NADP+-Specific Isocitrate Dehydrogenase from Oleaginous Yeast Yarrowia lipolytica CLIB122: Biochemical Characterization and Coenzyme Sites Evaluation

NADP⁺-dependent isocitrate dehydrogenase from Yarrowia lipolytica CLIB122 (YlIDP) was overexpressed and purified. The molecular mass of YlIDP was estimated to be about 81.3 kDa, suggesting its homodimeric structure in solution. YlIDP was divalent cation dependent and Mg²⁺ was found to be the most favorable cofactor. The purified recombinant YlIDP displayed maximal activity at 55 °C and its optimal pH for catalysis was found to be around 8.5. Heat inactivation studies revealed that the recombinant YlIDP was stable below 45 °C, but its activity dropped quickly above this temperature. YlIDP was absolutely dependent on NADP⁺ and no NAD-dependent activity could be detected. The K _m values displayed for NADP⁺ and isocitrate were 59 and 31 μM (Mg²⁺), 120 μM and 58 μM (Mn²⁺), respectively. Mutant enzymes were constructed to tentatively alter the coenzyme specificity of YlIDP. The K _m values for NADP⁺ of R322D mutant was 2,410 μM, being about 41-fold higher than that of wild type enzyme. NAD⁺-dependent activity was detected for R322D mutant and the K _m and k _cat values for NAD⁺ were 47,000 μM and 0.38 s^?1, respectively. Although the R322D mutant showed low activity with NAD⁺, it revealed the feasibility of engineering an eukaryotic IDP to a NAD⁺-dependent one.     

A Proof for Substitution of Endogenous Iron (II) in Lipoxygenase by Exogenous Cu2+

Soybean lipoxygenase (LOX) contains endogenous iron (II) at the active site, which is important for the enzyme activity. The activity of LOX can be accelerated by some exogenous metal ions including Cu²⁺. However, the mechanism of the activity improvement caused by exogenous metal ions remains unclear, not only for LOX but for most other metalloenzymes. Meanwhile, the possibility that exogenous metal ions can displace endogenous iron (II) is still in discussion for a lack of a direct and quantitative proof. In this paper, a quantitative proof of replacing iron (II) inside LOX by exogenous Cu²⁺ was provided, simply using UV-Vis spectrometry with two indicators p-carboxylantipyrylazo and 9-(4-carboxyphenyl)-2,3,7-trihydroxyl-6-fluorine. A 0.56 μM free iron (II) was observed in the bulk solution after incubating 9.45 μM Cu²⁺ with 16.10 μM LOX at 20°C for 5 min, which is in coincidence with the decrement of Cu²⁺ in the bulk solution (0.53 μM), implying that iron (II) was replaced by Cu²⁺.     

A Feruloyl Esterase (FAE) Characterized by Relatively High Thermostability from the Edible Mushroom Russula virescens

A monomeric feruloyl esterase (FAE) with a molecular mass of 62 kDa was acquired from fresh fruiting bodies of the edible mushroom Russula virescens. The isolation procedure involved ion exchange chromatography on CM-cellulose, Q-Sepharose, and SP-Sepharose and finally fast protein liquid chromatography–gel filtration on Superdex 75. Two amino acid sequences were obtained after tryptic digestion, and they both showed some homology with the esterase of some fungi. Maximal activity was observed at pH 5.0 and at 50 °C. The enzyme displayed relatively high thermostability as evidenced by over 70 % residual activity at 70 °C and about 34 % residual activity at 80 °C. The K _m and V _max for this enzyme on methyl ferulate were 0.19 mM and 1.65 U/mg proteins, respectively. The purified FAE prefers methyl ferulate over methyl caffeate and is least active on methyl p-coumarate. The FAE activity was not significantly affected by the presence of cations such as Mn²⁺, Ca²⁺, Cd²⁺, Zn²⁺, Mg²⁺, Cu²⁺, and K⁺ ions but inhibited by Al³⁺, Hg²⁺, Fe²⁺, and Pb²⁺ ions at a tested concentration of 2. 5 mM.     

Purification and Characterization of Agarase from Marine Bacteria <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. PS12B and Its Use for Preparing Bioactive Hydrolysate from Agarophyte Red Seaweed <Emphasis Type="Italic">Gracilaria verrucosa</Emphasis>

Acinetobacter strain PS12B was isolated from marine sediment and was found to be a good candidate to degrade agar and produce agarase enzyme. The extracellular agarase enzyme from strain PS12B was purified by ammonium sulfate precipitation followed by DEAE-cellulose ion-exchange chromatography. The specific activity of the crude enzyme which was 1.52 U increased to 45.76 U, after two-stage purification, with an enzyme yield of 9.76%. Purified enzyme had a molecular mass of 24 kDa. The optimum pH and temperature for activity of purified agarase were found to be 8.0 and 40 °C, respectively. The K_m and V_max values for agarase were 4.69 mg/ml and 0.5 μmol/min, respectively. Treatment with EDTA reduced the agarase activity by 58% at 5 mM concentration. The enzyme activity was stimulated by the presence of Fe²⁺, Mn²⁺, and Ca2⁺ ions while reducing reagents (β-mercaptoethanol and dithiothreitol, DTT) enhanced its activity by 30–40%. The purified agarase exhibited tolerance to both detergents and organic solvents. Major hydrolysis products of agar were DP4 and also a mixture of longer oligosaccharides DP6 and DP7. The enzyme hydrolysed seaweed (Gracilaria verrucosa) exhibited strong antioxidant activity in vitro. Successful hydrolysis of seaweed indicates the potential use of the enzyme to produce seaweed hydrolysate having health benefits as well as the industrial application like the production of biofuels.     

Production,Purification and Characterisation of a Potential Fibrinolytic Protease from Endophytic <Emphasis Type="Italic">Xylaria curta</Emphasis> by Solid Substrate Fermentation

The present investigation highlights the optimal conditions for production of a non-toxic, bi-functional fibrinolytic enzyme xylarinase produced by endophytic fungus Xylaria curta by solid substrate fermentation using rice chaff medium. The purified enzyme is a monomeric protein with a molecular mass of ～33 kDa. The enzyme exhibits cleavage of Aα and Bβ chains of fibrin(ogen) and has no effect on γ chain. The optimal fibrinolytic activity of the enzyme was observed at 35 °C and pH 8. The fibrinolytic activity was enhanced in the presence of Ca²⁺, whereas it was completely inhibited in the presence of Fe²⁺ and Zn²⁺ ions and inhibitors like EDTA and EGTA suggesting it to be a metalloprotease. The K _m and V _max of the enzyme for azocasein were 326 μM and 0.13 μM min^?1. The N-terminal sequence of the enzyme (SNGPLPGGVVWAG) was same when compared to xylarinase isolated from culture broth of X. curta. Thus, xylarinase could be exploited as a potent clot busting enzyme which could be produced on large scale using solid substrate fermentation.     

Exploring Thermophilic Cellulolytic Enzyme Production Potential of Aspergillus fumigatus by the Solid-State Fermentation of Wheat Straw

Cellulases can be used for biofuel production to decrease the fuel crises in the world. Microorganisms cultured on lignocellulosic wastes can be used for the production of cellulolytic enzymes at large scale. In the current study, cellulolytic enzyme production potential of Aspergillus fumigatus was explored and optimized by employing various cultural and nutritional parameters. Maximum endoglucanase production was observed after 72 h at 55 °C, pH 5.5, and 70 % moisture level. Addition of 0.3 % of fructose, peptone, and Tween-80 further enhanced the production of endoglucanase. Maximum purification was achieved with 40 % ammonium sulfate, and it was purified 2.63-fold by gel filtration chromatography. Endoglucanase has 55 °C optimum temperature, 4.8 optimum pH, 3.97 mM K _m, and 8.53 μM/mL/min V _max. Maximum exoglucanase production was observed at 55 °C after 72 h, at pH 5.5, and 70 % moisture level. Further addition of 0.3 % of each of fructose, peptone, and Tween-80 enhances the secretion of endoglucanase. It was purified 3.30-fold in the presence of 40 % ammonium sulfate followed by gel filtration chromatography. Its optimum temperature was 55 °C, optimum pH was 4.8, 4.34 mM K _m, and 7.29 μM/mL/min V _max. In the case of β-glucosidase, maximum activity was observed after 72 h at 55 °C, pH 5.5, and 70 % moisture level. The presence of 0.3 % of fructose, peptone, and Tween-80 in media has beneficial impact on β-glucosidase production. A 4.36-fold purification was achieved by 40 % ammonium sulfate precipitation and gel filtration chromatography. Optimum temperature of β-glucosidase was 55 °C, optimum pH was 4.8, K _m was 4.92 mM, and V _max 6.75 μM/mL/min. It was also observed that fructose is better than glucose, and peptone is better than urea for the growth of A. fumigatus. The K _m and V _max values indicated that endoglucanase, exoglucanase, and β-glucosidase have good affinity for their substrates.     

Purification and characterization of an extracellular lipolytic enzyme from the fermented fish-originated halotolerant bacterium, <Emphasis Type="Italic">Virgibacillus alimentarius</Emphasis> LBU20907

A halotolerant Virgibacillus alimentarius LBU20907 isolated from fermented fish (Budu) was found to be an efficient producer of extracellular halophilic lipase enzyme. The enzyme was purified 5.99-fold with a 0.15% final yield to homogeneity by ammonium sulfate precipitation, followed by dialysis, Toyopearl DEAE-650 M ion exchange chromatography, Toyopearl butyl-650 M hydrophobic interaction chromatography, and Toyopearl-HW 55 F gel filtration chromatography. SDS-PAGE of purified lipase exhibited a homogenous single band with a very high molecular weight of 100 kDa. The properties of purified lipase revealed maximum activity at pH 7.0 and 40 °C. It was also highly stable in a pH range of 6.0–7.0, retaining more than 90% activity for 24 h. It was stable at the temperature of 30–50 °C and maintained more than 80% activity for 16 h. The purified lipase performing the maximal activity in the presence of 20.0% NaCl indicated halophilic enzyme properties. Its lipolytic activity was highest against p-nitrophenyl palmitate. The lipase activity was found to be enhanced in hexane. The enzyme activity was stimulated in the presence of Zn²⁺, Ca²⁺, Mg²⁺, and Sr²⁺; while, it was completely inhibited by Ba²⁺ and Co²⁺. The enzyme had a K _m and V _max of 108.0 mg and 79.1 U mL^?1, respectively.     

Enhanced Resveratrol Production in Vitis vinifera Cell Suspension Cultures by Heavy Metals Without Loss of Cell Viability

The effects of heavy metal ions (Co²⁺, Ag⁺, Cd²⁺) on cell viability and secondary metabolite production, particularly anthocyanins and phenolic acids in Vitis vinifera cell suspension cultures, were investigated. Of these, Co at all three used concentrations (5.0, 25, and 50 μM), Ag, and Cd at low concentration (5.0 μM) were most effective to stimulate the phenolic acid production, increasing the 3-O-glucosyl-resveratrol up to 1.6-fold of the control level (250.5 versus 152.4 μmol/g), 4 h after the treatments. Meanwhile, the elicitors at effective concentrations did not suppress cell growth, while the cell viability maintained. In contrast, Ag and Cd at high concentrations (25 and 50 μM) remarkably reduced the cell viability, decreasing the cell viability up to about 15 % of the control level, 24 h after the treatments. The heavy metal ions did not affect the anthocyanin production. These observations show how, in a single system, different groups of secondary products can show distinct differences in their responses to potential elicitors. The 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, peroxidase activity, medium pH value, and conductivity were only slightly elevated by the heavy metal ions. The results suggest that some of the secondary metabolites production was stimulated by the used elicitors, but there was not a stress response of the cells.     

Expression and Characterization of a Novel Propionyl-CoA Dehydrogenase Gene from Candida rugosa in Pichia pastoris

The propionyl-CoA dehydrogenase (PACD) gene was firstly cloned from Candida rugosa by the cDNA RACE technique. The 6× His-tagged recombinant PACD gene was expressed in Pichia pastoris GS115 and purified with Ni-NTA affinity chromatography. SDS-PAGE analysis and Western blotting revealed that the molecular mass of the purified PACD was 49 kDa. The results showed that the recombinant protein had the activity of catalyzing propionyl-CoA to acrylyl-CoA. The K _m, k _cat, and V _max values of the purified PACD were calculated to be 40.86 μM, 0.566 s^?1 and 0.693 U?mg^?1 min^?1. The optimal temperature and pH of the purified PACD were 30 °C and 7.0, respectively. The recombinant PACD maintained 76.3%, 30.1%, and 4.3% of its original activity after 2 h incubation in standard buffer at 30, 40, and 50 °C, respectively. Mg²⁺ had an activating effect on the enzyme, while Mn²⁺, Ca²⁺, Zn²⁺, and Cu²⁺ had weak inhibition. Since PACD catalyzed the key step (from propionyl-CoA to acrylyl-CoA) in the modified β-oxidation pathway from glucose to 3-hydroxypropionic acid (3-HP), the integration of recombinant PACD could benefit the engineered strains for effective production of 3-HP from the most abundant biomass–sugars.     

Purification and biochemical characterization of two major thermophilic xylanase isoforms (T70 and T90) from xerophytic Opuntia vulgaris plant spp.

Thermostable xylanase isoforms T₇₀ and T₉₀ were purified and characterized from the xerophytic Opuntia vulgaris plant species. The enzyme was purified to homogeneity employing three consecutive steps. The purified T₇₀ and T₉₀ isoforms yielded a final specific activity 134.0 and 150.8 U mg^?1 protein, respectively. The molecular mass of these isoforms was determined to be 27 kDa. The optimum pH for the T₇₀ and T₉₀ xylanase isoforms was 5.0 and the temperature for optimal activity was 70 and 90 °C, respectively. The Km value of T₇₀ and T₉₀ enzyme isoforms was 3.49, 2.1 mg ml^?1, respectively when oat spelt xylan was used as a substrate. The T₇₀ had a Vmax of 10.4 μmol min^?1 mg^?1, and T₉₀ had a Vmax of 8.9 μmol min^?1 mg^?1, respectively. In the presence of 10 mM Co²⁺, and Mn²⁺ the activity of T₇₀ and T₉₀ isoforms increased, where as 90 % inhibition was noted with of the use 10 mM Hg²⁺, Cd²⁺, Cu²⁺, Zn²⁺ while partial inhibition was observed in the presence of Fe³⁺, Ni²⁺, Ca²⁺and Mg²⁺. The T₇₀ and T₉₀ isoforms retained nearly 50 % activity in the presence of 2.0 M urea, while use of 40 mM SDS lowered the activity nearly 38–41 %. The substrate specificity of both T₇₀ and T₉₀ isoforms showed maximum activity for oat spelt xylan. Western blot, immunodiffusion, and in vitro inhibition assays confirmed reactivity of the T₉₀ isoform with polyclonal anti-T₉₀ antibody raised in rabbit, as well as cross-reactivity of the antibody with the T₇₀ xylanase isoform.     

Purification and Characterization of a Glucosamine-Binding Antifungal Lectin from Phaseolus vulgaris cv. Chinese Pinto Beans with Antiproliferative Activity Towards Nasopharyngeal Carcinoma Cells

A lectin has successfully been isolated from Phaseolus vulgaris cv. Chinese pinto bean using affinity chromatography, ion exchange chromatography, and gel filtration in succession, with a 15.4-fold purification. Investigation of its characteristics revealed that Chinese pinto bean lectin (CPBL) was a 58-kDa dimeric glucosamine-binding protein. Its Mg²⁺-dependent hemagglutinating activity was stable at pH 7–8 and at or below 60 °C. When the purified lectin was tested against six fungal species including Phyllosticta citriasiana, Magnaporthe grisea, Bipolans maydis, Valsa mali, Mycosphaerella arachidicola, and Setosphaeria turcica, only the mycelial growth of V. mali was reduced by 30.6 % by the lectin at 30 μM. The lectin did not exert any discernible antiproliferative effects on breast cancer MCF-7 cells, but was able to suppress proliferation of nasopharyngeal carcinoma HONE-1 cells, with an IC₅₀ of 17.3 μM, as revealed by the MTT assay. Since few plant lectins demonstrate antifungal activity against V. mali, and not many others have inhibitory effects on HONE-1 cells, CPBL is a distinctive lectin which may be exploited for development into an agent against V. mali and HONE-1 cells.     

Monitoring 60Co activity for the characterization of the sorption process of Co2+ ions in municipal activated sludge

In large volumes produced activated sludges from wastewater treatment plants (WWTPs) with low concentrations of heavy metals can be utilized as agricultural fertilizers and soil conditioners. Increased contents of toxic xenobiotics are limiting factors that affect the utilization of these heterogeneous wastes. The main aim of our paper was to show the utilization of dried activated sludge (DAS) from municipal WWTP as potential Co²⁺ ions sorbent i.e. for non-agricultural purposes. The radio indicator method by radionuclide ⁶⁰Co and γ-spectrometry for characterization DAS sorption properties was used. DAS soluble and solid fractions were characterized by biochemical, ETAAS and CEC analysis. The sorption of Co²⁺ ions by DAS was rapid process and equilibrium was reached within 2 h. Sorption capacity of DAS (Q) increased with the initial concentration of CoCl₂ in the range from 100 to 4,000 μmol l^?1, reaching 20 and 160 μmol g^?1. Obtained Q values were depent on pH value from 2.0 to 8.0. The maximum sorption capacity (Q _max) of DAS at pH 6 calculated from mathematical model of Langmuir adsorption isotherm was 175 ± 9 μmol g^?1. FT-IR analyses showed the crucial role of carboxyl functional groups of DAS surfaces on cobalt uptake. For confirmation ion-exchange mechanism in sorption process of Co²⁺ ions by DAS scanning electron microscopy and EDX analysis were used.     

Production, Purification, and Characterization of Exoglucanase by Aspergillus fumigatus

Fungi are considered good producers of industrially valuable enzymes with higher enzymatic activities. Among these cellulases are group of extracellular enzymes commonly employed in many industries for the hydrolysis of cellulolytic material. Aspergillus fumigatus produced exoglucanase having high enzymatic activity (83 U/gds) during the solid-state fermentation of wheat straw under optimum physical and nutritional conditions. Maximum production was obtained after 72 h of fermentation, at 55 °C temperature, pH 5.5, 80 % moisture level, and 2 mL fungal inoculum. Production was further increased by the addition of fructose (0.3 %) as additional carbon source, peptone (0.4 %) as nitrogen source, Tween-80 (0.3 %) as surfactant, and ammonium sulfate (0.2 %) in media. Exoglucanase was 2.30-folds purified by adding 40 % ammonium sulfate with volumetric activity 95.4 U/gds and specific activity 14.74 U/mg. Further, it was 5.18-folds purified by gel filtration chromatography with volumetric activity 115.2 U/gds and specific activity 33.10 U/mg. Purified exoglucanase has maximum activity at 55 °C and pH 4.8 using 1 % Avicel aqueous solution as substrate. The K _m and V _max were 4.34 mM and 7.29 μM/min, respectively. Calcium, magnesium, and zinc ions have positive effect on exoglucanase activity.     

Gene Cloning,Expression, and Characterization of an Exo-inulinase from Paenibacillus polymyxa ZJ-9

An inulinase-producing strain, Paenibacillus polymyxa ZJ-9, was isolated from natural sources to produce R,R-2,3-butanediol via one-step fermentation of raw inulin extracted from Jerusalem artichoke tubers. The inulinase gene from P. polymyxa ZJ-9 was cloned and overexpressed in Escherichia coli BL21 (DE3), and the purified recombinant inulinase was estimated to be approximately 56 kDa by both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and gel filtration chromatography. This result suggests that the active form of the inulinase is probably a monomer. Terminal hydrolysis fructose units from the inulin indicate that enzymes are exo-inulinase. The purified recombinant enzyme showed maximum activity at 25 °C and pH 6.0, which indicate its extreme suitability for industrial applications. Zn²⁺, Fe²⁺, and Mg²⁺ stimulated the activity of the purified enzyme, whereas Co²⁺, Cu²⁺, and Ni²⁺ inhibited enzyme activity. The K _m and V _max values for inulin hydrolysis were 1.72 mM and 21.69 μmol min^?1 mg^?1 protein, respectively. The same parameters toward sucrose were 41.09 mM and 78.7 μmol min^?1 mg^?1 protein, respectively. Considering its substrate specificity and other enzymatic characteristics, we believe that this inulinase gene from P. polymyxa ZJ-9 could be transformed into other special bacterial strains to allow inulin conversion to other biochemicals and bioenergy through one-step fermentation.     

Purification and properties of a SDS-resistant xylanase from halophilic Streptomonospora sp. YIM 90494

An extracellular xylanase from halophilic Streptomonospora sp. YIM 90494 was purified to homogeneity from a fermentation broth by ammonium sulphate precipitation, gel filtration chromatography and ion exchange chromatography. The purified xylanase appeared as a single protein band on SDS-PAGE with a molecular mass of approximately 50 kDa. The xylanase had maximum activity at pH 7.5 and 55 °C. The enzyme was stable over a broad pH range (pH 4.0–10.0) and showed good thermal stability when being incubated at 60 °C for 2 h. Kinetic experiments indicated that the enzyme had K _m and V _max values of 19.24 mg/mL and 6.1 μmol/min/mg, respectively, using birch wood xylan as substrate. The inhibitory effects of various metal ions and chemical agents on the xylanase activity were investigated. It is greatly interesting to note that Ag⁺ ion and SDS, which strongly inhibited most xylanases reported previously increases the xylanase activity in this study. These characteristics suggest that the enzyme with new properties has considerable potential in industrial applications.     

Purification and Characterization of Hyaluronate Lyase from <Emphasis Type="Italic">Arthrobacter globiformis</Emphasis> A152

A hyaluronate lyase was obtained by cultivating Arthrobacter globiformis strain A152. The enzyme was purified to homogeneity from the supernatant by ammonium sulfate fractionation, Q Sepharose Fast Flow, and Sephadex G-100 chromatography. The purification resulted in a 32.78-fold increase in hyaluronate lyase activity with specific activity of 297.2 U/mg. The molecular weight of the enzyme determined by SDS-PAGE was approximately 73.7 kDa. Using hyaluronic acid (HA) as a substrate, the maximal reaction rate (V_max) and the Michaelis–Menten constant (K_m) of hyaluronate lyase were found to be 4.76 μmol/min/ml and 0.11 mg/ml, respectively. The optimum pH and temperature values for hyaluronate lyase activity were pH 6.0 and 42 °C, respectively. This enzyme was stable at pH 4–10, 5–7, and 5–7 at 4, 37, and 42 °C, respectively. Investigation about temperature effects on hyaluronate lyase displayed that it was stable at 30–37 °C and also showed high activity at 37 °C. The enzymatic activity was enhanced by Ca²⁺ and was strongly inhibited by Cu²⁺ and SDS. These properties suggested that the hyaluronate lyase in this study could bring promising prospects in medical and industry applications.     

Heavy Metal Removal from Synthetic Solutions with Magnetic Beads Under Magnetic Field

The aim of this study is to prepare magnetic beads which can be used for the removal of heavy metal ions from synthetic solutions. Magnetic poly(ethylene glycol dimethacrylate‐vinyl imidazole) [m‐poly(EGDMA‐VIM)] beads were produced by suspension polymerization in the presence of magnetite Fe₃O₄ nano‐powder. The specific surface area of the m‐poly(EGDMA‐VIM) beads was found to be 63.1 m²/g with a size range of 150–200 µm in diameter and the swelling ratio was 85%. The average Fe₃O₄ content of the resulting m‐poly(EGDMA‐VIM) beads was 12.4%. The maximum binding capacities of the m‐poly(EGDMA‐VIM) beads were 32.4 mg/g for Cu²⁺, 45.8 mg/g for Zn²⁺, 84.2 mg/g for Cd²⁺and 134.5 mg/g for Pb²⁺. The affinity order on mass basis is Pb²⁺>Cd²⁺>Zn²⁺>Cu²⁺. Equilibrium data agreed well with the Langmuir model. pH significantly affected the binding capacity of the magnetic beads. Binding of heavy metal ions from synthetic wastewater was also studied. The binding capacities were 26.2 mg/g for Cu²⁺, 33.7 mg/g for Zn²⁺, 54.7 mg/g for Cd²⁺ and 108.4 mg/g for Pb²⁺. The magnetic beads could be regenerated up to about 97% by treating with 0.1 M HNO₃. These features make m‐poly(EGDMA‐VIM) beads a potential candidate for support of heavy metal removal under magnetic field.     

Optimized solid phase extraction based on diethyldithiocarbamate-coated Fe3O4 magnetic nanoparticles followed by ICP-OES for determination of Cd(II) and Ni(II) in rice and water samples

In the present study, application of Fe₃O₄ magnetic nanoparticles (MNPs) coated with diethyldithiocarbamate as a solid-phase sorbent for extraction of trace amounts of cadmium (Cd²⁺) and nickel (Ni²⁺) ions by the aid of ultrasound was investigated. The analytes were determined by inductively coupled plasma-optical emission spectroscopy. Fe₃O₄ MNPs were prepared by solvothermal method and characterized with dynamic light scattering, scanning electron microscope and X-ray diffraction. Response surface methodology was used for optimization of the extraction process and modeling the data. The optimal conditions obtained were as follows: chelating agent, 1.2 g L^?1; pH, 6.13; sonication time, 13 min and Fe₃O₄ MNPs, 10.3 mg. The calibration curves were linear over the concentration range of 1–1,000 μg L^?1 for Cd²⁺ and 2.5–1,000 for Ni²⁺ with the determination coefficients (R ²) of 0.9997 and 0.9995, respectively. The limits of detection were 0.27 μg L^?1 for Cd²⁺ and 0.76 μg L^?1 for Ni²⁺. The relative standard deviations (n = 7, C = 200 μg L^?1) for determination of Cd²⁺ and Ni²⁺ were 2.0 and 2.7 %, respectively. The relative recoveries of the analytes from tap, river and lagoon waters and rice samples at the spiking level of 10 μg L^?1 were obtained in the range of 95–105 %.     

Biochemical Characterization and Antitumor Study of l-Glutaminase from Bacillus cereus MTCC 1305

l-Glutaminase (E.C.3.5.2.1) extracellularly produced by Bacillus cereus MTCC 1305 was purified to apparent homogeneity with a fine band. The molecular weight of native enzyme and its subunit were found to be approximately 140 and 35 kDa, respectively, which indicates its homotetrameric nature. The substrate specificity test of this enzyme showed its specificity for l-glutamine. The purified enzyme showed maximum activity at optimum pH 7.5 and temperature 35 °C. The enzyme retained stability up to 50 and 20 % even after treatment at 50 and 55 °C, respectively, for 30 min. Monovalent cations (Na⁺, K⁺) and phosphate ion activated the enzyme activity, while divalent cations (Mg²⁺, Mn²⁺, Zn²⁺, Pb²⁺, Ca²⁺, Co²⁺, Hg²⁺, Cd²⁺, Cu²⁺) inhibited its activity. Reducing agents (cysteine, glutathione, dithiothreitol, l-ascorbic acid, and β-mercaptoethanol) stimulated its activity, whereas thiol-binding agents (iodoacetamide, p-chloromercuribenzoic acid) resulted in the inhibition of this enzyme. Kinetic parameters, K _m, V _max, K _cat, of purified enzyme were found to be 6.25 mM, 100 μmol/min/mg protein and 2.22?×?10² M^?1s^?1, respectively. The gradual inhibition in growth of hepatocellular carcinoma (Hep-G2) cell lines was found with IC₅₀ value of 82.27 μg/ml in the presence of different doses of l-glutaminase (10–100 μg/ml).     

Optimization of operating parameters and rate of uranium bioleaching from a low-grade ore

In this study the bioleaching of a low-grade uranium ore containing 480 ppm uranium has been reported. The studies involved extraction of uranium using Acidithiobacillus ferrooxidans derived from the uranium mine samples. The maximum specific growth rate (µ ^max) and doubling time (t _d) were obtained 0.08 h^?1 and 8.66 h, respectively. Parameters such as Fe²⁺ concentration, particle size, temperature and pH were optimized. The effect of pulp density (PD) was also studied. Maximum uranium bio-dissolution of 100 ± 5 % was achieved under the conditions of pH 2.0, 5 % PD and 35 °C in 48 h with the particles of d ₈₀ = 100 μm. The optimum concentration of supplementary Fe²⁺ was dependent to the PD. This value was 0 and 10 g of FeSO₄·7H₂O/l at the PD of 5 and 15 %, respectively. The effects of time, pH and PD on the bioleaching process were studied using central composite design. New rate equation was improved for the uranium leaching rate. The rate of leaching is controlled with the concentrations of ferric and ferrous ions in solution. This study shows that uranium bioleaching may be an important process for the Saghand U mine at Yazd (Iran).