Influence of some trace metals and stimulants on citric acid production from brewery wastes by Aspergillus niger

The addition of increasing levels of Mn²⁺, Fe³⁺, Zn²⁺, Co²⁺, Cu²⁺, Ca²⁺, sodium monofluoracetate and methanol during citric acid surface fermentation of spent grain liquor by Aspergillus niger (ATCC 9142) was investigated. For spent grain liquor the addition of 51 ppb Mn²⁺, 5 ppb Fe³⁺, 75 ppb Zn²⁺ and 4% (v/va) methanol caused a 4.9, 1.9, 10.9 and 16.8% increase in citric acid yield respectively. In all other fermentations the yield of citric acid was decreased whereas the biomass production in some cases was increased.     

Novel synthesis of mannosamine conjugated magnetic nanoparticles for purification and stabilization of human lysosomal β-mannosidase

Human β-mannosidase (MANB) was purified to homogeneity directly from lysosomes by using mannosamine conjugated magnetic (Fe₃O₄) nanoparticles, DE-52 cellulose, and sephadex G-200 chromatography. Fe₃O₄ nanoparticles were synthesized and utilized ammonia to attach the amino group on the nanoparticles. The particles were covalently attached with D-mannosamine by cross linker glutaraldehyde and confirmed by FTIR spectroscopy. In FTIR analysis, the peaks appeared at 2,356.6 cm⁻¹ for −N = CH linkage and at 3,378.4 cm⁻¹, 3,664.9 cm⁻¹ for −OH groups confirmed the conjugation of D-mannosamine with Fe₃O₄ nanoparticles. Results showed a single band of 97 kDa of purified MANB in SDS-PAGE. The isoelectric point was 4.5 and the K_m and Vmax values were 2.51 mM and 0.315 μM/min/mg, respectively. The purification fold was 329 with 68% yield. The optimal activity was at pH 5.0 and 75% activity was stable in 20% glycerol at 4°C. The enzyme activity was inhibited by Ni²⁺, Zn²⁺, Cd²⁺, Cu²⁺, Mo²⁺, Ag⁺¹, iodoacetate, SDS, DMF, DMSO, ethanol, and acetone; slightly reduced by Pb²⁺, Co²⁺, EDTA, DTT, and β-mercaptoethanol. The activity was not affected by Mg²⁺, Mn²⁺, Sn²⁺, Ca²⁺, Fe³⁺, PMSF, Triton X-100, D-mannosamine, D-mannose, D-mannitol, D-glucose, and D-fructose. The homogeneity of MANB enzyme was further confirmed by 2D-PAGE and immunoblot. This is the first novel report of conjugation of D-mannosamine with Fe₃O₄ nanoparticles for purification of human MANB enzyme.     

Photoluminescence and thermoluminescence properties of pure and rare-earth-doped ZrO2 prepared by Pechini-type sol–gel

In this article, photoluminescence (PL) and thermoluminescence (TL) properties of ZrO₂, ZrO₂:Dy³⁺, ZrO₂:Dy³⁺–Gd³⁺, ZrO₂:Dy³⁺–Yb³⁺, ZrO₂:Dy³⁺–Er³⁺, and ZrO₂:Dy³⁺–Sm³⁺ phosphors synthesized by the Pechini method were investigated. The crystal structure, thermal properties, morphology, PL and TL properties were investigated using X-ray powder diffraction (XRD), differential thermal analysis/thermogravimetric analysis (DTA/TGA), scanning electron microscopy (SEM), PL and TL, respectively. The room temperature emission bands corresponding to ⁴F_9/2 → ⁶H_J (J = 9/2, 11/2, 13/2 and 15/2) transitions of Dy³⁺ ions were measured. The phosphors were analysed using T_m–T_STOP, variable dose, and computerized glow curve fitting methods. Reusability, dose–response, and fading characteristics were investigated. The phosphors have a natural TL emission that vanished by heating treatment. Moreover, new peaks with similar properties to the natural emissions were observed after high-dose irradiation and long-term fading experiments. The glow curves of the phosphors have 13 individual peaks and many low- and high-temperature satellite peaks. The origin of the peaks is ZrO₂ host material and doping with rare-earth ions (Gd³⁺, Dy³⁺, Yb³⁺, Er³⁺ and Sm³⁺) does not lead to a new glow peak. The dopants cause drastic changes in individual peak intensities of ZrO₂.The initial fading rates of all the phosphors are relatively fast, but they slow down as time goes on.     

Purification and characterization of the α-glucosidase produced by thermophilic fungus <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> CBMAI 756

An α-glucosidase enzyme produced by the fungus Thermoascus aurantiacus CBMAI 756 was purified by ultra filtration, ammonium sulphate precipitation, and chromatography using Q Sepharose, Sephacryl S-200, and Superose 12 columns. The apparent molecular mass of the enzyme was 83 kDa as determined in gel electrophoresis. Maximum activity was observed at pH 4.5 at 70°C. Enzyme showed stability stable in the pH range of 3.0–9.0 and lost 40% of its initial activity at the temperatures of 40, 50, and 60°C. In the presence of ions Na⁺, Ba²⁺, Co²⁺, Ni²⁺, Mg²⁺, Mn²⁺, Al³⁺, Zn²⁺, Ca²⁺ this enzyme maintained 90–105% of its maximum activity and was inhibited by Cr³⁺, Ag⁺, and Hg²⁺. The enzyme showed a transglycosylation property, by the release of oligosaccharides after 3 h of incubation with maltose, and specificity for short maltooligosaccharides and α-PNPG. The K_m measured for the α-glucosidase was 0.07 μM, with a V_max of 318.0 μmol/min/mg.     

Purification and characterization of extracellular inulinase from a marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> and inulin hydrolysis by the purified inulinase

The extracellular inulinase of the marine yeast Pichia guilliermondii strain 1 was purified to homogeneity resulting in a 7.2-fold increase in specific inulinase activity. The molecular mass of the purified enzyme was estimated to be 50.0 kDa. The optimal pH and temperature for the purified enzyme were 6.0 and 60°C, respectively. The enzyme was activated by Mn²⁺, Ca²⁺, K⁺, Li⁺, Na⁺, Fe³⁺, Fe²⁺, Cu²⁺, and Co²⁺, but Mg²⁺, Hg²⁺, and Ag⁺ inhibited activity. The enzyme was strongly inhibited by phenylmethanesulphonyl fluoride (PMSF), iodoacetic acid, EDTA, and 1, 10-phenanthroline. The K _m and V _max values of the purified inulinase for inulin were 21.1 mg/mL and 0.08 mg/min, respectively. A large number of monosaccharides were detected after the hydrolysis of inulin. The deduced protein sequence from the cloned P. guilliermondii strain 1 inulinase gene contained the consensus motifs R-D-P-K-V-F-W-H and W-M-N-D-P-N-G, which are conserved among the inulinases from other microorganisms.     

Antimicrobial and DNA-Cleavage Studies of 22-Membered N4 Tetraaza Macrocyclic Triazoles: Template Synthesis and Physicochemical Characterization

A novel series of 22-membered macrocyclic complexes of the type [MLCl₂] (M?Co²⁺, Ni²⁺ and Cu²⁺) have been synthesized with newly derived biologically active ligands (L¹–L^IV). These ligands were synthesized by the condensation of ortho-phthalaldehyde and bis-(4-amino-5-mercapto-1, 2, 4-triazole-3-yl)alkanes. The mode of bonding and overall geometry of the complexes have been inferred through IR, EPR, electronic spectral studies, conductivity, magnetic, thermal, and electrochemical studies. All these complexes have been screened for their antibacterial (Escherichia coli, Staphylococus aureus, Salmonella typhi, Pseudomonas aeruginosa) and antifungal activities (Aspergillus niger, Aspergillus flavus, and Cladosporium) by the minimal inhibitory concentration (MIC) method. The DNA cleavage study was done by agarose gel electrophoresis technique.     

Redox-active complexes formed during the interaction between glutathione and mercury and/or copper ions

Prompted by the recently reported capacity of the physiologically occurring Cu(I)-[glutathione]₂ complex (Cu(I)-[GSH)]₂) to reduce oxygen, the effect of various GSH-binding metals (Co²⁺, Cd²⁺, Zn²⁺, Pb²⁺, Al³⁺, Hg²⁺ and Ni²⁺) on the superoxide-generating capacity of such complex was investigated. Amongst all tested metals, only Hg²⁺ was able to substantially affect the capacity of Cu(I)-[GSH]₂ to generate superoxide. When Hg²⁺ and Cu(I)-[GSH]₂ were mixed equimolarly, the superoxide formation, assessed through the cytochrome c reduction and dihydroethidium oxidation, was increased by over 50%. Such effect was totally inhibitable by SOD. Based on the reportedly higher affinity of Hg²⁺ for GSH and the observed ability of Hg²⁺ to lower the concentration of Cu(I)-[GSH]₂ (spectroscopically assessed), we suggest that Hg²⁺ displaces Cu(I) from Cu(I)-[GSH]₂, to release Cu(I) ions and form a Hg(II)-[GSH]₂ complex. The latter species would account for the Hg²⁺-induced exacerbation of the superoxide production. In fact, the present study provides first time evidence that a preformed Hg(II)-[GSH]₂ complex is able to concentration-dependently reduce oxygen. Such redox-activity was evidenced using cytochrome c and confirmed by EPR studies using DMPO (5,5-dimethyl-1-pyrroline N-oxide, a spin-trapping agent). Considering this novel ability of Hg(II)-[GSH]₂ to generate superoxide, a further characterization of its redox-activity and its potential to affect superoxide-susceptible biological targets appears warranted.     

Persistent luminescence of CaMgSi2O6:Eu2+,Dy3+ and CaMgSi2O6:Eu2+,Ce3+ phosphors prepared using the solid‐state reaction method

CaMgSi₂O₆:Eu²⁺,Dy³⁺ and CaMgSi₂O₆:Eu²⁺,Ce³⁺ phosphors were synthesized using the solid‐state reaction method. X‐Ray diffraction (XRD) and photoluminescence (PL) analyses were used to characterize the phosphors. The XRD results revealed that the synthesized CaMgSi₂O₆:Eu²⁺,Dy³⁺ and CaMgSi₂O₆:Eu²⁺,Ce³⁺ phosphors were crystalline and are assigned to the monoclinic structure with a space group C2/c. The calculated crystal sizes of CaMgSi₂O₆:Eu²⁺,Dy³⁺ and CaMgSi₂O₆:Eu²⁺,Ce³⁺ phosphors with a main (221) diffraction peak were 44.87 and 53.51 nm, respectively. Energy‐dispersive X‐ray spectroscopy (EDX) confirmed the proper preparation of the sample. The PL emission spectra of CaMgSi₂O₆:Eu²⁺,Dy³⁺ and CaMgSi₂O₆:Eu²⁺,Ce³⁺ phosphors have a broad band peak at 444.5 and 466 nm, respectively, which is due to electronic transition from 4f⁶5d¹ to 4f⁷. The afterglow results indicate that the CaMgSi₂O₆:Eu²⁺,Dy³⁺ phosphor has better persistence luminescence than the CaMgSi₂O₆:Eu²⁺,Ce³⁺ phosphor. Copyright © 2015 John Wiley & Sons, Ltd.     

Extraction,purification, and biochemical characterization of serine protease from leaves of Abrus precatorius

A protease from fresh leaves of Abrus precatorius was purified using two classical chromatography techniques: ion-exchange (DEAE-Sepharose) and Gel filtration (Sephadex G-75). The purified protease showed a molecular weight of ～?28?kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH and temperature for the purified protease was 8 and 40°C, respectively. The purified protease was stable throughout a wide temperature range from 10 to 80°C and pH from 2 to 12. Protease activity was inhibited in the presence of Co²⁺, Ni²⁺, Hg²⁺, and Zn²⁺ while its activity has increased in the presence of Ca²⁺ and Mg²⁺. The protease was highly specific to casein when compared to its specificity for gelatin, bovine serum albumin, hemoglobin, and defatted flour of Ricinodendron heudelotii. Its V_max and K_m determined using casein as a substrate were 94.34?U/mL and 349.07?µg/mL respectively. Inhibition studies showed that this purified protease was inhibited by both phenylmethane sulfonyl fluoride and aprotinin which are recognized as competitive inhibitors of serine proteases.     

Complexation of metal ions by pseudotripeptides with different functionalized N-alkyl residues

Seven pseudotripeptides with the common structure Bz-His-[CO-N(CH₂)n-X]Gly-His-NH₂ were synthesized on the solid phase using the Fmoc-strategy, trityl protection for both His residues and Boc- or -OBu^t-protection for N-aminoalkyl- and N-carboxyalkyl residues, respectively. Functionalized N-alkyl glycyl peptides were formed on the solid phase by amination of a bromoacetyl dipeptide. All seven pseudotripeptides are able to form chelate complexes with the metal ions Zn²⁺, Ni²⁺, Cu²⁺ and Co²⁺. The existence of monomeric 1:1 complexes for these pseudopeptides was calculated from the MW estimated by MALDI-MS and from the isotope distribution pattern estimated by ESI.     

Synthesis and photoluminescence enhancement of the LiLa(MoO4)2:Sm3+ red phosphors by co-doping with Bi3+

The LiLa(MoO₄)₂:Sm³⁺ and LiLa(MoO₄)₂:Sm³⁺,Bi³⁺ phosphors were prepared using the citric-acid-fueled combustion method and the influence of concentrations of Bi³⁺ dopant on LiLa(MoO₄)₂:Sm³⁺ red luminescence was investigated. The LiLa(MoO₄)₂:Sm³⁺ and LiLa(MoO₄)₂:Sm³⁺,Bi³⁺ samples matched well with the scheelite structure and I4₁/a space group and did not detect structural changes. Under an excitation of 403 nm, the prepared LiLa(MoO₄)₂:Sm³⁺,Bi³⁺ phosphor was excited and produced orange-red emission. When compared with the LiLa(MoO₄)₂:Sm³⁺ phosphor, the LiLa(MoO₄)₂:Sm³⁺,Bi³⁺ phosphor exhibited enhanced fluorescence intensity because the Bi³⁺ dopant ions are doped as a sensitizer. The optimal doping concentrations of Sm³⁺ and Bi³⁺ were 5 and 1 mol%, respectively. Furthermore, the energy transfer from Bi³⁺ to Sm³⁺ is effective (³P₁ → ⁴K_11/2). Subsequently, the electrons in an unstable excited state were transferred to a stable ground state (⁴G_5/2 → ⁶H_5/2, ⁶H_7/2, ⁶H_9/2). The Commission Internationale de L'Eclairage (CIE) chromaticity coordinates of the optimized LiLa(MoO₄)₂:Sm³⁺,Bi³⁺ phosphor were situated in the orange-red region. The luminescence of the LiLa(MoO₄)₂:Sm³⁺,Bi³⁺ phosphor generated under near-ultraviolet (UV) irradiation could be used to produce a warm white light, indicating its possible applications in white light-emitting diodes.     

Purification and properties of oxaloacetate decarboxylase fromCorynebacterium glutamicum

Oxaloacetate (OAA) decarboxylase (E.C. 4.1.1.3) was isolated fromCorynebacterium glutamicum. In five steps the enzyme was purified 300-fold to apparent homogeneity. The molecular mass estimated by gel filtration was 118 ± 6 kDa. SDS-PAGE showed a single subunit of 31.7 KDa, indicating an ₄ subunit structure for the native enzyme. The enzyme catalyzed the decarboxylation of OAA to pyruvate and CO₂, but no other -ketoacids were used as substrate. The cation Mn²⁺ was required for full activity, but could be substituted by Mg²⁺, Co²⁺, Ni²⁺ and Ca²⁺. Monovalent ions like Na⁺, K⁺ or NH ₄ ⁺ were not required for activity. The enzyme was inhibited by Cu²⁺, Zn²⁺, ADP, coenzyme A and succinate. Avidin did not inhibit the enzyme activity, indicating that biotin is not involved in decarboxylation of OAA. Analysis of the kinetic properties revealed a K _m for OAA of 2.1 mM and a K _m of 1.2 mM for Mn²⁺. The V _max was 158 µmol of OAA converted per min per mg of protein, which corresponds to an apparent k _cat of 311 s^–1.Abbreviations OAA oxaloacetate - LDH lactate dehydrogenase     

Purification of glucose‐6‐phosphate dehydrogenase from rat (Rattus norvegicus) erythrocytes and inhibition effects of some metal ions on enzyme activity

Glucose‐6‐phosphate dehydrogenase (G6PD) is the first enzyme on which the pentose phosphate pathway was checked. In this study, purification of a G6PD enzyme was carried out by using rat erythrocytes with a specific activity of 13.7 EU/mg and a yield of 67.7 and 155.6‐fold by using 2′,5′‐ADP Sepharose‐4B affinity column chromatography. For the purpose of identifying the purity of enzyme and molecular mass of the subunit, a sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was carried out. The molecular mass of subunit was calculated 56.5 kDa approximately. Then, an investigation was carried out regarding the inhibitory effects caused by various metal ions (Fe²⁺, Pb²⁺, Cd²⁺, Ag⁺, and Zn²⁺) on G6PD enzyme activities, as per Beutler method at 340 nm under in vitro conditions. Lineweaver–Burk diagrams were used for estimation of the IC₅₀ and K_i values for the metals. K_i values for Pb⁺², Cd⁺², Ag⁺, and Zn⁺² were 113.3, 215.2, 19.4, and 474.7 μM, respectively.     

The high thermal stability of white emitting phosphor Mg2Y2Al2Si2O12:Dy3+,Eu3+,La3+/Lu3+ for white light emitting diodes

A series of Mg₂Y₂Al₂Si₂O₁₂:Dy³⁺,Eu³⁺ was prepared using a solid-state method, and the phosphor emitted white light by tuning the ratio of Dy³⁺/Eu³⁺. The effects of La³⁺/Lu³⁺ on the structure and luminescence properties of Mg₂Y₂Al₂Si₂O₁₂:Dy³⁺,Eu³⁺ were explored. Under the influence of bond length and twist, the luminescence intensity of the materials increased first and then decreased under excitation with ultraviolet light. The lattice distortion of the trivalent cation La³⁺-substituted Mg₂Y₂Al₂Si₂O₁₂:Dy³⁺ and Eu³⁺ phosphors was reduced, the symmetry of polyhedron occupied by the luminescence centre improved, and the thermal stability of the luminescence centre improved to a certain extent. White light emitting diodes (LEDs) were fabricated by combining a 370 nm LED chip and the Mg₂Y₂Al₂Si₂O₁₂:Dy³⁺,Eu³⁺,La³⁺ (Mg₂Y₂Al₂Si₂O₁₂:Dy³⁺,Eu³⁺,Lu³⁺) phosphor. The results showed that Mg₂Y₂Al₂Si₂O₁₂:Dy³⁺,Eu³⁺,La³⁺/Lu³⁺ may have potential application in the area of white LEDs.     

Spatial and temporal variation of soil solution chemistry and ion fluxes through the soil in a mature Norway Spruce (Picea abies (L.) Karst.) stand

In this study we investigated the spatial and temporal variation in soil solution chemistry and of water and ion fluxes through the soil in a forest ecosystem. Our aim was to evaluate the relevance of these variations for the accuracy of average areal soil solution concentrations and ion fluxes with seepage at 90 cm depth.Twenty spatially distinct subcompartments of approximately 1 m² were established within a mature stand of Norway spruce and ceramic suction lysimeters were installed at depths of 20, 35 and 90 cm. A tensiometer was placed close to each suction lysimeter, and one throughfall sampler was established for each subcompartment.Soil solution samples were analysed for major ions (H⁺, Na⁺, K⁺, Ca²⁺, Mg²⁺, Mn²⁺, Fe³⁺, Al³⁺, Cl^-, NO ₃ ^- , SO ₄ ^2- . We calculated water fluxes for each subcompartment separately by a numeric simulation of the soil water flux close to the lysimeters. The ion fluxes at each lysimeter were calculated by multiplying the simulated water fluxes with the ion concentrations on a fortnightly base. Averaging these 20 independent ion fluxes gave the areal average flux and an estimate of its statistical accuracy. The spatial variation of ion concentrations in the soil solution was high with coefficients of variance ranging from 5% to 128%. Part of the spatial variation was related to stem distance. Temporal variation of the concentrations was less than spatial for most ions. The spatial variation of water and ion fluxes with seepage was also substantial; for example the fluxes of SO ₄ ^2- -S calculated for each subcompartment ranged from 21 to 119 kg ha^-1 yr^-1, with an arithmetic average of 47 kg ha^-1 yr^-1. For H₂O, Mg²⁺, Cl^-, and SO ₄ ^2- , the spatial heterogeneity of seepage fluxes was largely explained by the heterogeneity of throughfall fluxes. No such relationship was found for nitrogen.Despite using 20 replicates, the 95% confidence intervals of the average annual areal fluxes with seepage were found to be 20–30% for most ions.     

兔肌3-磷酸甘油脱氢酶的提纯及性质研究

目的:研究兔肌3-磷酸甘油脱氢酶的分离纯化方法及其酶学性质,为测定血清甘油三酯所用酶联试剂的开发提供试验基础和理论依据。方法:通过硫酸铵分级沉淀、DEAE-Sepharose、Blue-Sepharose和羟磷灰石纯化兔肌3-磷酸甘油脱氢酶,利用凝胶过滤和梯度PAGE(5%～15%)法测定酶分子量,采用常规酶学动力学分析方法,考察pH、温度、底物浓度以及部分金属离子与有机化合物对酶促反应的影响。结果 纯化后的兔肌3-磷酸甘油脱氢酶经PAGE(12%)分析为单一条带;酶分子量为115～122 kDa;酶最适温度45℃,最适pH 9;酸碱稳定范围pH6～9,低于45℃时热稳定性好;最适条件下,以3-磷酸甘油和NAD⁺为底物,测得酶的K_m分别为7.4×10^-3mol/L和1.47×10^-4mol/L;Ba²⁺、Mn²⁺、Fe²⁺、Al³⁺、Cu²⁺、Ni²⁺、Ag⁺、Hg²⁺、NaN₃、EDTA对酶有不同程度的抑制作用,Mg²⁺、Ca²⁺、Co²⁺、Zn²⁺有一定程度的激活作用,其中Co²⁺和Zn²⁺对酶的激活作用能达到200%以上,有机化合物NaF对酶的活性没有影响。     

Purification and characterization of a novel plant-type carbonic anhydrase from Bacillus subtilis

Carbonic anhydrase enzyme, one of the fastest known enzymes, remains largely unexplored in prokaryotes when compared to its mammalian counterparts despite its ubiquity. In this study, the enzyme has been purified from Bacillus subtilis SA3 using sequential Sephadex G-75 chromatography, DEAE cellulose chromatography, and sepharose-4B-L-tyrosinesulphanilamide affinity chromatography and characterized to provide additional insights into its properties. The apparent molecular mass of carbonic anhydrase obtained by SDS-PAGE was found to be approximately 37 kDa. Isoelectric focusing of the purified enzyme revealed an isoelectric point (pI) of around 6.1 when compared with marker. The presence of metal ions such as Zn²⁺, Co²⁺, Cu²⁺, Fe³⁺, Mg²⁺, and anion SO₄⁻ increased enzyme activity while strong inhibition was observed in the presence of Hg²⁺, Cl⁻, HCO₃⁻, and metal chelator EDTA. The optimum pH and temperature for the enzyme were found to be 8.3 and 37°C, respectively. Enzyme kinetics with p-nitrophenyl acetate as substrate at pH 8.3 and 37°C determined the V_max and K_m values of the enzyme to be 714.28 μmol/mg protein/min and 9.09 mM, respectively. The K_i value for acetazolamide was 0.22 mM, compared to 0.099 mM for sulphanilamide. The results from N-terminal amino acid sequencing imply the purified protein is a putative beta-carbonic anhydrase with close similarities to CAs from plants, microorganisms.     

Substrate Profile and Metal-ion Selectivity of Human Divalent Metal-ion Transporter-1

Divalent metal-ion transporter-1 (DMT1) is a H⁺-coupled metal-ion transporter that plays essential roles in iron homeostasis. DMT1 exhibits reactivity (based on evoked currents) with a broad range of metal ions; however, direct measurement of transport is lacking for many of its potential substrates. We performed a comprehensive substrate-profile analysis for human DMT1 expressed in RNA-injected Xenopus oocytes by using radiotracer assays and the continuous measurement of transport by fluorescence with the metal-sensitive PhenGreen SK fluorophore. We provide validation for the use of PhenGreen SK fluorescence quenching as a reporter of cellular metal-ion uptake. We determined metal-ion selectivity under fixed conditions using the voltage clamp. Radiotracer and continuous measurement of transport by fluorescence assays revealed that DMT1 mediates the transport of several metal ions that were ranked in selectivity by using the ratio I_max/K_0.5 (determined from evoked currents at −70 mV): Cd²⁺ > Fe²⁺ > Co²⁺, Mn²⁺ ≫ Zn²⁺, Ni²⁺, VO²⁺. DMT1 expression did not stimulate the transport of Cr²⁺, Cr³⁺, Cu⁺, Cu²⁺, Fe³⁺, Ga³⁺, Hg²⁺, or VO⁺. ⁵⁵Fe²⁺ transport was competitively inhibited by Co²⁺ and Mn²⁺. Zn²⁺ only weakly inhibited ⁵⁵Fe²⁺ transport. Our data reveal that DMT1 selects Fe²⁺ over its other physiological substrates and provides a basis for predicting the contribution of DMT1 to intestinal, nasal, and pulmonary absorption of metal ions and their cellular uptake in other tissues. Whereas DMT1 is a likely route of entry for the toxic heavy metal cadmium, and may serve the metabolism of cobalt, manganese, and vanadium, we predict that DMT1 should contribute little if at all to the absorption or uptake of zinc. The conclusion in previous reports that copper is a substrate of DMT1 is not supported.     

IMMOBILIZATION OF CARBONIC ANHYDRASE ENZYME PURIFIED FROM Bacillus subtilis VSG-4 AND ITS APPLICATION AS CO2 SEQUESTERER

The purification, immobilization, and characterization of carbonic anhydrase (CA) secreted by Bacillus subtilis VSG-4 isolated from tropical soil have been investigated in this work. Carbonic anhydrase was purified using ammonium sulfate precipitation, Sephadex-G-75 column chromatography, and DEAE-cellulose chromatography, achieving a 24.6-fold purification. The apparent molecular mass of purified CA obtained by SDS-PAGE was found to be 37 kD. The purified CA was entrapped within a chitosan–alginate polyelectrolyte complex (C-A PEC) hydrogel for potential use as an immobilized enzyme. The optimum pH and temperature for both free and immobilized enzymes were 8.2 and 37°C, respectively. The immobilized enzyme had a much higher storage stability than the free enzyme. Certain metal ions, namely, Co²⁺, Cu²⁺, and Fe³⁺, increased the enzyme activity, whereas CA activity was inhibited by Pb²⁺, Hg²⁺, ethylenediamine tetraacetic acid (EDTA), 5,5′-dithiobis-(2-nitrobenzoic acid (DTNB), and acetazolamide. Free and immobilized CAs were tested further for the targeted application of the carbonation reaction to convert CO₂ to CaCO₃. The maximum CO₂ sequestration potential was achieved with immobilized CA (480 mg CaCO₃/mg protein). These properties suggest that immobilized VSG-4 carbonic anhydrase has the potential to be used for biomimetic CO₂ sequestration.     

Beneficial Effects of Nickel on Pseudomonas saccharophila under Nitrogen-Limited Chemolithotrophic Conditions

Addition of nickel stimulated growth and nitrogenase activity of Pseudomonas saccharophila under nitrogen-limited chemolithotrophic conditions, apparently because of a significant increase in expression of uptake hydrogenase activity. Inhibition of hydrogenase expression by 50 μM EDTA was relieved by nickel over a wide concentration range (1 to 200 μM). Co²⁺, Zn²⁺, Mn²⁺, and Cu²⁺ stimulated expression of hydrogenase activity, but to a much lesser degree than nickel, and Fe²⁺, Mg²⁺, SeO₄²⁻, and SeO₃²⁻ did not increase expression. Nickel in individual combination with Mg²⁺, Fe²⁺, SeO₃²⁻, and SeO₄²⁻ resulted in activities that were essentially the same as that with nickel alone. Hydrogenase synthesis required the presence of nickel, and repression by O₂ was alleviated by increasing the concentration of added nickel. Cells placed under hydrogenase derepression conditions showed progressive incorporation of radioactive nickel to a much greater extent than did cells which were not derepressed.