbox which allows an almost fully automated creation of sMOMENT-enhanced stoichiometric metabolic models. In particular, this includes the automatic read-out and processing of relevant enzymatic data from different databases and the reconfiguration of the stoichiometric model with embedded enzymatic constraints. Additionally, tools have been developed to adjust (kcat and enzyme pool) parameters of sMOMENT models based on given flux data. We finally applied the new sMOMENT approach and the AutoPACMEN toolbox to generate an enzyme-constrained version of the E. coli genome-scale model iJO1366 and analyze its key properties and differences with the standard model. In particular, we show that the enzyme constraints improve flux predictions (e.g., explaining overflow metabolism and other metabolic switches) and demonstrate, for the first time, that these constraints can markedly change the spectrum of metabolic engineering strategies for different target products.ConclusionsThe methodological and tool developments presented herein pave the way for a simplified and routine construction and analysis of enzyme-constrained metabolic models.