Call number: LD2668 .T4 1985 Z58 / Master of Science

Submitted in fulfilment of the requirements of the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2011. / Eremothecium gossypii has the ability to utilize vegetable oils as a carbon source to produce riboflavin. This organism has been known to produce as much as 40 000 times more riboflavin than it requires after genetic modification on simple sugars. Adaptation of this organism to various oil substrates for riboflavin production has been poorly investigated. The aim of this research was thus to investigate the production of riboflavin by Eremothecium gossypii, on various oils and to improve production by mutating the organism and optimising media components using Design of Experiments (DOE). Nine overproducing mutants were obtained after mutating with various concentrations of ethylmethane sulphonate (EMS), n-methyl-n‟-nitro-n-nitrosoguanidine (MNNG) and Ultraviolet light. Riboflavin overproducing mutants were screened on an itaconate-containing medium; the colonies appeared yellow instead of white in the case of the wild-type. The itaconate screening medium isolated mutants with an isocitrate lyase that was insensitive to feedback inhibition. Mutations performed using EMS increased the ability of E. gossypii to produce riboflavin by 611% (7-fold) compared to the wild-type. This was achieved with soybean oil as a carbon source and was better than the other five oils used. Using DOE, fractional factorial experiments were carried out to optimise media components for riboflavin production on soybean oil. The total riboflavin produced by E. gossypii mutant EMS30/1 increased from 59.30 mg l-1 on a standard O&K medium using soybean oil as a carbon source to 100.03 mg l-1 on a DOE improved O&K medium, a 69% increase. The final optimised growth medium was determined from a central composite design using response surface plots together with a mathematical point-prediction tool and consisted of 5.0 g l-1 peptone, 5.0 g l-1 malt extract, 5.1 g l-1 yeast extract, 0.64 g l-1 K2HPO4, 0.6 g l-1 MgSO4 and 20 g l-1 soybean oil. Fractional factorial and central composite media optimization designs increased riboflavin production by several fold over their iterations. There was an overall increase of 1099% (12-fold) in riboflavin production by the mutant grown in an optimized medium compared to the initial riboflavin produced by the wild-type.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

The objective of this dissertation was to study the disposition and absorption of riboflavin and ascorbic acid in healthy male subjects as a function of age. Deficiencies of these vitamins have been found in the elderly and malabsorption is suspected in this population. Absorption of riboflavin was examined by administration of an oral dose of 200 mg of riboflavin-5'-phosphate (FMN) and disposition was studied following a 30 min constant rate IV infusion of FMN equivalent to 25 mg of riboflavin. Ascorbic acid absorption was examined by administration of a 5 g oral dose and disposition was studied following a 1 g IV dose. Test doses of these vitamins were administered to subjects ranging in age from 33 to 85 years. High performance liquid chromatographic assays were developed for the determination of riboflavin and ascorbic acid in plasma and urine. The method for riboflavin separated FMN and flavin adenine dinucleotide under isocratic conditions. Quantification of the three flavins was achieved by fluorescence detection. The assay system for ascrobic acid used a postcolumn reaction for the fluorometric detection of a dehydroascorbic acid derivative. This method used isoascorbic acid as the internal standard. Finally, similar specificity between a "methoxyaniline" colorimetric method and the chromatographic method was observed for the quantification of ascorbic acid in plasma and urine. Albumin plasma concentration and riboflavin protein binding were decreased with age (p < 0.05). The in vitro stability of ascorbic acid in whole blood and plasma decreased with age (p < 0.05, n = 17). The results of this study indicate no age-related alterations in parameters associated with the gastrointestinal absorption of these vitamins. Increased relative renal excretion of riboflavin with age may explain the deficiency of this vitamin. On the other hand, ascorbic acid deficiency with age may be explained by decreased stability of the vitamin in blood.

Les oligonucléotides antisens et leurs analogues, tels que les Peptide Nucleic Acids (PNA), ont la capacité d'inhiber ou de moduler l'expression d'un gène cible, de manière spécifique de séquence. Leur utilisation pour des applications thérapeutiques est cependant limitée par leur faible internalisation cellulaire ou leur mauvaise localisation intracellulaire, etnécessite le développement de stratégies efficaces de vectorisation. La Riboflavine, ou vitamine B2, est une vitamine essentielle qui a les caractéristiques requises pour être potentiellement utilisée en tant qu'agent ciblant de vectorisation. Le travail accompli au cours de ce projet de thèse a permis de démontrer la capacité de deux dérivés de la Riboflavine, la Flavine et le Lumichrome, à induire une internalisation par endocytose de PNA conjugués, dans plusieurs lignées cellulaires. En outre, un phénomène d'internalisation photochimique induit par la Rhodamine a été mis en évidence avec des double-conjugués Flavine ou Lumichrome - PNA - Rhodamine et conduit à une sortie efficace des endosomes de ces conjugués. Ce travail de thèse a donc permis de caractériser un conjugué trifonctionnel pour l'internalisation et lalibération cytoplasmique de molécules bioactives. / Antisense oligonucleotides and analogs, such as Peptide Nucleic Acids (PNA), are valuable tools to inhibit or modulate gene expression in a sequence-specific manner. Their successful application for therapeutic purposes is however hampered by poor cellular uptake or incorrect intracellular localization, and requires the development of efficient delivery strategies.Riboflavin, or vitamin B2, is an essential vitamin that could potentially be used as a targeting delivery ligand. We demonstrate in this PhD work the ability of two Riboflavin derivatives, Flavin and Lumichrome, to promote endocytosis-mediated internalization of PNA conjugates in several cell lines. Furthermore, a phenomenon of Photochemical Internalization produced by Rhodamine has been characterized using Flavin or Lumichrome - PNA - Rhodamine conjugatesand is responsible for an efficient release of these conjugates from endosomes. This PhD work has thus led to the characterization of a trifunctional conjugate for the internalization and cytoplasmic delivery of bioactive molecules.

Zugl.: Dortmund, Techn. University, Diss., 2008.

Thesis (Ph. D.)--Ohio State University, 2005. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 Nov 29

München, Techn. Univ., Diss., 2002.

A simple and economical fluorometer using blue LEDs excitation sources and simple PMT detection had been built, assembled, optimized, and employed for measurement of fluorescence from riboflavin (vitamin B2). Surfactants have been known to enhance the intensity of fluorescence of fluorescent compounds. Fluorescence analysis of riboflavin in the presence of various anionic, cationic, and nonionic surfactants was also conducted to determine if they could improve analysis. However, the surfactants employed did not seem to have any meaningful enhancement; in fact, some actually diminished the fluorescence intensity of riboflavin. The procedure was linear for riboflavin from 0.01 to 2.5 μg/mL. Reproducibility expressed as relative standard deviation was about 2%. The recoveries obtained range from 91.3% to 100.21% for the samples determined. The proposed method was successfully applied to the analysis of riboflavin in commercial vitamin tablets and cereal products. The results obtained were consistent with expected values as provided by the manufacturers. The method is simple, sensitive, economical, and rapid.

Master of Science