Thesis (M.S.)--Oregon State University, 2006. / Printout. Includes bibliographical references. Also available on the World Wide Web.

Thesis (M.S.)--University of Delaware, 2006. / Principal faculty advisor: John F. Wehmiller, Dept. of Geological Sciences. Includes bibliographical references.

No description available.

Thesis (M.A.)--Boston University

Inductively coupled plasma mass spectrometry (ICP-MS), a comparatively new multi-element analytical technique was employed for elemental determination in human hair, placenta and brain samples. The performance criteria of ICP-MS such as precision, accuracy and detection limits were assessed. For most elements sub ng ml-1 limits of detection were achieved. Amino acid analysis of human foetal brain extracts was performed using reverse phase high performance liquid chromatography. Prior to separation, samples were derivatised using o-phthaldialdehyde derivatising reagent. Precision and limits of detection were assessed. For most amino acids detection limits of sub nmoles ml-1 were achieved. Hair is commonly used as a diagnostic and monitoring tool to observe the effects of mineral supplementation on an individuals bodily status, especially prior and after pregnancy. Some of the factors influencing the elemental content of hair were investigated. Analysing scalp hair of thirteen female individuals showed that longitudinal variations exist in elemental composition of human scalp hair. Radial and longitudinal diffusion of Co, Zn and Cd were investigated in scalp hair, using tresses of "virgin" hair. Both radial and longitudinal diffusions occurred for all three elements. Analysis of scalp hair of thirty-seven females and twenty male subjects from United Kingdom showed that there were significant differences in concentrations of Mg, K, Ca, Mn and Fe between males and females. Effects of six months mineral supplementation (18.6 mg Mg, 31.0 mg Ca, 1.2 mg Cr, 4.0 mg Mn and 7.5 mg Zn daily) on elemental content of hair was investigated in six female subjects and no significant changes were observed in the levels of supplemented elements (Mg, Ca, Cr, Mn and Zn). For elemental analysis of placenta, the importance of defining the location of the sampling site was demonstrated by investigating levels of Mg, Ca, Mn, Fe, Cu, Zn and Pb in different regions of a placental disc. Significant variations were found in the elemental content of the various regions. The peripheral region of the placental discs from fifty black females from Soweto, South Africa, were analysed for their elemental content in relation to birthweight and gestational age of their neonates. Concentrations of Mg, Al, K, Ca, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Sr, Mo, Cd and Pb were determined. Positive correlation was found for Mg, K, Fe, Cu and As with both medical parameters and Zn with gestational age. Lower concentrations of Mg (P < 0.10), Cu (P < 0.05), Zn (P < 0.10) and As (P < 0.001) were found in low birthweight cases compared to high birthweight groups. Comparison with an English population showed that significant differences existed in the placental elemental levels of the two communities. The contributing factors are fully discussed. Analysis of Mg, P, Ca, Fe, Cu, Zn, Rb, Sr, Mo and Pb in foetal brain tissues of eleven stillbirths and ten socially terminated neonates showed that stillbirth brains contained significantly (P < 0.001) higher levels of Pb compared to the controls. Analysis of four different regions of the brains; hippocampus, cerebrum, cerebellum and basal ganglia, also showed higher concentrations of Pb in all the four regions in comparison with the controls. Most elements showed significant differences in their regional concentrations within each study group and the patterns were different in stillbirths compared to the social terminations. However, concentration of Zn was predominantly higher in hippocampus compared to the other regions, in both study groups. The concentrations of fifteen amino acids were measured in the same foetal brain samples used for elemental analysis. Stillbirth brain tissues had lower levels of alanine (P < 0.05), phenylalanine (P < 0.05) and Isoleucine (P < 0.05) compared to social terminations. Regional brain analysis of amino acids revealed higher concentrations of glutamic acid, aspartic acid and serine in the hippocampal region. The significance of these results and any relationship found between the amino acid and elemental content of the analysed foetal brain samples are discussed in detail. Overall this work demonstrates and supports the importance of an adequate balance of elements and amino acids for a successful outcome of pregnancy.

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No description available.

There is widespread interest in the biocatalytic production of enantiomerically pure D-amino acids for use in the synthesis of antibiotics, insecticides, herbicides, drug carriers and many other pharmaceuticals. Hydantoin-hydrolysing enzyme systems can be successfully utilised to stereoselectively convert racemic hydantoins into enantiomerically pure amino acid products. In fact, the use of microbial D-hydantoinase and D-stereoselective N-carbamoyl amino acid amidohydrolase activity to produce D-p-hydroxyphenylglycine from D,L-5-phydroxyphenylhydantoin has been described as one of the most successful biotechnological applications of enzyme technology developed to date. A need to utilise the novel biodiversity of South African microorganisms for the development of an indigenous process to produce enantiomerically pure amino acids was identified in 1995. Subsequently, the Rhodes Hydantoinase Group was established and several local hydantoin-hydrolysing microorganisms were isolated. The research in this study describes the isolation and selection of Agrobacterium tumefaciens RU-OR, which produced D-stereoselective hydantoinhydrolysing activity. Characterisation of the hydantoin-hydrolysing enzyme system of RU-OR revealed novel biocatalytic properties, and potential for the application of this strain for the biocatalytic production of D-amino acids. A fundamental understanding of the regulation of hydantoin-hydrolysing enzyme activity in A. tumefaciens RU-OR was established, and utilised to produce mutant strains with altered regulation of hydantoin-hydrolysing activity. These strains were used to further elucidate the mechanisms regulating the production of hydantoins-hydrolysing activity in A. tumefaciens RU-OR cells. Overproduction of hydantoinase and N-carbamoyl-D-amino acid amidohydrolase activity in selected mutant strains resulted in efficient conversion of D,L-5-p-hydroxyphenylhydantoin to D-p-hydroxyphenylglycine. Thus the establishment of a primary understanding of the hydantoin-hydrolysing enzyme system in A. tumefaciens RU-OR could be used to manipulate the hydantoin-hydrolysing activity in RU-OR cells to produce an improved biocatalyst. The isolation of A. tumfecaiens RU-OR genes encoding for hydantoin-hydrolysing activity revealed two separate N-carbamoyl-D-amino acid amidohydrolaseencoding genes (ncaR1 and ncaR2) in this bacterium with distinct chromosomal locations, nucleotide coding sequence and predicted primary amino acid sequence. The novel biocatalytic properties of the hydantoin-hydrolysing enzyme system in A. tumefaciens RU-OR and mutant derivatives present fascinating opportunities for further elucidation of the natural function, regulation and biocatalytic potential of hydantoin-hydrolysing enzymes.

This thesis describes a series of investigations into the hydantoin-hydrolysing activity of bacterial strains RU-KM1 and RU-OR, which were previously isolated for their ability to hydrolyse hydantoins to amino acids. The main aim of the study was to develop biotransformations with potential application in the production of enantiomerically pure amino acids using a bioreactor based system utilising the hydantoin hydrolysing enzymes of the two isolated microorganisms. Different substituted hydantoins may be used as substrates by these enzymes for the production of a variety of amino acids. These are not only important for amino acid production, but they may be used for production of other industrially important compounds, such as semisynthetic penicillin/ampicillin, L-aspartame (sweetener), Fluvalinate (insecticide), Enalapril (ACE inhibitor). Thus, the ability of the above-mentioned strains to hydrolyse these substrates was investigated, with the view to utilizing the maximum potential of these biocatalysts. Hydantoin conversion involves a two-step hydrolysis reaction which yields, initially, an N-carbamylamino acid intermediate, and subsequently, an amino acid. The hydantoin-hydrolysing enzymes of a Pseudomonas sp. RU-KM1, and an Agrobacterium sp. RU-OR were characterised as whole cells and in a crude extract preparation, and reaction conditions for its biocatalytic application were optimised. The optimum conditions for conversion of hydantoin to glycine were found to be 1 hour at 40 °C, with conversion yields greater than 30 % achieved. The enzymes of RU-KM1 demonstrated considerable stability, retaining 80 % of their activity after storage for 2 weeks at 4 °C. The activities of the enzymes were increased by the addition of a detergent to the extraction medium, suggesting that the enzymes might be membrane-bound. The results of the determination of the metal-dependence of the hydantoinase and N-carbamoylase of RU-KM1 suggested that these enzymes required metal ions for activity, with metal ions such as Cu[superscript (2+)], Fe[superscript (2+)], and Co[superscript (2+)] resulting in no significant change in enzyme activity, however there was an activation of the enzymes when Mn[superscript (2+)] was added to the enzymes. The stereoselectivity of the enzymes was investigated, and the results suggested that the hydantoinase was D-selective, whereas the N-carbamoylase was shown to be L-selective by other researchers. The hydantoin substrate selectivity of RU-KM1 and RU-OR was investigated, and the organisms were shown to be able to hydrolyse all of the seven substrates tested. However, there was a difference in activity levels between crude extract preparations and whole cells, with crude extracts generally showing slightly lower activity than whole cells in RU-KM1, and the whole cells or RU-OR showing the lower activity than its crude extract. Some difference was also observed in the order of preference of substrates between whole cells and crude extracts. The preferred substrate for RU-KM1 whole cells was isopropylhydantoin, whereas the crude extract preparation preferentially hydrolysed p-hydroxyphenylhydantoin, achieving 57 % and 52 % conversions respectively. RU-OR whole cells preferred methylhydantoin where as the crude extract preferred isopropylhydantoin, and showed 49 % and 51 % conversions respectively. The enzymes were characterised in terms of their temperature and pH optima, inducer requirements, and product inhibition studies. The hydantoinase of RU-KM1 was shown to be inducible with low levels of hydantoin, and thermostable upto 75 °C with its optima between 60 and 70 °C. The N-carbamoylase was shown to have its optima at 50 °C. The addition of ATP (0.5 mM), DTT (1 mM) and a protease inhibitor (2 mg.mL[superscript (-1)]) all increased the hydantoinase activity of RU-KM1 crude extract, however they had very little effect on the N-carbamoylase activity. The hydantoinase enzyme from extracts of RU-KM1 was partially purified by development of cell disruption methods using mechanical and lysing enzymes, followed by precipitation and chromatographic resolution. The results obtained showed a hydantoinase enzyme of between 48 and 66 kDa. RU-KM1 was grown under fermentation conditions using different minimal media. The activity and yields under these conditions were low. Previous attempt to grow the organism in a rich medium had resulted in an increase in biomass but no hydantoinase activity. A rich medium was developed by carbon and nitrogen optimisation and yielded biomass up to 30 g.L[superscript (-1)] dry cell weight. The hydantoinase activity was restored by nitrogen starvation in stationary phase. This resulted in high biomass with increased activity. This data is currently in press. Crude extract and whole cells were immobilised on flat sheet membranes, hollow fibre membranes and in alginate beads. Low hydantoinase activity was measured in bioreactors using membranes in different configurations. A significant increase in hydantoinase activity was measured when the crude extract was immobilised in sodium alginate, as a result of stabilisation of the N-carbamoylase. Temperature and pH optima were unaffected by the immobilisation procedure, however the durability of the enzymes increased 2-fold. Different configurations of the bioreactor were investigated, as well as a hydroxyphenylhydantoin as an alternative substrate in this study. The bioreactors showed a near 95 % conversion of the hydantoin to glycine, and a 99 % conversion using HPG. In conclusion, the hydantoin-hydrolysing enzymes of RU-KM1 have been shown to be possibly membrane associated, which is a novel finding. This study has shown that the hydantoinase of RU-KM1 is D-stereoselective, with high temperature stability. A growth medium was developed for the rapid production of active biomass. A bioreactor was developed using a single and a dual biocatalyst configuration, which was capable of hydrolysing hydantoin and monosubstituted hydantoins to produce amino acids. To our knowledge this system is the first such dual biocatalyst system reported for the production of amino acids.