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Jul 2020 DOI 10.14302/issn.2377-2549.jndc-20-3460
Laccase enzyme production is important and more beneficial for environment, because it has many roles like, involved in bioremediation, biodegradation, decolorization of environmental polluted dyes and pharmaceutical sector also. Production of laacse enzyme from bacillus sp as using of Agro waste (rice bran) as a substrate. The Agricultural soil sample was collected, after the sample were processed for the preliminary and biochemical tests to identification of Bacillus organism. The Guiacol inducer were used for microbial screening of laccase enzyme production. After that microbial screening, various optimization parameters (pH, Temperature, Inducers, carbon and nitrogen sources) are checked for that production of laccase enzyme in mass level. Based on that optimization the bulk fermentation (large scale) (solid state fermentation) were done as a rice bran substrate. The fermentation product was subjected to analyzed the physiochemical properties and purification based on that techniques of Gel filtration chromatography, Dialysis, Ammonium sulfate preciptation. The protein estimation of that product to analysed by lowry’s method.
Jan 2020 DOI 10.14302/issn.2379-7835.ijn-19-3139
In this study, a peroxidase from new source was purified using ion exchange and gel filtration techniques. The recovery for peroxidase activity was 19% with 11-fold purification and specific activity of 749 unit/mg protein. Purified peroxidase demonstrated a molecular mass of 39 kDa using gel filtration and was confirmed as a single band on SDS-PAGE. The purified peroxidase revealed a broad optimum pH activity at 6.0-6.5 and 50°C temperature. The kinetic parameters for purified peroxidase toward H2O2 and guaiacol as substrates were found to be Km = 3.355, 5.395 mM, Kcat = 99.52, 79.56 s-1 and Vmax =1.531, 1.242 µmole ml-1 min-1, respectively. The catalytic efficiency (kcat/Km) of the purified peroxidase was 14.75 and 29.66 s−1 mM−1 for guaiacol and H2O2, respectively. Peroxidase activity was observed to be enhanced by Cu2+, Co2+, Ni2+ and inhibited in the presence of Sn2+, Al3+, Hg2+, NaN3, EDTA and urea. Characterization showed that peroxidase purified from C. forskohlii has the ability to be used for food industrial applications.
Mar 2019 DOI 10.14302/issn.2471-2140.jaa-19-2699
Xanthine oxidase is a commercially important enzyme with wide area of medical applications to develop diagnostic kits. Xanthine oxidase was extracted, purified and characterized from sheep liver (SLXO). The purification procedure involved acetone precipitation and chromatography on DEAE-cellulose and Sephacryl S-300 columns. The sheep liver xanthine oxidase was homogeneously purified 31.8 folds with 3.5 U/mg specific activity and 24.1% recovery. SLXO native molecular weight was 150 kDa and on SDS-PAGE appeared as single major band of 75 kDa representing a homodimer protein. Isoelectric focusing of the purified SLXO resolved into two closely related isoforms with pI values of 5.6 and 5.8. The apparent Km for xanthine oxidase at optimum pH 7.6 was found to be 0.9 mM xanthine. FeCl2 and NiCl2 increased the activity of SLXO, while CuCl2 and ZnCl2 were found to be potent inhibitors of the purified enzyme. Allopurinol inhibits SLXO competitively with one binding site on the purified molecule and Ki value of 0.06 mM.
Dec 2012 DOI 10.14302/issn.2326-0793.jpgr-12-100
Defining protein-protein interactions is essential for understanding the mechanisms by which cells regulate basic functions, such as metabolism, transcription, and signal transduction. Affinity purification followed by tandem mass spectrometry (AP-MS) has application for discovery of new interactors regulating various cellular processes. Here we optimize the purification method for AP-MS and develop a simplified unbiased analytical tool, Z-score plus prey occurrence and reproducibility (ZSPORE) for data analysis. Using this pipeline we achieve a higher efficiency of AP-MS and enhanced identification of high confidence interacting proteins (HCIP) in mammalian cells. When applied to analysis of the innate immune interactome, these methods enhanced HCIP identification. In addition, we investigated the GRB2 complex, which is associated with signal transduction and cell growth. Twenty-four known GRB2 interacting proteins were identified plus 26 new GRB2 binding partners. Thus, these straightforward methods recapitulate known protein interactions, discover novel complexes, and allow mapping of protein interaction networks.
Aug 2023 DOI 10.14302/issn.2639-3166.jar-23-4682
Objectives The solid-liquid separation (SLS) process generally separates solid and liquid fractions in wastewater and livestock manure. The solid-liquid separation process is an essential pretreatment step for the recycling and purification of pig manure. This study has assessed the separation and/or reduction efficiency by various SLS processes used in pig farms. Methods Seven types of SLS processes (centrifuge, centrifuge (+coagulation agent), belt press (+ coagulation agent), drum screen, inclined screen, vibration screen, and screw press) were used on 11 pig farms and conducted a comparative analysis. As for the sample in this study, the pig raw manure before treatment, the separated liquid and solid manure after treatment of the SLS process collected, respectively. These samples were provided for pH, EC(electrical conductivity) moisture content, CODMn, BOD5, TN, TP, K, TS, SS, NaCl, and heavy metals analysis. Results and Discussion The belt press (+coagulation agent) process had the highest TS and SS reduction rate of 78.8% and 96.9%, respectively. The highest removal efficiency of TN and TP was41.0% and 94.2% by belt press (+coagulation agent) and centrifuge (+coagulation agent),respectively. The belt press (+coagulation agent) removed 59.4% and 66.0% of BOD5 and CODMn,respectively. The Zn and Cu were removed 100% and 98.6% by centrifuge (+coagulation agent).However, the drum screen, inclined screen, vibration screen, screw press, and centrifuge showed lower removal efficiency of nutrient contents, solids, Zn, and Cu than centrifugal and belt press processes with chemical coagulation. Conclusions The centrifugal and belt press separation processes that used chemical coagulation showed much more removal efficiency of nutrient contents, solids, and metals like Zn and Cu. Although SLS with chemical coagulants is an effective pre-treatment process for liquid manure treatment and helps removal effect for suspended solids, nutrients, and heavy metals, further studies are needed on how it affects biological or chemical processing processes that are linked.
Nov 2021 DOI 10.14302/issn.2690-4837.ijip-21-4015
Copper (Cu) has a strong impact on the function of the immune system through several different pathways. These impacts include helping the function of monocytes, neutrophils, and macrophages, and enhancing Natural Killer cells’ activities. Cu also has a role in antimicrobial properties and inflammatory response. It is also important for IL-2 production and response, which is a component of adaptive immune cells. Additionally, Cu has multiple roles in both proliferation and differentiation of T cells and is involved in the production of antibodies. Cu deficiency can even lead to "increased viral virulence"1. Copper has a long history of use in medicine, and has continued to be used for purification of water, including use in hospitals to prevent legionnaires disease. The CDC pre released information on a study completed in March 2020 on the lifespan of COVID-19 on different surfaces which included its lifespan on copper, where it was completely dead within 4 hours. In addition, "Several reports demonstrated that Cu deficiency weakens the human immune response" 2. Given the multiple avenues of impact, it has been suggested that Cu supplementation, within recommended levels, be given to individuals who are low in Cu to help them fight off COVID-19. It is also possible that Cu supplementation, within recommended levels, may help prevent COVID-19 infection, or help individuals who are not low in Cu to fight off COVID-19 infection. However, dosage would have to be carefully managed, as excess levels of Cu can lead to both neurodegenerative and neurodevelopmental diseases.
Jul 2020 DOI 10.14302/issn.2690-4829.jen-20-3480
Conversion of biomass into fermentable sugars is a major requirement for successful and cost-effective biofuels production. The conversion of xylan to sugars requires multiple enzymes including α-glucuronidase. Here we report the cloning, expression, purification and characterization of the α-glucuronidase from Dictyoglomusturgidum(DtuAgu). DtuAgu is an intracellular protein of 685 amino acids and a predicted molecular weight of 79.4 kD. Enzymatic activity was optimum between pH 7.0 and 8.0 and at 85°C. The specific activity of the enzyme was 10 u/mg when measured using mixed aldouronic acids. The specific activity on isolated glucuronoxylan was approximately 20% of the value obtained with xylooligosaccharides. DtuAgu significantly improved xylan conversion to xylose when evaluated using two mixtures of thermostable bacterial enzymes and two sources of xylan. DtuAgu has the potential to be a key player in thermostable enzyme cocktails for the conversion to biomass to biofuels.α
Dec 2018 DOI 10.14302/issn.2377-2549.jndc-18-2430
Introduction: Analysis of 8-oxodG is usually conducted by either chromatography-based methods or by immunochemical methods commonly used based upon their low cost and high-throughput. However, concern regarding the accuracy of ELISA methods has complicated their use. We directly compare the levels of urinary 8-oxodG obtained by HPLC-MS/MS with three commercially available ELISA kits in this report. Methods: In the current study, a total of 9 human urine samples were analyzed by LC-MS/MS and three commonly used commercial available ELISA kits. Results: We found that urinary 8-oxodG levels analyzed by HPLC-MS/MS [1.4 ± 0.3 nmol/mmol creatinine) were 7.6- to 23.5-fold lower than those detected by ELISA. Overall, the correlations between ELISA and HPLC-MS/MS were poor but were improved after SPE purification for kits from ENZO (P = 0.2817 without SPE; P = 0.0086 with SPE) and Abcam (P = 0.0596 without SPE; P = 0.0473 with SPE). Discussion and Conclusion: While we confirmed that SPE purification can improve the correlation between the selected ELISA kits and HPLC-MS/MS, HPLC-MS/MS is still the method of choice to accurately assess the levels of 8-oxodG in human urine.
Oct 2018 DOI 10.14302/issn.2328-0182.japst-18-2344
In recent years, the consumption of dietary supplements (DS) has increased worldwide. In Argentina, approximately 14 million DS units were sold between 2015 and 2017. The adulteration of DS with active pharmaceutical ingredients or their analogues has been reported. This represents an alarming emerging risk to public health. The aim of this work was to detect the possible adulteration of a DS marketed in Argentina for the treatment of erectile dysfunction. Initially, thin layer chromatography analysis of the DS capsules content suggested the presence of a major compound. For the isolation and purification of this compound, an easy method consisted of a liquid-liquid extraction (water/CH2Cl2) followed by re-crystallisation from ethanol, is reported. Spectroscopic techniques such as mono- and bidimensional nuclear magnetic resonance, Fourier transform infrared spectroscopy and mass spectrometry allowed its identification as tadalafil. A rapid and reliable method was developed for the quantification of tadalafil in this DS by high performance liquid chromatography-mass spectrometry (HPLC-MS/MS). The mean content of tadalafil per capsule was 21.2 mg which represents a slightly higher value than that found in approved products in Argentina (5 or 20 mg per tablet). In addition, an undeclared alga was identified in the DS by microscopic techniques.