Formation and characterization of iron-binding phosphorylated human-like collagen as a potential iron supplement.

Iron incorporated into food can induce precipitation and unwanted interaction with other components in food. Iron-binding proteins represent a possibility to avoid these problems and other side effects, as the iron is protected. However, there are several technical problems associated with protein-iron complex formation. In this paper, the iron-binding phosphorylated human-like collagen (Fe-G6P-HLC) was prepared under physiological conditions through phosphorylated modification. One molecule of Fe-G6P-HLC possesses about 24 atoms of Fe. Spectroscopy analysis, differential scanning calorimetry (DSC) and equilibrium dialysis techniques were employed to investigate the characteristics of the Fe-G6P-HLC. The binding sites (nb) and apparent association constant (Kapp) between iron and phosphorylated HLC were measured at nb=23.7 and log Kapp=4.57, respectively. The amount of iron (Fe(2+) sulfate) binding to phosphorylated HLC was found to be a function of pH and phosphate content. In addition, the solubility and thermal stability of HLC were not significantly affected. The results should facilitate the utilization of HLC as a bioactive iron supplement in the food and medical industry and provide an important theoretical evidence for the application of HLC chelates.

[Sorption and desorption characteristics of different structures of organic phosphorus onto aluminum (oxyhydr) oxides].

The sorption and desorption characteristics of four kinds of organic phosphorus with different molecular structures (glycerophosphate (GP), glucose-6-phosphate (G6P), adenosine triphosphate (ATP), and myo-inositol hexakisphosphate (IHP)) on three kinds of aluminum (oxyhydr)oxides (amorphous Al(OH)3, boehmite, and alpha-Al2O3) were studied. The underlying mechanisms were also illustrated. Results showed that the maximum sorption amounts of OP onto Al (oxyhydr)oxides, on a per gram dry weight basis, decreased as following: amorphous Al(OH)3 > boehmite > alpha-Al2O3. This mainly related to the mineral crystallinity and surface heterogeneity. With the exception of sorption of IHP on amorphous Al (OH)3, the maximum sorption density decreased with increasing molecular weight (MW) of OP, following the order: GP > G6P > ATP > IHP. However, the sorption amount of IHP on amorphous Al (OH)3 was much higher than those of other OP, due to the transformation of surface complexes of IHP to surface precipitation and thus enhancing the sorption. The sorption kinetics results showed that sorption of OP underwent the first onset rapid sorption, i. e. a certain amount of sorption occurred within an onset extremely short period, and a following long and slow sorption process. Amorphous Al (OH)3 had the greatest onset rapid sorption density, and the onset rapid sorption density of OP on Al (oxyhydr) oxides decreased with increasing MW. Desorption capacities of OP by KCl and citrate solutions related to the surface affinity between OP and boehmite. Initial desorption percentages by KCl decreased in the order: G6P (10.53%) > GP(6.91%) > ATP (3.06%) > IHP (0.8%). The maximum desorption percentages of OP by citrate were 4-5 times greater than those by KCl. During resorption process of P by KCl, the maximum desorption rate achieved after a fast desorption in a few hours, followed by diffusion-resorption during which the desorption percentage gradually decreased. Specially, both diffusion-resorption and surface precipitation promoted the resorption of IHP on mineral surface. Conclusively, the strong specific sorption of OP occurs on the surface of Al (oxyhydr) oxides, and molecular structure and size of OP as well as the crystallinity and crystal structure of minerals are the key factors affecting the interfacial reactions and environmental behaviors of OP.

A mass spectrometric method for quantifying C3 and C6 phosphorylation of starch.

The glucosyl residues comprising starch can be phosphorylated at either the C3 or the C6 position of the molecule because of the activities of two distinct dikinase enzymes. After hydrolysis of the starch, the C6 phosphorylation is easy to measure using a routine enzyme assay for glucose 6-phosphate, but the C3 phosphorylation is more difficult to assay. A mass spectrometric (MS) method has been developed that, in a single run, can distinguish and quantify the glucose 3-phosphate and glucose 6-phosphate produced by hydrolysis of starch and can also measure the glucose content to give an accurate estimate of the starting material. The MS method involves quantification by LC/MS with external standards, using normal-phase hydrophilic interaction liquid chromatography and selective reaction monitoring. The MS method has been used to determine degrees of starch phosphorylation in a diverse group of potato lines, revealing threefold differences in phosphorylation between high- and low-phosphate lines. The method was also used to show that cold storage of potato tubers for up to 24weeks had little substantive effect on the levels of starch phosphorylation. MS provided an effective and efficient means of determining both the C6 and the C3 phosphorylation of starch.

Cloning and characterization of a novel phytase from wastewater treatment yeast Hansenula fabianii J640 and expression in Pichia pastoris.

Phosphohydrolysis of organic phosphorus compounds by acid phosphatases (EC 3.1.3.1 and EC 3.1.3.2) is an important method for efficient removal of phosphorus from high concentration organic wastewater. Another important method is supplementation of animal feed with phytase (EC 3.1.3.8 and EC 3.1.3.26), which improves the availability of phytate-phosphates (phosphate that are hydrolyzed by phytases), making it possible to add less phosphate to animal feed and resulting in the excretion of less phosphorus by the animals. In the present study, we purified a novel phytase from the wastewater treatment yeast Hansenula fabianii J640 (Hfphytase), cloned the 1456 bp open reading frame (ORF) encoding Hfphytase, and characterized Hfphytase. The molecular weight of Hfphytase after deglycosylation by PNGaseF was 49 kDa. The optimal pH and temperature for enzyme activity were 4.5 and 50 degrees C, respectively. Hfphytase exhibits 40% identity with Debaryomyces castellii phytase, 37% identity with Aspergillus niger PhyB, and 34% identity with Saccharomyces cerevisiae Pho5p. Recombinant Hfphytase was transformed and expressed in Pichia pastoris. The yield was 23 g/l by jar fermenter cultivation. The marked phosphohydrolysis activity exhibited by Hfphytase on six substrates (pNP-P, sodium phytate, glucose-1 phosphate, glucose-6 phosphate, alpha-glycerophosphate and beta-glycerophosphate) indicated that it is a non-specific acid phosphatase.

Infrared multiphoton dissociation and electron-induced dissociation as alternative MS/MS strategies for metabolite identification.

A major challenge encountered in mass spectrometric metabolite analysis is the identification and structural characterization of metabolites. Fourier transform ion cyclotron resonance mass spectrometry is a valuable technique for metabolite structural determination because it provides accurate masses and allows for multiple MS/MS fragmentation strategies, including infrared multiphoton dissociation (IRMPD) and electron-induced dissociation (EID). Collision activated dissociation (CAD) is currently the most commonly used MS/MS technique for metabolite structural characterization. In contrast, IRMPD and EID have had very limited, if any, application for metabolite characterization. Here, we explore IRMPD and EID of phosphate-containing metabolites and compare the resulting fragmentation patterns to those of CAD. Our results show that CAD, IRMPD, and EID provide complementary structural information for phosphate-containing metabolites. Overall, CAD provided the most extensive fragmentation for smaller (<600 Da) phosphate-containing metabolites; however, IRMPD generated more extensive fragmentation for larger (>600 Da) phosphate-containing metabolites, particularly for species containing increased numbers of phosphate groups. EID generally provided complementary fragmentation to CAD and showed extensive fragmentation with relatively evenly abundant product ions, regardless of metabolite size. However, EID fragmentation efficiency is lower than those of CAD and IRMPD.

[Stability of glucose 6-phosphate dehydrogenase complexed with its substrate and/or cofactor in aqueous and micellar environment]. / Stabil'nost' gliukozo-6-fosfat-degidrogenazy v sostave kompleksov s kofaktorom i substratom v vodnykh i mitselliarnykh sredakh.

Inactivation of glucose 6-phosphate dehydrogenase (G6PDH) complexed with its substrate, glucose 6-phosphate (GP), and/or cofactor, NADP+, has been studied within the range 20-40 degrees C in three media: (a) 0.04 M NaOH-glycine buffer (pH 9.1); (b) Aerosol OT (AOT) reversed micelles in octane; and (c) Triton X-100 micelles in octane supplemented with 10% hexanol. The enzyme inactivation was characterized quantitatively by first order rate constants, kin (s-1). In the case of G6PDH-NADP+ complexes, the values of kin were independent of the initial concentrations of G6PDH, either in aqueous medium or AOT micelles. The values of kin for the complex G6PDH-GP were inversely related to the initial concentration of the enzyme, in both aqueous and micellar media. When inactivation of both complexes were studied in AOT micelles, minimum values of kin corresponded to the degree of hydration W0 = 16.7; at W0 > 16.7 and W0 < 16.7, kin increased. Within the range 20-40 degrees C, the values of kin measured for both complexes in aqueous medium were significantly lower than those measured in AOT micelles. Temperature dependences of kin were characterized by inflections in Arrhenius plots, which corresponded, depending on the medium, to certain temperatures from 33.6 degrees C to 40 degrees C. In all media studied, NADP+ complexes of the enzyme exhibited higher stability than their GP counterparts. The parameters of G6PDH and G6PDH-NADP+ melting, measured by differential scanning microcalorimetry (maximum temperature and half-width of the transition, enthalpy of denaturation, and van't Hoff enthalpy), provided unequivocal evidence of the higher stability of the complex as compared to that of the enzyme. In addition, this approach demonstrated that G6PDH undergoes destabilization in AOT micelles.

Excretion of a phosphorus-containing carbohydrate by Streptomyces sp. A50.

A new phosphorus-containing compound (1) was detected by (31)P NMR spectroscopy in Streptomyces sp. A50. Compound 1, 1(alpha)-O-methyl-2-(N-acetyl)glucoseamine-6-O-phosphate-1(alpha)-2-(N-acetyl)glucosamine, exhibited a pK(a) value around zero. The compound was found both in the extracellular culture broth and in the cells. While very low concentrations of 1 were found in the culture broth of other species of Streptomyces, its presence in high concentrations was specific to Streptomyces sp. A50. The highly acidic compound was isolated from the broth, and its structure was elucidated by a combination of 1D-, 2D-homonuclear, and inverse heteronuclear NMR techniques and mass spectroscopy.

The distribution of covalently bound phosphate in the starch granule in relation to starch crystallinity.

Five selected starches with a 60-fold span in their content of monoesterified starch phosphate were investigated with respect to distribution of glucose 6-phosphate and glucose 3-phosphate residues, amylopectin chain length distributions and gelatinisation properties. The distribution of starch phosphate in the starch granules was determined by preparation of Nägeli dextrins followed by quantitative 31P-nuclear magnetic resonance spectroscopy. Total starch phosphate content was positively correlated to the unit chain lengths of the amylopectin as well as to the chain lengths of the corresponding Nägeli dextrins. The major part (68-92%) of the total starch phosphate content was partitioned to the hydrolysed (amorphous) parts. Starch-bound glucose 6-phosphate per milligram of starch was 2-fold enriched in the amorphous parts, whereas phosphate groups bound at the 3-position were more evenly distributed. The gelatinisation temperatures of the native starches as determined by differential scanning calorimetry were positively correlated (R(2)=0.75) to starch phosphate content, while crystallinity (gelatinisation enthalpy) and crystal heterogeneity (endotherm peak width) showed no correlations to starch phosphate content. The relations between starch molecular structure, architecture and functional properties are discussed.