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    Studies in protein structure : the structure and properties of the iron superoxide dismutase from Methanobacterium thermoautotrophicum : the structures of [beta]-lactoglobulin in two new crystal forms : a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Institute of Fundamental Sciences at Massey University
    (Massey University, 2002) Adams, Julian James
    The crystal structure of Methanobacterium thermoautotrophicum iron superoxide dismutase (Mt-FeSOD) has been determined by X-ray diffraction to a resolution of 2.6 Å. The crystals were grown from PEG 6000 at a pH of 5.5, and the structure was solved by molecular replacement. The structure, in concert with structural and functional data from other Fe and Mn SODs, provides insights into aspects of metal specificity, reactivity of superoxide dismutase towards toward the inhibitor azide and deactivator hydrogen peroxide, and how the primary structure is involved in subtle tuning of these properties. The structure reveals how the protein is designed for thermal and chemical stability, yet retains moderate superoxide dismutase activity at ambient temperature. Bovine β-lactoglobulin (BLG) has been studied for many decades; numerous X-ray and NMR structures are available. Here we present two new X-ray structures, one from a crystal grown at very low ionic strength, and at the lowest pH (~5.2) of any X-ray structure. This structure provides validation of the other, high ionic strength X-ray structures. The core elements of BLG, an eight-stranded β-barrel, a three-turn α-helix external to the barrel, and an external β-strand (that forms the dimeric interface), are almost invariant across all structures. Four flexible loops have a variety of positions in the known structures and this represents a set of snapshots of the in vivo states of BLG. These flexible loops play an important role in the entropic stabilization of the β-barrel.
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    Backbone dynamics of bovine [beta]-lactoglobulin by ¹⁵N NMR spectroscopy : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry, Institute of Fundamental Sciences and Institute of Molecular Biosciences, Massey University, New Zealand
    (Massey University, 2011) Baker, Kristy
    Bovine β-lactoglobulin (β-Lg) is a small 162 residue protein of unknown function from the whey component of milk, constituting ~50 % by dry mass. The protein is of great interest to the dairy industry due, in part, to its role in the fouling of dairy plants during heat treatment, and the significant operational costs this incurs. The structure of this protein is an eight stranded β-barrel with one long and two short flanking  helices. It is dimeric at neutral pH but dissociates at pH < 3. In New Zealand herds there are three genetic variants, with variants A and B of bovine β-Lg predominating, while the C variant occurs at low levels in Jersey cows. However, despite the structural similarities of the three variants, milks containing one of A, B or C behaves differently when subjected to thermal processing. A greater understanding of factors that differentiate these protein variants is therefore important. In this study, 15N nuclear magnetic (NMR) spectroscopy methods have been used to study the backbone dynamics of β-Lg A and B, at one temperature, and the hitherto unstudied C variant, at three temperatures. For follow-up functional studies a mutant protein, a covalently linked Ala34Cys dimer, was produced.
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    Effects of milk protein ingredients on physico-chemical properties of rice starch : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University Palmerston North, New Zealand
    (Massey University, 2009) Noisuwan, Angkana
    The overall aim of this thesis is to determine if the interactions between normal and waxy rice starch and milk proteins from four milk protein ingredients, namely skim milk powder (SMP), milk protein concentrate (MPC), sodium caseinate (NaCAS) and whey protein isolate (WPI) do occur, and to identify the mechanisms underlying these interactions. Different milk protein ingredients at various concentrations (0 to 10%, w/w) affected markedly and differently the pasting behaviour of 10% (w/w) rice starches. SMP delayed the pasting of both rice starches by increasing the onset temperature (Tonset) and the peak viscosity temperature (Tpeak) of pasting. This was mainly due to the presence of lactose and ions, which was further supported by the investigation of the effects of UFSMP (a solution of salts and lactose present in SMP at their proper concentration) and lactose. The addition of NaCAS also delayed the pasting of rice starch; Tpeak in the case of both starches was increased. For normal rice starch paste, MPC and WPI decreased the Tpeak. MPC had no affect on Tpeak of waxy rice starch paste. The qualitative viscoelastic behaviour of rice starch/milk protein ingredient gels obtained from the above pastes was dominated by the continuous phase made of the starch molecules. There was evidence, as indicated by confocal microscopy, of phase separation between the milk proteins of SMP and MPC and the two starches. The phase separation was not observed in the addition of either NaCAS or WPI. Studies on the thermal behaviour of rice starch/milk protein ingredient mixtures by differential scanning calorimetry (DSC) showed that SMP, similarly to UFSMP, delayed the gelatinization of both starches. NaCAS also delayed the gelatinisation of both starches but had a greater effect on waxy than normal rice starch. The addition of NaCAS did not affect Tonset but increased Tpeak for normal rice starch, whereas the gelatinisation temperature of waxy rice starch was highly affected by the addition of NaCAS with both Tonset and Tpeak shifted to higher temperatures. MPC had no affect on the gelatinization temperature of normal rice starch, whereas the gelatinization temperature of waxy rice starch was increased by the addition of MPC. The addition of WPI to both rice starches showed two thermal transitions. The first of these was due to the gelatinisation of the starches and the second to the denaturation of ß-lactoglobulin (ß-lg). The addition of WPI to normal rice starch showed that the thermal behaviour of normal starch and protein were independent from each other. In contrast, the thermal behaviour of waxy rice starch was modified by the addition of WPI; both Tonset and Tpeak were increased. SMP decreased the Tonset of swelling, swelling ratio and the amount of starch leaching from both starches. These observed changes were due to the presence of lactose and ions in SMP. NaCAS slightly increased Tonset of swelling but the amount of starch leaching was reduced for both rice starches. The rigidity of both starches tended to increase in the presence of NaCAS. MPC and WPI affected the swelling behaviour of normal and waxy rice starch differently. A dramatic increase in the swelling of normal rice starch/MPC or WPI mixtures was observed, whereas this trend was not evident for waxy rice starch/ MPC or WPI mixtures. The difference in the water holding ability and gelatinization peak temperatures of the two starches over the temperature range at which whey proteins denature and form gels are believed to be responsible for the observed differences. The results from confocal microscopy showed that milk proteins, such as a-casein, ß- casein, ß-lg and a-lactalbumin (a-la), were adsorbed onto the granule surface of both normal and waxy rice starch. The mechanism for this adsorption is the hydrophilic interactions; hydrogen bonds between hydroxyl group from terminated glucan molecule that protrude around starch granule surface-hydroxyl; amino, or other electron-donation or electron-accepting groups of the added proteins. Using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) it was found that for SMP and MPC the adsorbed as- to ß-casein ratio on both starches was similar to the as-casein to ß- casein ratio in the casein micelle at low SMP and MPC concentrations. But at high concentrations of SMP or MPC, this ratio decreased indicating that more ß-casein was adsorbed preferentially to as-casein. In the case of NaCAS, as-casein was adsorbed preferentially to ß-casein. Moreover, there was evidence of multilayer adsorption of ascasein into the surface of rice starch granules. Compared to the other milk protein ingredients, very small amounts of the ß-lg and a-la from WPI were adsorbed onto starch granules. However, the adsorbed amounts of ß-lg and a-la from WPI continuously increased with increasing WPI concentration, suggesting that these two proteins, particularly ß-lg, adsorbed in multilayers too.