Enzyme Fraction Research Articles

Anesthetic Preconditioning of the Small Intestine in Hemorrhagic Hypotension

Aim of the study. To investigate the preconditioning effect of sevoflurane on small intestinal mucosa in experimental hemorrhagic hypotension.Material and methods. The study was performed on a cohort of 106 male rats that included two experimental groups: one exposed to ether (Group 1, n=40) and another one exposed to sevoflurane (Group 2, n=40); two control groups included 20 intact animals, of which 10 were anesthetized with ether and 10 with sevoflurane. Six animals were excluded from the study because they died by the 2nd hour of hemorrhagic hypotension under ether anesthesia. The study parameters were measured at 15 min, 30 min, 1 h, and 2 h of hemorrhagic hypotension. Amylolytic activity of the small intestine mucosa was determined by E. A. Zabelinsky, B. W. Smith and I. M. Roe technique modified by A. M. Ugolev. The data were statistically analyzed using the nonparametric Mann-Whitney method.Results. By 15 min of hemorrhagic hypotension, the activity of amylase fractions in all small intestine regions in Group 2 animals was significantly lower vs the Group 1 rats. By 30 min of hemorrhagic hypotension, the activity of the enzyme fractions in all small intestine regions in Group 2 animals remained significantly lower than in Group 1, by an average of 2 to 9 times (P=0.01; P<0.001), and after 1 h of hemorrhagic hypotension, it was 2 and 4 times lower (P=0.02; P<0.001). By the 2nd hour of hemorrhagic hypotension, the activity of nearly all duodenal amylase fractions in the Group 2 animals remained 3-4 times lower compared to Group 1. Meanwhile, a significantly higher activity of slowly desorbing and intracellular amylase fractions vs the control group was observed in jejunum and ileum.Conclusion. In hemorrhagic hypotension under sevoflurane anesthesia, a decrease of the pancreas excretory function, stabilization of the brush border of the mucosa of all small intestine regions, including enterocyte membranes, was found during the first hour of experiment. Two hours after the hemorrhage, the biochemical evidence of brush border damage in the jejunum and ileum was revealed.

Individual and joint effects of phthalate metabolites on biomarkers of oxidative stress among pregnant women in Puerto Rico

Exposures to phthalate compounds have been linked to adverse birth outcomes, potentially through oxidative stress mechanisms. We explored associations between mixtures of biomarkers of phthalate and phthalate replacement metabolites and oxidative stress using lipid peroxidation biomarker 8-iso-prostaglandin-F2α (8-iso-PGF2α). As 8-iso-PGF2α can be generated via both chemical (nonenzymatic) and enzymatic lipid peroxidation pathways, we calculated the ratio of 8-iso-PGF2α/prostaglandin F2α in an attempt to distinguish the potential contributions of the two pathways. Urinary biomarker measurements were taken from 775 pregnant women in the Puerto Rico Testsite for Exploring Contamination Threats (PROTECT) longitudinal birth cohort at up to three time points during gestation (16–20, 20–24, and 24–28 weeks gestation). Adaptive elastic net with pairwise linear interaction terms (adENET-I) was used to determine individual phthalate metabolites and phthalate interactions that were predictive of lipid oxidative stress biomarkers, and to subsequently create environmental risk scores (ERS) to represent weighted sums of phthalate exposure for each individual at each study visit. Repeated ERS were then used in linear mixed effects models to test for associations between biomarkers of phthalate mixtures and biomarkers of oxidative stress. We also used Bayesian kernel machine regression (BKMR) to explore nonlinearity and interactions between phthalate metabolites within the mixture. An increase from the first to fourth quartile of phthalate ERS derived from adENET-I was associated with a 96.7% increase (95% CI: 74.0, 122) in the hypothesized chemical fraction of 8-iso-PGF2α and a 268% increase (95% CI: 139, 465) in the hypothesized enzymatic fraction of 8-iso-PGF2α. BKMR analyses also suggested strong linear associations between the phthalate mixture and biomarkers of lipid oxidative stress. Various phthalates displayed nonlinear relationships with both chemical and enzymatic fractions of 8-iso-PGF2α, and we observed some evidence of interactions between metabolites in the mixture. In conclusion, exposure to phthalate mixtures was strongly associated with linear increases in biomarkers of lipid oxidative stress, and differences observed between hypothesized chemical and enzymatic lipid peroxidation outcomes highlight the need to critically assess pathways of 8-iso-PGF2α generation in relation to environmental exposures.

A standard operating procedure for an enzymatic activity inhibition assay.

This Standard Operating Protocol (SOP) describes the key steps of experimental setup for an inhibition assay of enzymatic activity. The protocol begins with the design of an experiment, including the choice of a catalytic reaction, optimal conditions, fraction and concentration of the active enzyme, substrate and inhibitor concentrations and the positive and negative controls. The protocol ends with the data analysis followed by a typical example of an experiment. Despite an apparently standard procedure, the assay has a number of possible pitfalls such as low fraction of the active enzyme or errors in the analysis such as application of an improper model or incorrect determination of the inhibition constant while not recognizing the dependence on enzyme concentration. The protocol provides examples of necessary steps and controls to avoid these problems and obtain highly reliable results.

Keeping cGAS silent

Innate Immunity Cells detect microbial and self-DNA in the cytosol as a danger signal that triggers immune and inflammatory responses. Paradoxically, a large fraction of a DNA-sensing enzyme called cGAS is tightly associated with the chromatin, especially during mitosis. Li et al. uncovered two mechanisms that prevent cGAS from being activated by the chromatin DNA (see the Perspective by Ablasser). First, cGAS is hyperphosphorylated as cells enter mitosis, thereby inhibiting its DNA-binding and liquid-liquid phase separation, which promotes cGAS activation. Second, chromatin-bound cGAS is unable to oligomerize, a process required for its activation. Together, these mechanisms ensure cGAS inactivation during cell division to prevent autoimmune reactions. Science , this issue p. [eabc5386][1]; see also p. [1204][2] [1]: /lookup/doi/10.1126/science.abc5386 [2]: /lookup/doi/10.1126/science.abg7422

Characteristic features of molybdenum, copper, tungsten and rhenium accumulation in the environment

The article deals with the assessment of biogenic migration of molybdenum and other trace elements in ore and background conditions. The purpose of this study was to determine the specific features of copper and/or molybdenum accumulation rates in animals against the background of various tungsten and rhenium content levels in the environment and animal feeds, as well as possible metals incorporation into XO-XDH of cow milk. The field biogeochemical studies were performed in summer 2009, 2012, and 2018 in ore landscapes of W-Mo deposits (Tyrnyauz) and background areas of the North Caucasus with the selection of breeds, soils, natural water, plants, animal blood, and dairy products. For comparison, dairy products from the Moscow region were also examined. It was established that metals accumulated not only in soils but also in pasture plants. High content of molybdenum in the blood of cows in ore regions accompanied by a sharp decrease in the concentration of copper was noted. The concentration of studied metals in buttermilk in ore regions increased by 5–10 times in comparison with the control zone. When studying W-Mo ore landscapes of the North Caucasus (Tyrnyauz), the tungsten and rhenium were found to be capable of incorporation into the xanthine enzyme fractions (oxidase) in animals. This fact was discovered for the first time. In the case of an increase in the content of molybdenum and/or tungsten (rhenium) in the environment, the migration of the latter metals is significantly enhanced in ore conditions.

Potential of corncobs (Zea mays) fraction as tyrosinase inhibitor and natural antioxidant in vitro

Corncobs (Zea mays) are beneficial to human health as they contain tyrosinase inhibitors and natural antioxidants, but they are not used as they are considered as waste. This research evaluated the inhibition test towards tyrosinase enzyme and antioxidant activity of corncob fraction using in-vitro DPPH method and its correlation to phenolic and flavonoids. Corncob fraction was extracted using the maceration method applying 70% ethanol solvent. The ethanol extract of corncob was suspended by water and then partitioned with chloroform, ethyl acetate, and aquadest to produce three fractions (chloroform, ethyl acetate, and aquadest fractions). These fractions were analyzed through the tyrosinase inhibition test, applying in vitro tyrosinase enzyme inhibition and antioxidant activity using radical scavenging test DPPH (2 2,2-diphenyl-1-picrylhydrazyl). Meanwhile, the total phenolic and flavonoids content tests were determined spectroscopically. The results showed ethyl acetate fraction had the highest tyrosinase activity with IC50 values of 185.76 µg/mL, followed by the aquadest fraction (IC50 676.44µg/ml) and the chloroform fraction (IC50 709.26 µg/mL). The antioxidant activity using DPPH radical scavenging method exhibited that ethyl acetate fraction had the highest antioxidant activity with IC50 of 25.79 µg/mL followed by the chloroform fraction (IC50 of 29.15 µg/mL) and the aquadest fraction (IC50 of 32.41 µg/mL). The total phenolic content of the corncob fraction ranged between 1.73 to 7.43% (w/w) gallic acid equivalents (GAE), while the entire flavonoid content ranged between 0.01 to 1.34% (w/ w) quercetin equivalent (QE). The tyrosinase activity and antioxidants of the corncob fractions correlated with the total phenolic and flavonoid contents.

Phosphorylation and chromatin tethering prevent cGAS activation during mitosis.

The cyclic GMP-AMP synthase (cGAS) detects microbial and self-DNA in the cytosol to activate immune and inflammatory programs. cGAS also associates with chromatin especially after nuclear envelope breakdown when cells enter mitosis. How cGAS is regulated during cell cycle transition is not clear. Here we found direct biochemical evidence that cGAS activity was selectively suppressed during mitosis, and uncovered two parallel mechanisms underlying this suppression. Firstly, cGAS was hyperphosphorylated at the N-terminus by mitotic kinases, including Aurora kinase B. The N-terminus of cGAS was critical for sensing nuclear chromatin, but not mitochondrial DNA. Chromatin sensing was blocked by hyperphosphorylation. Secondly, oligomerization of chromatin-bound cGAS, which is required for its activation,was prevented. Together, these mechanisms ensure that cGAS is inactive when associated with chromatin during mitosis, which may help to prevent autoimmune reaction.

Enzyme biotechnology development for treating polymers in hydraulic fracturing operations.

Carboxymethyl cellulose (CMC) is a polymer used in many different industrial sectors. In the oil and gas industry, CMC is often used during hydraulic fracturing (fracking) operations as a thickening agent for effective proppant delivery. Accumulations of CMC at fracture faces (known as filter cakes) can impede oil and gas recovery. Although chemical oxidizers are added to disrupt these accumulations, there is industrial interest in developing alternative, enzyme-based treatments. Little is known about CMC biodegradation under fracking conditions. Here, we enriched a methanogenic CMC-degrading culture and demonstrated its ability to enzymatically utilize CMC under the conditions that typify oil fields. Using the extracellular enzyme fraction from the culture, significant CMC viscosity reduction was observed between 50 and 80˚C, at salinities up to 20% (w/v) and at pH 5-8 compared to controls. Similar levels of viscosity reduction by extracellular enzymes were observed under oxic and anoxic conditions. This proof-of-concept study demonstrates that enzyme biotechnology holds great promise as a viable approach to treating CMC filter cakes under oilfield conditions.

Small-molecule control of neurotransmitter sulfonation

Controlling unmodified serotonin levels in brain synapses is a primary objective when treating major depressive disorder—a disease that afflicts ∼20% of the world’s population. Roughly 60% of patients respond poorly to first-line treatments and thus new therapeutic strategies are sought. To this end, we have constructed isoform-specific inhibitors of the human cytosolic sulfotransferase 1A3 (SULT1A3)—the isoform responsible for sulfonating ∼80% of the serotonin in the extracellular brain fluid. The inhibitor design includes a core ring structure, which anchors the inhibitor into a SULT1A3-specific binding pocket located outside the active site, and a side chain crafted to act as a latch to inhibit turnover by fastening down the SULT1A3 active-site cap. The inhibitors are allosteric, they bind with nanomolar affinity and are highly specific for the 1A3 isoform. The cap-stabilizing effects of the latch can be accurately calculated and are predicted to extend throughout the cap and into the surrounding protein. A free-energy correlation demonstrates that the percent inhibition at saturating inhibitor varies linearly with cap stabilization — the correlation is linear because the rate-limiting step of the catalytic cycle, nucleotide release, scales linearly with the fraction of enzyme in the cap-open form. Inhibitor efficacy in cultured cells was studied using a human mammary epithelial cell line that expresses SULT1A3 at levels comparable with those found in neurons. The inhibitors perform similarly in ex vivo and in vitro studies; consequently, SULT1A3 turnover can now be potently suppressed in an isoform-specific manner in human cells.

Thermal inactivation of pectin methylesterase from different potato cultivars (Solanum tuberosum L.)

Pectin methylesterase (PME) is an important enzyme that can alter the texture of potato products during processing and storage. To manage the textural properties of potato based products, low and/or high temperature blanching can be used to promote or inactivate PME, respectively. However, the many potato cultivars being used for processing may contain PMEs with a different thermal stability. Therefore, the thermal inactivation of PME extract from three different potato cultivars (Fontane, Royal and Innovator) was studied. A thermostable and thermolabile enzyme fraction were found in all three cultivars, being inactivated at temperatures from 45 °C and 57.5 °C on, respectively. Innovator may contain a lower level of thermostable PME than the other cultivars tested. The inactivation rate constant of the thermostable PME fraction was lower than the rate constant of the labile fraction at a reference temperature of 55 °C, but no significant differences between the respective rate constants of the different cultivars were found. The thermal inactivation kinetics of PME extract from Fontane were investigated in detail and represented by a first order model that allows the calculation of temperature-time combinations to reach a target residual PME activity.

Alkaline protease production by halophilic Bacillus sp. strain SP II-4 and characterization with special reference to contact lens cleansing

This study aims to screening of protease producing bacterial strain from saltpan located in Kanyakumari. Proteolytic bacterial strains were isolated by using skim milk agar plates. The potent bacterial isolate (SP II-4) was typically identified as Bacillus sp. SP II-4 by studying phenotypic characteristics. The purified enzyme fraction showed 591.04 (U/mg) of specific activity. The protease enzyme liberated from this strain illustrated better stability and activity in pH 10.0, 40 °C and stable upto 7.5% salt. The protease enzyme demonstrated better content lens cleansing effectiveness along with common chemical disinfectant. These above properties of the enzyme clearly stating that may use in various industrial processes which are performed even at extreme conditions.

Optimizing enzymatic catalysts for rapid turnover of substrates with low enzyme sequestration.

Enzymes are central to both metabolism and information processing in cells. In both cases, an enzyme's ability to accelerate a reaction without being consumed in the reaction is crucial. Nevertheless, enzymes are transiently sequestered when they bind to their substrates; this sequestration limits activity and potentially compromises information processing and signal transduction. In this article, we analyse the mechanism of enzyme-substrate catalysis from the perspective of minimizing the load on the enzymes through sequestration, while maintaining at least a minimum reaction flux. In particular, we ask: which binding free energies of the enzyme-substrate and enzyme-product reaction intermediates minimize the fraction of enzymes sequestered in complexes, while sustaining a certain minimal flux? Under reasonable biophysical assumptions, we find that the optimal design will saturate the bound on the minimal flux and reflects a basic trade-off in catalytic operation. If both binding free energies are too high, there is low sequestration, but the effective progress of the reaction is hampered. If both binding free energies are too low, there is high sequestration, and the reaction flux may also be suppressed in extreme cases. The optimal binding free energies are therefore neither too high nor too low, but in fact moderate. Moreover, the optimal difference in substrate and product binding free energies, which contributes to the thermodynamic driving force of the reaction, is in general strongly constrained by the intrinsic free-energy difference between products and reactants. Both the strategies of using a negative binding free-energy difference to drive the catalyst-bound reaction forward and of using a positive binding free-energy difference to enhance detachment of the product are limited in their efficacy.

Evolving trend of Boletus versicolor IBL-04 by chemical mutagenesis to overproduce laccase: Process optimization, 3-step purification, and characterization

In this study, Boletus versicolor IBL-04 was exploited to produce laccase in solid-state fermentation of wheat straw. Wild-type strain of Boletus versicolor (Bv IBL-04) produced laccase with maximum activity of 118.89 ± 11.32 U/mL, which was further improved by manipulating the fungal genome through random chemical mutagenesis to gain a maximum enzyme productivity of 403.34 ± 13.79 U/mL. The maximum laccase-producing mutant strain (Bv EB-75ʺ) was selected by the double-screening process and proceeded to optimize the culture conditions for laccase production using response surface methodology (RSM). Through optimization, laccase activity was improved to 695.78 ± 6.7 U/mL, indicating a 3-fold relative increase in the production yield. The optimally produced enzyme fraction from mutant isolates was eventually purified using 3-step purification system of: (i) (NH4)2SO4 precipitation; (ii) ion-exchange chromatography and (iii) gel-filtration. By purification, a final purification fold of 44.07 was obtained with total enzyme and laccase specific activities of 9406 ± 15.094 IU/mL and 855.09 ± 22.065 U/mg, respectively. Characterization studies revealed the comparative improvement in pH and temperature profiles of laccases from mutant isolates than the wild-type strain. In conclusion, the mutagenesis improved the stability of laccase at higher pH and temperature values, indicating BvEB-75ʺ a potent fungal strain for laccases production for various biotechnological applications.

Adsorption kinetics, conformational change, and enzymatic activity of β-glucosidase on hematite (α-Fe2O3) surfaces

Substantial fractions of extracellular enzymes are intimately associated with soil minerals, which may protect enzymes from denaturation, precipitation, proteolysis, or microbial consumption in soil environments. However, how mineral surface properties affect enzyme-mineral interactions and enzymatic activity of enzymes associated to mineral surface is still unclear. In the present study, adsorption behavior, conformational change, and enzymatic activity of β-glucosidase (BG) on hematite (001) face and (104) face, respectively, were investigated using in situ attenuated total reflectance FTIR spectroscopy and batch experiments. β-glucosidase undergoes greater conformational changes upon adsorption onto hematite (104) face than on hematite (001) face, probably due to the stronger protein-surface interactions on (104) face with the relatively higher surface hydroxyl density. On the other hand, the amount of BG sorbed on hematite (001) face was nearly two times higher than that on hematite (104) at the end of the 150-min adsorption experiments, due to the higher extent of conformational change in β-glucosidase on hematite (104) face. Correspondingly, the initial rate of cellobiose hydrolysis by per gram of β-glucosidase adsorbed on hematite (104) face was 1.7 times higher than that on hematite (001) face. However, when the density of hematite particles was same, the extent of cellobiose hydrolysis was 1.2 times higher on hematite (001) face than that on the (104) face, because of the higher adsorbed amount of β-glucosidase on the former. This study decoupled the effects of mineral surface properties on adsorption kinetics and conformational changes of soil enzymes bound to soil minerals and provided new insights into the correlation between mineral surface properties and catalytical activity of mineral-associated enzymes in soil environments.

Collagenolytic proteases from Bacillus subtilis B13 and B. siamensis S6 and their specificity toward collagen with low hydrolysis of myofibrils

To obtain proteases with high collagenolytic activity, 400 bacteria isolates were screened. The secreted proteases from Bacillus subtilis subsp. subtilis B13 (isolated from beef butchery) and B. siamemsis S6 (isolated from soil) which showed the two highest activity to hydrolyze collagen from bovine Achilles tendon were partially characterized. After enzyme fractionation, both enzymes had the optimum pH for the collagenolytic activity at pH 7.5. However, the optimum temperature of enzyme produced by S6 (60 °C) was higher than B13 (50 °C). The inhibitor study revealed that these proteases were mainly a member of serine proteases and some metalloproteases. Both enzymes were able to degrade collagen with multiple cleavage sites as observed by electrophoretic patterns. In contrast to papain and bromelain, these collagenolytic proteases showed strong hydrolysis toward collagen and elastin as well as beef intramuscular collagen with low beef myofibrillar protein degradation. Moreover, the lower digestion of myofibrillar protein and casein by these enzymes as compared with collagenase from Clostridium histolyticum were observed. These proteases could be applied as a meat tenderizer when collagen or connective tissue protein was responsible for the meat toughness.

A Mesohaline Thraustochytrid Produces Extremely Halophilic Alpha-Amylases.

Halophilic bacteria are well known to produce highly salt-tolerant enzymes that have unusual applications in biotechnology. Production of halophilic proteins is generally not expected in mesohaline microorganisms. Ulkenia sp. AH-2, a mesohaline, marine straminipilan thraustochytrid isolated from waters of a mangrove ecosystem, produces halophilic alpha-amylases. Four enzyme fractions, viz.., A, B, C, and D, were obtained upon ammonium sulfate fractionation and gel filtration. These had a broad salinity tolerance ranging from 0 to 4M NaCl, with an optimum at 3M NaCl. Pools A, C, and D each resolved as a single band on PAGE and zymogram analysis, and the purified proteins were designated Amy a, Amy c, and Amy h. The major activity resided in "pool B," consisting of several amylases which could not be further resolved into pure fractions. Together, these had an optimum at 2M NaCl. All the enzymes were stable to storage for 2 to 24h at 4°C in a range of salt concentrations and even showed enhanced activity following such incubations. True to halophilic enzymes, the complex of "pool B" amylases showed improved activity in the presence of a wide range of organic solvents at 20% concentration. These enzymes are of particular interest by virtue of their constitutive nature as well as production under culture conditions that do not require salinity beyond that of seawater.

Evaluation of Cardioprotective Effect of a Single Oral Dose of Nicorandil before Primary PCI in Patients presenting with Anterior STEMI

Abstract Background Nicorandil in conjunction with primary percutaneous coronary intervention (PPCI) has been reported to exert cardioprotective effect in patients with ST segment elevation myocardial infarction (STEMI). Intravenous and intracoronary Nicorandil administration with PPCI have been reported to reduce reperfusion injury consequences and improve cardiac function in patients with STEMI even on the long term .However, there is limited information on the cardioprotective effect of oral Nicorandil in STEMI patients . Objective The Aim of this study was to evaluate Cardioprotective effect of a single oral dose of Nicorandil 20 mg given before primary PCI in patients presenting with a first anterior STEMI within 6 hours from symptoms onset. Material and Methods This study is a Prospective multicenter Study that included 80 Patients who were presenting with first anterior STEMI and undergone primary PCI as a myocardial reperfusion method in the period between June 2016 and May 2018. Patients were divided into two groups: Group A (40 patients) was the Study group which received Nicorandil 20mg single oral dose immediately on admission with the loading doses of aspirin and clopidogril prior to the PPCI procedure and Group B (40 patients) was the Control group that did not receive Nicorandil . Results There were no statistically significant differences between two groups as regards demographic data, risk factors for coronary artery disease and procedural data . There was statistically significant lower peak levels of cardiac enzymes and higher left ventricle ejection fraction (LVEF%) in group (A) compared to group (B). The peak level of CKT in group (A) was 1899.1± 601.000 compared to 2386.300 ± 844.318 in group (B), (P = 0.004) .The peak level of CK MB in group (A) was 264.100 ± 86.982 compared to 329.325 ± 147.404 in group (B), (P = 0.018) .The LVEF% in group (A) was (43.550±5.114) compared to (40.325±5.753) in group (B), (P = 0.010). Conclusion Single dose of Nicorandil 20 mg administered orally and given to patients who were presenting with a first anterior STEMI within 6 hours from symptoms onset prior to reperfusion with primary PCI had a significant cardioprotective effect in the form of higher left ventricular ejection fraction and lesser release of cardiac enzymes (CKT and CK MB) compared to control group which reflects more myocardial salvage of the area at risk and lesser reperfusion induced myocardial damage.

Degradation of rice straw at low temperature using a novel microbial consortium LTF-27 with efficient ability

In this study, a novel psychrotrophic lignocelluloses degrading microbial consortium LTF-27 was successfully obtained from cold perennial forest soil by successive enrichment culture under facultative anaerobic static conditions. The microbial consortium showed efficient degradation of rice straw, which cellulose, hemicelluloses and lignin lost 71.7%, 65.6% and 12.5% of its weigh, respectively, in 20 days at 15 °C. The predominant liquid products were acetic acid and butyric acid during degrading lignocellulose in anaerobic digestion (AD) process inoculated with the LTF-27. The consortium mainly composed of Parabacteroides, Alcaligenes, Lysinibacillus, Sphingobacterium, and Clostridium, along with some unclassified uncultured bacteria, indicating powerful synergistic interaction in AD process. A multi-species lignocellulolytic enzyme system working cooperatingly on lignocelluolse degradation was revealed by proteomics analysis of cellulose bound fraction of the crude extracellular enzyme, which provides key theoretical base for further exploration and application of LTF-27.

The effects of PMSF and cysteine addition into partially purified bromelain from pineapple (Ananas comosus [L] Merr) cores to its kinetics behaviour

Bromelain found in various tissues in the pineapple plant (Ananas Comusus [L] Merr. The bromelain will be taken from pineapple cores for kinetic study. The effect of addition organic compounds, such as phenylmethylsulfonyl fluoride (PMSF) and cysteine on its kinetics behaviour will be determined by proteolytic activities of the bromelain. The stages of this study were started from isolation of crude enzyme, fractionation with ammonium sulphate salt, dialysis, and ion-exchange column chromatography with CM sephadex C-50 for enzyme purification. The obtained enzyme fractions from crude enzyme, ammonium sulphate, dialysis, and CM-sephadex C-50 ion exchange chromatography have specific activities of 45.39, 238.31, 311.16, and 427.13 U/mg, respectively. The purification stages result in increasing of the bromelain specific activities by 9 times compared to the crude enzyme. The Michaelis-Menten constant (Km) value for the purified bromelain was 0.85 % casein and the Vmax value was 0.41 U/min. Addition of PMSF and cysteine compound effect on its Vmax and Km with various trends. The Vmax value will be decreased by addition of PMSF and increased by addition of cysteine. Thus, proteolytic activity of bromelain from pineapple cores can be significantly increased by the addition of cysteine compounds and inhibited by the addition of PMSF.

Amalgamation of stress degradation and metabolite profiling in rat urine and feces for characterization of oxidative metabolites of flibanserin using UHPLC-Q-TOF-MS/MS, H/D exchange and NMR technique

Flibanserin (FLB) is the first FDA approved drug showed to have significant activity against sexual desire disorder of premenopausal and postmenopausal women. Unfortunately, FLB is used as an adulterant in dietary supplement products as a performance enhancer in sports. Identification of FLB and its metabolites in the biological samples requires an authenticated analytical technique. The aim of this study was to identify N-oxide metabolite of FLB in microsomal and S9 human liver enzyme fractions, rat urine and feces. There are several N-oxide reported as genotoxic impurity or reactive metabolites based on position of N-oxide in piperazine ring. This study also describes the strategy to utilize degradation chemistry for isolation of N-oxide and its step-wise characterization. An LC-MS method has been developed and employed for identifying the N-oxide metabolite of FLB. The targeted N-oxide metabolite in the extracted ion chromatogram of the in vitro and in vivo samples has been confirmed by analyzing the changes in observed mass at m/z 407.1693. Major distinguished abundant ions at m/z 243.1104, 190.0974, 161.0705, 119.0601 confirmed the structure of the metabolite. This study will help to understand the oxidative potential of FLB in toxicokinetic study. The developed method can be useful to identify FLB or its N-oxide metabolite in dope testing in future. This is the first time to report a strategy to utilize degradation chemistry for N-oxide metabolite characterization. In this study, isolated N-oxidative degradation product was used to confirm N-oxide metabolite which was characterized by LC-MS through H/D exchange and structure was ensured by NMR spectroscopy (1H, COSY).