Mulholland, Dulcie Aca.Dutton, Michael Francis.Gengan, Robert Moonsamy.2012-09-122012-09-1219961996http://hdl.handle.net/10413/6391Thesis (Ph.D.)-University of Natal, Durban, 1996.The biosynthesis of Aflatoxin B1 (AFB1) has been the subject of conflicting speculation and numerous reviews. The currently accepted scheme for the aflatoxin pathway is based on data obtained from feeding studies using isotopically labelled precursors. In these studies the conversion of possible intermediate metabolites to AFBl by mutants of Aspergillus parasiticus illustrated their role as biogenetic precursors. Currently there is now agreement on the identity of most of the intermediate Illetabolites involved in the biosynthesis of AFB1. However, there is a lack of clarity on the details of AFB1 biosynthesis including the conversion of sterigmatocystin (ST) to AFB1 via the metabolite O-methylsterigmatocystin (OMST). There is no clear cut evidence of the metabolic role of OMST, i.e., either it is a compulsory intermediate or a shunt metabolite and hence part of a metabolic grid. In order to investigate this step in AFBl biosynthesis, ST was isolated from surface cultures of A. versicolor (M1101) and purified by silica gel column chromatography and repeated recrystallisation. Sterigmatocystin was characterised by thin layer chromatography (t.1.c.), low resolution mass spectrometry (M.S) and nuclear magnetic resonance spectroscopy (N.M.R). A series of seven derivatives of the free hydroxyl group of ST were synthesised by known chemical reactions, purified by silica gel column chromatography and characterised by high resolution mass spectrometry and proton nuclear magnetic resonance spectroscopy. A high pressure liquid chromatography (HPLC) method was developed using a fluorescence detector. The optimum parameters for the separation of the four major aflatoxins, namely AFBl, AFB2, AFGl and AFG2, using trifluoroacetic acid as the derivatising reagent, were obtained for a reversed phase Prodigy C18 column with a mobile phase of water: acetonitrile: isopropanol: acetic acid (8: 1: 0.5: 0.5, v/v). Feeding studies, using whole cells of A. parasiticus (WhI-11-105), showed that ST and the ST derivatives were converted to AFB1. A time courser study for the conversion of ST and selected ST derivatives to AFB1 indicated a decrease in the rate of conversion in the order: a-propyl sterigmatocystin (OPROST) > a-ethyl sterigmatocystin > a-methylsterigmatocystin > Sterigmatocystin> a-benzoyl sterigmatocystin (OBzST). It was apparent that the "enzyme" responsible for the conversion of the derivatives to AFB1 did not display a high degree of substrate specificity, since it was unable to recognize the difference between the various alkyl groups, either as ether or ester functional groups. An HPLC method was developed using a diode array detector. The optimum parameters for the separation of aflatoxin metabolites and the synthesised derivatives were obtained for a reversed phase Lichrosphere RP-I8 column with a 30 minute gradient elution program with water and acetonitrile as the mobile phase. Crude cell-free extracts were prepared by lyophilisation of the mycelia of A. parasiticus (Whl-11l-105) with phosphate buffer. The temperature and pH for the conversion of ST to AFB1, were found to be optimum at 28°C and 7.2, respectively. The addition of SAM (1.5 mM) and NADPH (1.5 mM) increased the conversion of ST to AFBl from 11.21 % to 27.10 %. A time course study with ST, OMST and OPROST showed that the rate of conversion to AFBl was close to linear for an incubation time of up to 60 minutes. Approximation of the reaction rate indicated a decrease in the order: OMST > ST > OPROST. This indicated that the time course reaction using whole cells was in part a measure of membrane permeability rather than substrate specificity. Molecular exclusion chromatography was used to separate enzymatic protein from primary and secondary metabolites, small biomolecules and indigenous co-factors (MW < 10 000) and the partially purified "enzyme" was concentrated by dialysis against solid sucrose. The "enzyme" was subjected to non-denaturing polyacrylamide gel electrophoresis and was found to be made of sub-units ranging from 58 kDa to over 200 kDa. Enzymatic investigations with ST, as substrate, indicated that OMST is a compulsory intermediate in the biosynthesis of AFBl. Also, enzymatic investigations of selected ST derivatives showed that the partially purified "enzyme" displayed relative specificity for these substrates, viz., OMST, OPROST and OBzST. Three xanthones, namely, 1-hydroxy-,6-dimethylxanthone, I-methoxy-3,6-dimethylxanthone and l-acetyl-3,6-dimethylxanthone were synthesised, purified and characterised spectroscopically. Whole cell studies of A. parasiticus (CMI 91019b) and A. parasiticus (Wh1-11-105) showed that these xanthones inhibited AFBl production to varying extents. Kinetic studies of cell-free extracts revealed that the 1-methoxy-3,6-dimethylxanthone derivative was a non-competitive inhibitor. The Michaelis Menten constant (Km) of approximately 5.60 uM (for OMST) was determined for a cell-free reaction at pH 7.2 and 28 QC. A Clark oxygen electrode was used to carry out oxygen consumption studies in a partially purified "enzyme" preparation. A calibration system was designed and the enzymatic conversion of OMST to AFB1 and NADPH consumption were monitored by HPLC and UV spectroscopy, respectively. From the results of these enzymatic reactions, the following stoichiometric relationship was determined: 2 mole oxygen consumed = 1 mole NADPH consumed = 1 mole AFB1 produced A tentative mechanism is discussed for the conversion of OMST to AFB1 which utilizes a monooxygenase and a dioxygenase.en-ZATheses--Human physiology.Aflatoxins.Xanthone.Enzymatic analysis.Organic compounds--Synthesis.Thesis