Heat-induced, metal-catalyzed oxidative degradation of quercetin and rutin (Quercetin 3-O-rhamnosylglucoside) in aqueous model systems

The oxidative degradation of quercetin and rutin in phosphate buffer solutions, pH 8.0, at 97 degrees C, was studied by means of UV-vis spectroscopy and reversed-phase high-performance liquid chromatography (HPLC). The effect of the transition metal ions Fe(2+) and Cu(2+) on degradation rate and browning development was also assessed. It was shown that both flavonols are very labile to thermally induced degradation under oxidative conditions. Fe(2+) and Cu(2+) caused an increase in the degradation rate, as well as an increase in browning (A(420)). Significant differences were observed in the degradation mechanisms, as implied by HPLC analyses. It is postulated that metal ions promote flavonol oxidation through reactive oxygen species formation, whereas increases in browning could be ascribed to oxidation and metal-polyphenol interactions.     

Thermal stabilities of lupin seed conglutin gamma protomers and tetramers

Various experimental approaches have been used in this work to assess the thermal stabilities of lupin seed conglutin gamma at two pH values, 4.5 and 7.5, at which the protein exists as a protomer and a tetramer, respectively. The patterns of thermal unfolding at the two pH values differed significantly; the tetramer aggregated and became insoluble, whereas the protomer was still soluble after thermal treatment. Also, the midpoint transition temperatures were dramatically different, being 60.3 and 75.1 degrees C for the protomer and tetramer, respectively. The behavior of conglutin gamma at neutral pH was also affected by disulfide formation/interchange, in that some unfolded protein molecules became covalently stabilized. More detailed analyses by differential scanning calorimetry and indirect fluorescence measurements, using 8-anilino-1-naphthalenesulfonic acid as a probe, confirmed the remarkable differences observed in the thermal stabilities of the two protein forms and allowed models for their unfolding patterns to be drawn.     

Calcium ions make phytohemagglutinin resistant to trypsin proteolysis

To investigate the mechanism of phytohemagglutinin (PHA) susceptibility or resistance to the action of proteolytic enzymes, its in vitro proteolysis by trypsin was studied. It was found that Ca (2+) gives resistance to the native PHA molecule to trypsin proteolysis. In the absence of Ca (2+) trypsin performs a thorough hydrolysis of PHA. At the first stage of trypsin hydrolysis of PHA the formation of a relatively stable high molecular mass product occurs (PHA-T) as a result of non-co-operative proteolysis. At the second stage, the degradation of PHA-T occurs, and this degradation is performed by parallel co-operative proteolysis. This type of proteolysis differs from the action of trypsin on phaseolin, the main storage protein from common bean ( Phaseolus vulgaris L.). The implications of Ca (2+)influence of PHA hydrolysis by trypsin are discussed.     

Computer simulation of the interactions of glyphosate with metal ions in phloem

Essential nutrients such as trace metal ions, amino acids, and sugars are transported in the phloem from leaves to other parts of the plant. The major chelating agents in phloem include nicotianamine, histidine, cysteine, glutamic acid, and citrate. A computer model for the speciation of metal ions in phloem has been used to assess the degree to which the widely used herbicide glyphosate binds to Fe(3+), Fe(2+), Cu(2+), Zn(2+), Mn(2+), Ca(2+), and Mg(2+) in this fluid over the pH range of 8 to 6.5. The calculations show that glyphosate is largely unable to compete effectively with the biological chelating agents in phloem. At a typical phloem pH of 8, 1.5 mM glyphosate binds 8.4% of the total Fe(3+), 3.4% of the total Mn(2+), and 2.3% of the total Mg(2+) but has almost no effect on the speciation of Ca(2+), Cu(2+), Zn(2+), and Fe(2+). As the pH decreases to 6.5, there are some major shifts of the metal ions among the biological chelators, but only modest increases in glyphosate binding to 6% for Fe(2+) and 2% for Zn(2+). The calculations also indicate that over 90% of the glyphosate in phloem is not bound to any metal ion and that none of the metal-glyphosate complexes exceed their solubility limits.     

辣根风味形成优化

对辣根硫代葡萄糖苷转化为风味成分异硫氰酸酯的最优条件及挥发成分的保留方法进行了研究。酶解产物的类型和浓度受pH值、酶解温度、时间、含水量及其它因子的影响。优化了酶解条件，探讨了金属离子、抗坏血酸及β-环糊精、变性淀粉、大豆蛋白对风味成分AIT（烯丙基异硫氰酸酯）和PEIT（苯乙基异硫氰酸酯）形成及保留的效果，比较了热风干燥和冷冻干燥对AIT和PEIT形成及保留的效果。研究结果表明：辣根风味形成的最适条件是：10 g辣根添加5 mL pH 6的缓冲液在温度30℃条件下酶解30 min。添加抗坏血酸、Mg²⁺、Zn²⁺或外源黑芥子硫苷酶能明显增加AIT和PEIT含量。加入β-环状糊精及采用冷冻干燥或流化床干燥有利于AIT和PEIT的保留。     

Changes in trout hemoglobin conformations and solubility after exposure to acid and alkali pH

The effect of different acid and alkali treatments followed by pH readjustment on solubility and conformation of trout hemoglobins was investigated. At low pH (1.5-3.5) hemoglobin was unfolded at faster rates as the pH was lowered. Inclusion of 500 mM NaCl at low pH significantly increased the rate of unfolding. At alkaline pH (10-12) the conformation of hemoglobin was much less affected than at acid pH, and the presence of salt had little additional effect. When hemoglobin solutions were adjusted to neutrality at different stages of unfolding, the recovery of native structure on refolding was proportional to the extent of unfolding prior to pH readjustment: the more unfolded the protein, the less was the recovery of native structure. The presence of salt led to a smaller recovery of native structure. The more improperly unfolded the hemoglobin was (and hydrophobic), the lower was its solubility. Results suggest that the presence of NaCl (25-500 mM) may not only interfere with the refolding process but also enhance the hydrophobic interactions of improperly refolded hemoglobin, possibly due to charge screening. These results show that proper control of unfolding and refolding time and ionic strength in processes using highly acidic or alkaline conditions can minimize loss of hemoglobin solubility.     

Highly effective adsorption of heavy metal ions from aqueous solutions by macroporous xylan-rich hemicelluloses-based hydrogel

Xylan-rich hemicelluloses-based hydrogel was developed as a novel porous bioadsorbent by graft co-polymerization of acrylic acid (AA) and xylan-rich hemicelluloses for adsorption of heavy metal ions (Pd(2+), Cd(2+), and Zn(2+)) from aqueous solutions. The chemical structure, the interaction between the hydrogel and metal ions, and the porous structure of xylan-rich hemicelluloses-g-AA hydrogel were revealed by Fourier transform infrared spectroscopy and scanning electron microscopy. The effects of AA and cross-linker dosage, pH value, contacting time, and initial concentration of metal ion on the adsorption capacity were studied. The adsorption equilibrium time was about 60 min from the adsorption kinetics study. The maximum adsorption capacities of Pd(2+), Cd(2+), and Zn(2+) were 859, 495, and 274 mg/g, respectively. Furthermore, xylan-rich hemicelluloses-g-AA hydrogel also exhibited highly efficient regeneration and metal ion recovery efficiency and can be reused without noticeable loss of adsorption capacity for Pd(2+), Cd(2+), and Zn(2+) after quite a number of repeated adsorption/desorption cycles.     

Cloning, expression, and characterization of a D-psicose 3-epimerase from Clostridium cellulolyticum H10

The noncharacterized protein ACL75304 encoded by the gene Ccel_0941 from Clostridium cellulolyticum H10 (ATCC 35319), previously proposed as the xylose isomerase domain protein TIM barrel, was cloned and expressed in Escherichia coli . The expressed enzyme was purified by nickel-affinity chromatography with electrophoretic homogeneity and then characterized as d-psicose 3-epimerase. The enzyme was strictly metal-dependent and showed a maximal activity in the presence of Co(2+). The optimum pH and temperature for enzyme activity were 55 °C and pH 8.0. The half-lives for the enzyme at 60 °C were 6.8 h and 10 min when incubated with and without Co(2+), respectively, suggesting that this enzyme was extremely thermostable in the presence of Co(2+) but readily inactivated without metal ion. The Michaelis-Menten constant (K(m)), turnover number (k(cat)), and catalytic efficiency (k(cat)/K(m)) values of the enzyme for substrate d-psicose were estimated to be 17.4 mM, 3243.4 min(-1), and 186.4 mM min(-1), respectively. The enzyme carried out the epimerization of d-fructose to d-psicose with a conversion yield of 32% under optimal conditions, suggesting that the enzyme is a potential d-psicose producer.     

Leucine aminopeptidase from red sea bream (Pagrus major) skeletal muscle: purification, characterization, cellular location, and tissue distribution

A leucine aminopeptidase was purified for the first time from marine fish red sea bream ( Pagrus major) skeletal muscle to homogeneity with 4850-fold and a yield of 7.4%. The purification procedure consisted of ammonium sulfate fractionation and chromatographies including DEAE-Sephacel, Sephacryl S-200, hydroxyapatite, and phenyl-Sepharose. The enzyme was approximately 96 kDa as estimated by SDS-PAGE and gel filtration and preferentially hydrolyzed substrate Leu-MCA. The enzymatic activity was optimal at 45 degrees C and pH 7.5. The K m and k cat values of the enzyme for Leu-MCA were 1.55 microM and 26.4 S (-1) at 37 degrees C, respectively. Activation energy ( E a) of the enzyme was 59.6 kJ M (-1). The enzyme was specifically inhibited by metal-chelating agents, and Zn (2+) and (or) Mn (2+) seemed to be its metal cofactor(s). In addition, bestatin strongly inhibited its activity, and K i was 1.44 microM. Using a highly specific polyclonal antibody, the location of enzyme was demonstrated intracellularly and distributed in different tissues.     

Proteomic analysis of lupin seed proteins to identify conglutin Beta as an allergen, Lup an 1

Lupin products may be valuable as human foods because of their high protein content and potential anticholesterolemic properties. However, a small percentage of the population is allergic to lupin. In this study, we use in vitro IgE binding and mass spectrometry to identify conglutin beta, a major storage protein, as an allergen in seeds of Lupinus angustifolius and Lupinus albus. Purification of conglutin beta from L. angustifolius flour confirmed that serum IgE binds to this protein. Where IgE in sera recognized lupin proteins on Western blots, it recognized conglutin beta, suggesting this protein is a major allergen for lupin. The L. angustifolius conglutin beta allergen has been designated Lup an 1 by the International Union of Immunological Societies (IUIS) allergen nomenclature subcommittee.     

Interactive Sorption of Metal Ions and Humic Acids onto Mineral Particles

The sorption of metal ions (Pb2+, Zn2+ and Cu2+) and soil humic acids (HA) from aqueous solutions onto mineral particles (sand, calcite and clay) was investigated using a batch equilibrium system. The sorption reactions in two- component systems (heavy metals-mineral particles and humic acids- mineral particles), as well as interactions in three-component system (heavy metals-humic acids-mineral particles) were examined. Results showed that the presence of humic acids, dissolved or bound onto mineral surfaces, considerably influenced the fixation of heavy metals. The various effects, depending on mineral type, humic concentration and specific metal-ion, were observed in three- component system. Sorption of Cu2+-ions on all minerals studied rapidly increased as the concentration of dissolved HA increased. The amount of Pb2+-ions sorbed on sand slightly decreased, while on kaolin increased between 15 and 20%. Sorption of Zn2+-ions on all minerals studied decreased at pH 4. At pH 5.5 the sorption of Zn2+-ions onto calcite decreased, while on kaolin and sand increased as a function of the humic acid concentration giving the curve with maximum at c(HA) = 2.5 mmol C L-1. At pH 6.5 sorption onto kaolin and sand increased. This effect occurs as a result of the conditional stability constant of Zn-HA complexes increasing at higher pH which in turn promotes the chelation of Zn2+-ions to mineral- bound humic substances. The enhanced sorption of metal ions from the aqueous phase in three-component systems is not only the result of mineral sorption of free metals but also the result of chelation with HA sorbed on the mineral surface.     

Zinc ion enhances GABA tea-mediated oxidative DNA damage

GABA tea is a tea product that contains a high level of γ-aminobutyric acid (GABA). Previous study has demonstrated a synergistic effect of GABA tea and copper ions on DNA breakage. This study further explored whether zinc (Zn), a nonredox metal, modulated DNA cleavage induced by GABA tea extract. In a cell-free system, Zn(2+) significantly enhanced GABA tea extract and (-)-epigallocatechin-3-gallate (EGCG)- or H(2)O(2)-induced DNA damage at 24 h of incubation. Additionally, low dosages of GABA tea extract (1-10 μg/mL) possessed pro-oxidant activity to increase H(2)O(2)/Zn(2+)-induced DNA cleavage in a dose-dependent profile. By use of various reactive oxygen scavengers, it was observed that glutathione, catalase, and potassium iodide effectively inhibited DNA degradation caused by the GABA tea extract/H(2)O(2)/Zn(2+) system. Moreover, the data showed that the GABA tea extract itself (0.5-5 mg/mL) could induce DNA cleavage in a long-term exposure (48 h). EGCG, but not the GABA tea extract, enhanced H(2)O(2)-induced DNA cleavage. In contrast, GABA decreased H(2)O(2)- and EGCG-induced DNA cleavage, suggesting that GABA might contribute the major effect on the antioxidant activity of GABA tea extract. Furthermore, a comet assay revealed that GABA tea extract (0.25 mg/mL) and GABA had antioxidant activity on H(2)O(2)-induced DNA breakage in human peripheral lymphocytes. Taken together, these findings indicate that GABA tea has the potential of both pro-oxidant and antioxidant. It is proposed that a balance between EGCG-induced pro-oxidation and GABA-mediated antioxidation may occur in a complex mixture of GABA tea extract.     

营养物质及金属离子对DLL-E4菌降解对硝基苯酚的影响

研究了酵母膏、葡萄糖、蛋白胨、土壤浸液及Ca2 、Mg2 、Fe2 、Fe3 、Al3 、Mn2 、Co2 、Zn2 、Li 、Cu 、Cu2 、Ba2 、Ni2 等 13种金属离子对甲基对硫磷降解菌DLL E4降解对硝基苯酚的影响。结果表明 :适量添加酵母膏、葡萄糖和蛋白胨都能有效提高菌株对对硝基苯酚的降解 ,土壤浸液没有影响 ;金属离子中 ,除Li 和 0 1mmolL-1Fe3 外均对DLL E4降解对硝基苯酚的性状有一定的影响 ,其中Ca2 、Mg2 、Mn2 影响不大 ,Fe3 、Fe2 、Al3 、Ba2 、Zn2 高浓度时影响较大 ,Co2 、Cu2 、Cu 、Ni2 对DLL E4降解对硝基苯酚有较大的影响     

Purification and characterization of ferulic acid esterase from malted finger millet (Eleusine coracana, Indaf-15)

Ferulic acid esterase (EC 3.1.1.73) cleaves the feruloyl groups substituted at the 5'-OH group of arabinosyl residues of arabinoxylans and is known to modulate their functional properties. In this study, ferulic acid esterase from 96 h finger millet malt was purified to apparent homogeneity by three-step purification with a recovery of 3% and a fold purification of 22. The substrate p-nitrophenylferulate (PNPF) was synthesized and used to assay this enzyme spectrophotometrically. The products liberated from ragi and wheat water-soluble polysaccharides by the action of purified ragi ferulic acid esterase were identified by ESI-MS. The pH and temperature optima of the enzyme were found to be 6.0 and 45 degrees C, respectively. The pH and temperature stabilities of the enzyme were found to be in the range of 5.5-9.0 and 30 degrees C, respectively. The activation energy of the enzymatic reaction was found to be 4.08 kJ mol(-1). The apparent K m and V max of the purified ferulic acid esterase for PNPF were 0.053 microM and 0.085 unit mL(-1), respectively. The enzyme is a monomer with a molecular mass of 16.5 kDa. Metal ions such as Ni(2+), Zn(2+), Co(2+), and Cu(2+) and oxalic and citric acids enhanced the enzyme activity. The enzyme was completely inhibited by Fe(3+). Group specific reagents such as p-chloromercuric benzoate and iodoacetamide inhibited the enzyme, indicating the possible presence of cysteine residues in the active site pocket.     

Purification and characterization of a novel extracellular tripeptidyl peptidase from Rhizopus oligosporus

A novel extracellular tripeptidyl peptidase (TPP) was homogenously purified from the culture supernatant of Rhizopus oligosporus by sequential fast protein liquid chromatography. The purified enzyme was a 136.5 kDa dimer composed of identical subunits. The effects of inhibitors and metal ions indicated that TPP is a metallo- and serine protease. TPP was activated by divalent cations, such as Co(2+) and Mn(2+), and completely inhibited by Cu(2+). Enzyme activity was optimal at pH 7.0 and 45 °C with a specific activity of 281.9 units/mg for the substrate Ala-Ala-Phe-pNA. The purified enzyme catalyzed cleavage of various synthetic tripeptides but not when proline occupied the P1 position. Purified TPP cleaved the pentapeptide Ala-Ala-Phe-Tyr-Tyr and tripeptide Ala-Ala-Phe, confirming the TPP activity of the enzyme.     

Production, purification, and properties of an endoglucanase produced by the hyphomycete Chalara (Syn. Thielaviopsis) paradoxa CH32

The hyphomycete Chalara (syn. Thielaviopsis) paradoxa produces endoglucanase activity during the late trophophase. The low molecular mass (35 kDa) endoglucanase purified from cultured broths works optimally at 37 degrees C and pH 5.0. The enzyme inactivates at pH below 3.0 and also at temperatures of 50 degrees C or higher, but it is stable at lower temperatures, including refrigeration temperature and freezing. The enzyme is inhibited by detergents, by EDTA, and by the divalent cations Hg(2+) and Ag(2+). It is also inhibited to some extent by 10 mM Zn(2+), Fe(2+), and Mg(2+), but it is stimulated by Mn(2+). Enzyme activity is not affected by reducing agents. In the presence of low concentrations of water miscible organic solvents (20%) endoglucanase activity is inhibited by 7% (for methanol) to 50% (for acetonitrile), and it is totally inhibited at higher solvent concentrations (50%). Enzyme activity is not affected by the water immiscible solvent ethyl acetate. Carboxymethylcellulose is the preferred substrate (K(m(app)) = 8.3 g/L; V(max(app)) = 1.1 microM/min). Hydrolysis of crystalline cellulosic substrates is very limited, but it is greatly enhanced by phosphoric acid swelling. The purified enzyme shows no activity toward disaccharides or aryl-glucosides. Its activity is inhibited by cellobiose.     

Microbial biomass, enzyme and mineralization activity in relation to soil organic C, N and P turnover influenced by acid metal stress

This study focused on the potential of using soil microbial biomass, enzyme and mineralization activities involved in organic C, N and P turnover, to evaluate the quality of a subtropical agricultural soil affected by long-term acid metal stress. Fractions of C, N and P involved in soil organic matter, microbial biomass and mineralization processes were estimated. Total enzyme activity (FDA) and eight hydrolase activities (xylanase, amylase, β-glucosidase, invertase, N-acteyl-glucosaminidase, urease, alkaline and acid phosphatases) in different decomposition stages of organic C, N and P were selected to characterize the soil functional diversity. These biological datasets were compared with soil metal variables (total contents and free and ligand-complexed ions of Cu, Pb, Zn, Cd, Al and Mn), using principal component analyses, co-inertia and discriminant analyses. The multiple statistics indicate that the metal variables were significantly related with not only general biological factors, but also respective datasets of biomass, enzyme activities and mineralization rates (all P < 0.001). In general, metal variables were inversely related to parameters and indices of microbial biomass C, N and P, FDA and C-related polysaccharidase and heterosidase activities, and P mineralization. As comparison, metal variables exhibited positive relationships with parameters and indices of N-related N-acteyl-glucosaminidase, urease, ammonification, total N mineralization and metabolic quotient, compared with inhibited nitrification. Specifically, free and complexed metal cations showed higher bioavailability than total contents in most cases. Cu, Pb, Al and Mn had different ecotoxicological impacts than Cd and Zn did. Stepwise regression models demonstrated that metal variables are key stress factors, but most of them excluded soil pH. Furthermore, spatial distribution in land uses and of sampling sites clearly separated the soil samples in these models (P < 0.001). We conclude that such a statistical analysis of microbiological and biochemical indices can provide a reliable and comprehensive indication of changes in soil quality and organic nutrient cycling, after exposure to long-term acid metal stress.     

Adsorption of heavy metals by a porous bioadsorbent from lignocellulosic biomass reconstructed in an ionic liquid

A novel porous bioadsorbent for metal ion binding (Pd(2+) and Cd(2+)) was successfully prepared from lignocellulosic biomass in ionic liquid by homogeneous succinoylation and sequent chemical cross-linking. The morphology of the bioadsorbent and the interaction between bioadsorbent and metal ions was revealed by scanning electron microscopy and Fourier transform infrared spectroscopy. Results showed that the adsorption mechanism of the bioadsorbent was an ion exchange. A lower dose of cross-linker or higher carboxyl content increased the adsorption capacities of Pd(2+) and Cd(2+). The adsorption capacities of Pd(2+) and Cd(2+) remarkably increased as the pH of metal ion solutions increased. The pores in the bioadsorbent greatly favored the diffusion and adsorption of metal ions, and the adsorption equilibrium time was about 50 min. The adsorption of metal ions could be well explained by the Langmuir model, and the maximum adsorption capacities of Pd(2+) and Cd(2+) were 381.7 and 278.6 mg/g.     

不同金属离子对稻田自然生物膜磷酸酶活性的影响

以稻田自然生物膜为研究对象,研究不同浓度金属离子对生物膜酸性和碱性磷酸单酯酶(ACPase和ALPase)活性与酶反应动力学参数的影响。发现K~+和Na~+在0～1.0 mmol/L浓度下均对ACPase和ALPase无明显影响,Ca~(2+)和Mg~(2+)对ACPase和ALPase活性均有激活效应,可使磷酸酶活性最大提高14.1%和46.7%;Zn~(2+)、Cu~(2+)、Mn~(2+)、Al~(3+)和Ag~+对ACPase和ALPase活性均有抑制作用。Co~(2+)在低浓度时对ACPase和ALPase活性有一定的激活作用,高浓度时变为抑制作用;Cr~(6+)在0.1 mmol/L时对ACPase活性有促进作用,而在离子浓度0.25～1 mmol/L的范围内,均对ACPase活性产生抑制作用。通过酶反应动力学分析发现,Mg~(2+)的加入使ACPase与底物的亲和力与催化效率增强,Cu~(2+)对ACPase和ALPase的抑制作用主要是非竞争性抑制作用,Zn~(2+)虽然提高ACPase与底物的亲和力,但是降低了ALPase与底物的亲和力与催化效率。金属离子对酶活性的影响因金属离子种类、浓度和磷酸酶类型而异,主要通过与酶活性位点或底物结合,改变酶的活性、对底物的亲和力,以及影响酶基因的表达等实现的。研究结果将为评估自然生物膜在稻田磷的生物地球化学循环中的作用,以及稻田受重金属污染的可能风险提供理论依据。     

THE SPECIFIC ADSORPTION OF DIVALENT Cd, Co, Cu, Pb, AND Zn ON GOETHITE

The specific adsorption of divalent Cd, Co, Cu, Pb, and Zn on goethite is measured as a function of pH. For each mole of heavy metal adsorbed approximately two moles of H+ ions are displaced from the interface. Using these results the heavy metal adsorption data are expressed as functions of the solution concentrations of both H+ and metal ions, and the interfacial reaction is described by the equation, ₂SH+M²⁺= S₂M+₂H⁺. The adsorption data are consistent with an electrochemical model of the simultaneous adsorption of H⁺ ions and divalent metal ions on to the oxide. The intrinsic affinities of the metal ions for the oxide surface increase in the order, Cd < Co < Zn < Pb < Cu. However, besides the affinity of the metal ion for the surface, the adsorption curves are considered to be influenced by surface charge, the adsorption density of the metal ions and their size. The analysis of the data in terms of H⁺ and M²⁺ ion adsorption is considered to be complementary to the hydrolysis model for heavy metal adsorption.