Interaction between dry starch and plasticisers glycerol or ethylene glycol, measured by differential scanning calorimetry and solid state NMR spectroscopy

The interaction of crystalline amylose and of crystalline and amorphous amylopectin with the plasticisers glycerol or ethylene glycol in the absence of water was studied, by using differential scanning calorimetry (DSC) and solid state nuclear magnetic resonance (NMR) spectroscopy. Upon heating starch freshly mixed with plasticisers, a strong exothermal interaction enthalpy of ΔH−35 J/g was detected by DSC. At room temperature glycerol interacts mainly with the amorphous starch regions, the interaction taking 8 days to reach equilibrium. For ethylene glycol the interaction is faster, taking four days to reach equilibrium, and the rate is not affected by crystallinity. Ethylene glycol interacts in a more ordered manner with amorphous than with crystalline material, resulting in a narrower ethylene glycol cross-polarisation magic angle spinning (CP/MAS) signal when equilibrium is reached at room temperature. Upon heating, more glycerol or ethylene glycol is immobilised, but in a less ordered manner than upon storage at room temperature. This results in a more intense, but broader plasticiser CP/MAS signal upon heating. Interaction in a more ordered manner probably implies interaction with more of the hydroxy groups of the plasticiser. The polysaccharide mobility is increased more when the plasticiser interacts in a more ordered manner, as observed by small starch signals in HP/DEC spectra.     

The protein glass transition as measured by dielectric spectroscopy and differential scanning calorimetry

The glass transition and its related dynamics of myoglobin in water and in a water–glycerol mixture have been investigated by dielectric spectroscopy and differential scanning calorimetry (DSC). For all samples, the DSC measurements display a glass transition that extends over a large temperature range. Both the temperature of the transition and its broadness decrease rapidly with increasing amount of solvent in the system. The dielectric measurements show several dynamical processes, due to both protein and solvent relaxations, and in the case of pure water as solvent the main protein process (which most likely is due to conformational changes of the protein structure) exhibits a dynamic glass transition (i.e. reaches a relaxation time of 100 s) at about the same temperature as the calorimetric glass transition temperature T_g is found. This glass transition is most likely caused by the dynamic crossover and the associated vanishing of the α-relaxation of the main water relaxation, although it does not contribute to the calorimetric T_g. This is in contrast to myoglobin in water–glycerol, where the main solvent relaxation makes the strongest contribution to the calorimetric glass transition. For all samples it is clear that several proteins processes are involved in the calorimetric glass transition and the broadness of the transition depends on how much these different relaxations are separated in time.     

Observation of slow dynamic exchange processes in Ras protein crystals by 31P solid state NMR spectroscopy

The folding, structure and biological function of many proteins are inherently dynamic properties of the protein molecule. Often, the respective molecular processes are preserved upon protein crystallization, leading, in X-ray diffraction experiments, to a blurring of the electron density map and reducing the resolution of the derived structure. Nuclear magnetic resonance (NMR) is known to be an alternative method to study molecular structure and dynamics. We designed and built a probe for phosphorus solid state NMR that allows for the first time to study static properties as well as dynamic processes in single-crystals of a protein by NMR spectroscopy. The sensitivity achieved is sufficient to detect the NMR signal from individual phosphorus sites in a 0.3mm(3) size single-crystal of GTPase Ras bound to the nucleotide GppNHp, that is, the signal from approximately 10(15) phosphorus nuclei. The NMR spectra obtained are discussed in terms of the conformational variability of the active center of the Ras-nucleotide complex. We conclude that, in the crystal, the protein complex exists in three different conformations. Magic angle spinning (MAS) NMR spectra of a powder sample of Ras-GppNHp show a splitting of one of the phosphate resonances and thus confirm this conclusion. The MAS spectra provide, furthermore, evidence of a slow, temperature-dependent dynamic exchange process in the Ras protein crystal.     

Use of solid and gel state 13C NMR spectroscopy for differentiation between agarophytes and carrageenophytes

Both solid state (CP-MAS) and gel state (using standard solution state conditions) ¹³C NMR spectroscopy have been used to characterize a range of red algae that produce either agar or carrageenan. These techniques allow rapid determination of phycocolloid type within the algal tissue before extensive and time-consuming extractions and fractionations are carried out.The gel state technique can be used on living or dried material. Gel state spectra give high resolution and, because of the expectation that they will be correlated with the extractable phycocolloid, provide promise of a powerful technique for screening potentially useful red algae.     

The effect of oxygen on methanol oxidation by an obligate methanotrophic bacterium studied by in vivo 13C nuclear magnetic resonance spectroscopy

¹³C NMR was used to study the effect of oxygen on methanol oxidation by a type II methanotrophic bacterium isolated from a bioreactor in which methane was used as electron donor for denitrification. Under high (35–25%) oxygen conditions the first step of methanol oxidation to formaldehyde was much faster than the following conversions to formate and carbon dioxide. Due to this the accumulation of formaldehyde led to a poisoning of the cells. A more balanced conversion of ¹³C-labelled methanol to carbon dioxide was observed at low (1–5%) oxygen concentrations. In this case, formaldehyde was slowly converted to formate and carbon dioxide. Formaldehyde did not accumulate to inhibitory levels. The oxygen-dependent formation of formaldehyde and formate from methanol is discussed kinetically and thermodynamically. Journal of Industrial Microbiology & Biotechnology (2001) 26, 9–14. Received 04 March 2000/ Accepted in revised form 07 November 2000     

The role of the anticancer drug vinorelbine in lipid bilayers using differential scanning calorimetry and molecular modeling

Differential scanning calorimetry (DSC) has been employed to investigate the thermal changes caused by the anticancer alkaloid drug vinorelbine in dipalmytoylphosphatidylcholine (DPPC) bilayers. The total enthalpy change was increased by the presence of the drug molecule, indicating a partial interdigitation of the lipid alkyl chains. The presence of cholesterol in DPPC bilayers including vinorelbine induced an obstruction of the interdigitation, since cholesterol interrupts the upraise of enthalpy change. Vinorelbine's interdigitation ability and stabilizing properties with the active site of the receptor have been compared with those of similar in structure amphipathic and bulky alkaloid vinblastine. The obtained results may in part explain their similar mechanism of action but different bioactivity.     

Characterisation of hydrogen bonding networks in RNAs via magic angle spinning solid state NMR spectroscopy

It is demonstrated that the spatial proximity of ¹H nuclei in hydrogen bonded base-pairs in RNAs can be conveniently mapped via magic angle spinning solid state NMR experiments involving proton spin diffusion driven chemical shift correlation of low gamma nuclei such as the imino and amino nitrogens of nucleic acid bases. As different canonical and non-canonical base-pairing schemes encountered in nucleic acids are characterised by topologically different networks of proton dipolar couplings, different base-pairing schemes lead to characteristic cross-peak intensity patterns in such correlation spectra. The method was employed in a study of a 100 kDa RNA composed of 97 CUG repeats, or (CUG)₉₇ that has been implicated in the neuromuscular disease myotonic dystrophy. ¹⁵N–¹⁵N chemical shift correlation studies confirm the presence of Watson–Crick GC base pairs in (CUG)₉₇.     

The influence of internuclear spatial distribution and instrument noise on the precision of distances determined by solid state NMR of isotopically enriched proteins

The relative merits of different isotopic enrichment strategies that might be used in solid state NMR protein structure determinations are explored. The basis for comparison of these merits is the determination of the relative uncertainties in rates measured by a generalized dipolar recoupling experiment. The different schemes considered use ¹³C, ¹⁵N and ²H labeling of ubiquitin with homonuclear magnetization-transfer type experiments under magic-angle spinning (MAS). Specific attention is given to the sensitivity of the predicted relative precisions to variation in natural nuclear density distribution and noise levels. A framework is suggested to gauge the precision of measurement of a given dipolar coupling constant, and the potential for a set of such measurements to constrain structure calculations is explored. The distribution of nuclei in homonuclear ¹⁵N and ¹H dipolar recoupling spin-exchange experiments appear to provide the most promising tertiary structure information for uniformly labeled ubiquitin.     

The diffusional water permeability in the halotolerant alga Dunaliella as measured by nuclear magnetic resonance

T₁ nuclear relaxation measurements of ¹H and ¹⁷O of water have been applied to study the kinetics of the diffusional transport of water across the cytoplasmic cell membrane of Dunaliella salina and Dunaliella bardawil. The water permeability coefficients at 25°C were found to be 1.5·10⁻³ cm/s and 1.8·10⁻³ cm/s, respectively, with an activation energy of 3.7 kcal/mol. The results indicate that the cell membrane of Dunaliella exhibits high diffusional permeability to water, similar in magnitude to that found for other cells and model membranes, and a relatively low activation energy. This regularity is in contrast to the exceptionally low glycerol permeability of the membrane (Brown, F.F., Sussman, I., Avron, M. and Degani, H. (1982) Biochim. Biophys. Acta 690, 165–173).     

Slow molecular mobility in the crystalline and amorphous solid states of pentitols: a study by thermally stimulated depolarisation currents and by differential scanning calorimetry

The molecular mobility of the pentitol isomers (xylitol, adonitol, D-arabitol and L-arabitol) was studied by thermally stimulated depolarisation currents (TSDC) in the crystalline and in the amorphous solid states. Differential scanning calorimetry (DSC) was used to characterise the phase transformations, to detect polymorphism and to analyse the dynamics of the structural relaxation in the glassy state (from the heating rate dependence of the onset temperature of the glass transition signal). The mobility in crystalline xylitol and adonitol displays features that are different compared with crystalline arabitols. No difference of the dynamic behaviour seems to emerge from our results on the primary and secondary relaxations in the amorphous isomeric pentitols. The values of the steepness index or fragility obtained in this work by TSDC and DSC are compared with the values reported in the literature obtained from other experimental techniques, and with values predicted by empirical formulae.     

Production of pectinesterase and polygalacturonase by Aspergillus niger in submerged and solid state systems

Production of pectinesterase and polygalacturonase by Aspergillus niger was studied in submerged and solid-state fermentation systems. With pectin as a sole carbon source, pectinesterase and polygalacturonase production were four and six times higher respectively in a solid state system than in a submerged fermentation system and required a shorter time for enzyme production. The addition of glucose increased pectinesterase and polygalacturonase production in the solid state system but in submerged fermentation the production was markedly inhibited. A comparison of enzyme productivities showed that those determined for pectinesterase and polygalacturonase with pectin as a carbon source were three and five times higher by using the solid state rather than the submerged fermentation system. The productivities of the two enzymes were affected by glucose in both fermentation systems. The membranes of cells from the solid state fermentation showed increased levels of C18:1, C16:0 and C18:0 fatty acids. Differences in the regulation of enzyme synthesis by Aspergillus niger depended on the fermentation system, favoring the solid state over the submerged fermentation for pectinase production. Received 12 May 1997/ Accepted in revised form 19 September 1997     

Studies of mercurated derivatives of acetylacetone and ethyl acetoacetate by solid state nuclear magnetic resonance and vibrational spectroscopies

Seven new mono- and/or dimercurated compounds involving acetylacetone (2,4-pentanedione) or ethyl acetoacetate (3-ethyl ketobutanoate) were synthesized in aqueous medium. In all cases, mercuration occurred at methylene carbon atoms. All compounds were carefully analyzed by solid state carbon-13 nuclear magnetic resonance. Assignments were confirmed by using selective sequences, which allowed a total editing of the spectra. It was shown that deshielding of ¹³C mercurated sites occurred when the rate of mercuration increased. It was also possible to measure direct ¹J(¹⁹⁹Hg¹³C) coupling constants. The main bands of the vibrational spectra (infrared and Raman) were assigned. It was proved that v(C = O) and δ(C(γ)-H) could be directly related to the mercuration rate of molecules.     

Studies on the interaction of human erythrocyte band 3 with membrane lipids using deuterium nuclear magnetic resonance and differential scanning calorimetry

Human erythrocyte band 3, reconstituted into large unilamellar phospholipid vesicles, has been used as a model system for studying the interactions between membrane lipids and large transmembrane glycoproteins. Both 2H-nuclear magnetic resonance (2H-NMR) and differential scanning calorimetric techniques have been used to probe dimyristoylphosphatidylcholine-band 3 interactions over the temperature range 4-32 degrees C. Analysis of 2H-NMR spectra allowed the assignment of liquid crystal, gel phase and two-phase regions for several protein/lipid mole fractions in the range (1-20) X 10(-4). Sample size was limited by the amount of available glycoprotein and this precluded exact determination of the phase boundaries for this system. The sharp discontinuity in the spectral first moment, M1, seen at the phase transition of the pure phospholipid is progressively diminished by addition of protein, and at the highest protein concentration the first moment varies smoothly between the two phases. For T greater than 26 degrees C or less than 16 degrees C, the moments are relatively insensitive to protein concentration, while between 20 and 26 degrees C the moments increase with protein concentration up to the boundary of the two-phase region. Beyond this boundary, they remain constant or decrease slightly with increasing amount of protein. A preliminary phase diagram for band 3 in this lipid system is presented, based on 2H-NMR data. Differential scanning calorimetry (DSC) showed that addition of glycoprotein dramatically alters the scan shape and tends to extend the coexistence of two phases to higher temperatures.     

Study of hydration of cross-linked high amylose starch by solid state 13C NMR spectroscopy

Starch is subjected to chemical treatments such as cross-linking or hydroxypropylation to meet the material requirements for food uses or controlled release in the pharmaceutical industries. In this work, two types of cross-linking formulations have been employed for the preparation of high amylose starch for use as an excipient for sustained drug release. The structural differences and chain dynamics of the modified starches in the dry and hydrated states have been compared by the use of variable contact time cross polarization-magic angle spinning solid state (13)C NMR spectroscopy.     

Effect of protein structural integrity on cross-linking by tyrosinase evidenced by multidimensional heteronuclear magnetic resonance spectroscopy

Enzymatic cross-linking of proteins can be catalyzed either by transferase-type enzymes, e.g., transglutaminases, or by oxidoreductases, e.g., tyrosinases or laccases. Three-dimensional structure of protein substrate plays a key role in these reactions, that is, the reactivity and end product are strongly modulated by the accessibility of target amino acid residues to the cross-linking enzyme. Typically structural integrity of protein can be distorted by heat, pH, or mechanical action, as well as by varying ionic concentration of the solution. In this study we used partially unfolded protein (wild-type DrkN SH3) and its structurally stabilized mutant (T22G) to investigate the impact of folded/unfolded conformations on cross-linking by Trichoderma reesei tyrosinase. Our results clearly showed formation of intermolecular cross-links solely between unfolded conformations, making them superior substrates to folded proteins when using tyrosinase as a cross-linking enzyme. Multidimensional heteronuclear magnetic resonance experiments in solution state were employed to investigate cross-linked end-products. The results presented in this study form basis for application development in food, medical, cosmetic, textile, packing and other sectors. In addition, the outcome of this study has a high value for the basic understanding of reaction mechanism of tyrosinases on proteins.     

The fermentation of xylose: studies by carbon-13 nuclear magnetic resonance spectroscopy

Summary The fermentation ofd-xylose byPachysolen tannophilus, Candida shehatae, andPichia stipitis has been investigated by¹³C-nuclear magnetic resonance spectroscopy of both whole cells and extracts. The spectra of whole cells metabolizingd-xylose with natural isotopic abundance had significant resonance signals corresponding only to xylitol, ethanol and xylose. The spectra of whole cells in the presence of [1-¹³C]xylose or [2-¹³C]xylose had resonance signals corresponding to the C-1 or C-2, respectively, of xylose, the C-1 or C-2, respectively, of xylitol, and the C-2 or C-1, respectively, of ethanol. Xylitol was metabolized only in the presence of an electron acceptor (acetone) and the only identifiable product was ethanol. The fact that the amount of ethanol was insufficient to account for the xylitol metabolized indicates that an additional fate of xylitol carbon must exist, probably carbon dioxide. The rapid metabolism of xylulose to ethanol, xylitol and arabinitol indicates that xylulose is a true intermediate and that xylitol dehydrogenase catalyzes the reduction (or oxidation) with different stereochemical specificity from that which interconverts xylitol andd-xylulose. The amino acidl-alanine was identified by the resonance position of the C-3 carbon and by enzymatic analysis of incubation mixtures containing yeast and [1-¹³C]xylose or [1-¹³C]glucose. The position of the label from both substrates and the identification of isotope also in C-1 of alamine indicates flux through the transketolase/transaldolase pathway in the metabolism. The identification of a resonance signal corresponding to the C-1 of ethanol in spectra of yeast in the presence of [1-¹³C]xylose and fluoroacetate (but not arsenite) indicates the existence of equilibration of some precursor of ethanol (e.g. pyruvate) with a symmetric intermediate (e.g. fumarate or succinate) under these conditions.     

Characterization of the wound-induced material in Citrus paradisi fruit peel by carbon-13 CP-MAS solid state NMR spectroscopy

Grapefruit, Citrus paradisi, were injured, inoculated with Penicillium digitatum and incubated under conditions favourable for the accumulation of defence related material. Histochemical examination revealed that tissues adjacent to inoculated injuries contained phloroglucinol-HCl (PG-HCl) reactive material. Solvent washed cell wall preparations of intact and injured-inoculated peel were further purified using a mixture of cell wall degrading enzymes. Samples from injured inoculated tissue contained PG-HCl reactive globular material in addition to the fragments of xylem and cuticle found in controls. The principal chemical moieties of the material that accumulates in grapefruit injuries during wound-healing were studied by solid state 13C cross-polarization magic angle spinning NMR. A complete assignment of the NMR signals was made. From the analysis evidence was found that cellulose and hemicellulose are the biopolymers present in the intact peel samples, in addition, relevant quantities of cutin were found in the residues of enzyme digest. The NMR difference spectrum intact- wounded peels showed resonances which were attributed to all major functional groups of the aromatic-aliphatic suberin polyester of new material produced by the wounds. Information on the latter polyester was obtained by analyzing the T(1)rho (1H) relaxation.     

Production of raw-starch digesting amyloglucosidase by Aspergillussp GP-21 in solid state fermentation

Aspergillus sp GP-21 produced a raw-starch digesting amyloglucosidase which showed optimum activity at 65°C and pH 5.0–5.5. At 50°C the enzyme converted about 40% of raw corn starch to glucose within 48 h. Enzyme production was studied in solid state fermentation using wheat bran. Productivity was affected by the level of moisture, incubation temperature and the presence or absence of supplements. Maximum enzyme production was observed at a moisture level of 75% and at 30°C. Enzyme production was stimulated by supplementing wheat bran with 0.25% proteose peptone, 1% trace mineral solution, 0.01% CaCl₂ and 0.01% MgSO₄. Received 27 October 1998/ Accepted in revised form 15 March 1999     

The effects of cholera enterotoxin on intestinal tissue water as measured by nuclear magnetic resonance (NMR) spectroscopy.

Cholera enterotoxin has been postulated to change the configuration of the intracellular protein-water system, altering the permeability of the cell to water. Using nuclear magnetic resonance (NMR) spectroscopy, this protein-water relationship can be examined. Small intestinal loops in the rat were injected with 0.5 ml of either Schwarz/Mann cholera enterotoxin (40 mug/cc saline solution) or normal saline. Full thickness segments of intestine from each loop were taken and percentage water (using a gravimetric procedure which includes a correction for fat) and NMR relaxation times were determined. The mean value +/- S.D. for tissue water was 79.49 +/- 2.65% in the controls and 84.52 +/- 2.01% in the cholera specimens (p less than .001). T1 (spin-lattice) relaxation times were 521.22 +/- 69.5 msec in the controls and 667.96 +/- 119.25 msec in cholera tissue (p less than .001). T2 (spin-spin) relaxation times were 62.34 +/- 9.59 msec in controls and 80.35 +/- 21.46 msec in cholera tissue (p less than .02). These findings are consistent with the theory that cholera enterotoxin acts to alter intracellular protein-water relationship.     

Spatial distribution of oil in groundnut and sunflower seeds by nuclear magnetic resonance imaging

Abstract. The nuclear magnetic resonance imaging technique has been used to obtain images of different transverse and vertical sections in groundnut and sunflower seeds. Separate images have been obtained for oil and water components in the seeds. The spatial distribution of oil and water inside the seed has been obtained from the detailed analysis of the images. In the immature groundnut seeds obtained commercially, complementary oil and water distributions have been observed. Attempts have been made to explain these results.