Due to recent legislation requiring the determining of the pharmacokinetic effect of both enantiomers separately, of any new racemic drug before commercialisation, much research is done to improve and optimise methods for obtaining chirally pure compounds important for the pharmaceutical industry, for example epoxide precursors. To date most experiments regarding the biocatalytic chiral separation of 1,2-epoxyoctane has been done in batch processes. The aim of this study was to optimise the enantioselective hydrolysis of 1,2-epoxyoctane by Rhodosporidiurn tondoides in both a batch and continuous process. The batch process was optimised in terms of stir speed, biomass (cell) concentration and reaction time, while the flow-through reactor (continuous process) was optimised with regards to the flow rate as a function of the pressure and the amount of chitosan and biomass in the reactor. Initial inconsistencies of epoxide concentrations in preliminary studies were found to be due to adsorption by reaction and sampling vessels, and the lower than expected solubility of 1,2- epoxyoctane (3.85 mM instead of 6 mM as reported by previous investigators). The results from the batch process suggest that as the reaction time increases, the % ee-epox increases initially, but decreases after 40 minutes. Optimum yield in terms of % ee-epox were obtained at medium stir speed (400 rpm) and biomass (cell) concentration (13 %). Below these values the % ee-epox increases with an increase in stir speed and/or biomass concentration. Above these values however, the increased stir speed and/or biomass concentration causes abrasion between cells, which negatively affects the % ee-epox. The % ee-diol reached a steady state after 10 minutes, and the effect of the different operating conditions on % ee-diol was negligible. In the flow-through reactor chitosan was used as a spacer material (antifouling agent) to help decrease the fouling due to biomass deposition. The use of chitosan as a spacer ensured higher and stabilised flow rates for extended periods of time. In initial studies 0.5 g chitosan increased the flow rate by 34 % with a resistance removal of 25 %. For 1 g chitosan these values were 130 % flow increase and 57 % resistance removal. The flow rate was optimised in relation to the chitosan amount, biomass (cell) amount and pressure. The maximum flow rate was obtained at a pressure of 40 kPa, using the minimum amount of cells (0.4 g) and a maximum amount of chitosan (1.6 g) / Thesis (M.Sc.)--North-West University, Potchefstroom Campus, 2004.

Due to recent legislation requiring the determining of the pharmacokinetic effect of both enantiomers separately, of any new racemic drug before commercialisation, much research is done to improve and optimise methods for obtaining chirally pure compounds important for the pharmaceutical industry, for example epoxide precursors. To date most experiments regarding the biocatalytic chiral separation of 1,2-epoxyoctane has been done in batch processes. The aim of this study was to optimise the enantioselective hydrolysis of 1,2-epoxyoctane by Rhodosporidiurn tondoides in both a batch and continuous process. The batch process was optimised in terms of stir speed, biomass (cell) concentration and reaction time, while the flow-through reactor (continuous process) was optimised with regards to the flow rate as a function of the pressure and the amount of chitosan and biomass in the reactor. Initial inconsistencies of epoxide concentrations in preliminary studies were found to be due to adsorption by reaction and sampling vessels, and the lower than expected solubility of 1,2- epoxyoctane (3.85 mM instead of 6 mM as reported by previous investigators). The results from the batch process suggest that as the reaction time increases, the % ee-epox increases initially, but decreases after 40 minutes. Optimum yield in terms of % ee-epox were obtained at medium stir speed (400 rpm) and biomass (cell) concentration (13 %). Below these values the % ee-epox increases with an increase in stir speed and/or biomass concentration. Above these values however, the increased stir speed and/or biomass concentration causes abrasion between cells, which negatively affects the % ee-epox. The % ee-diol reached a steady state after 10 minutes, and the effect of the different operating conditions on % ee-diol was negligible. In the flow-through reactor chitosan was used as a spacer material (antifouling agent) to help decrease the fouling due to biomass deposition. The use of chitosan as a spacer ensured higher and stabilised flow rates for extended periods of time. In initial studies 0.5 g chitosan increased the flow rate by 34 % with a resistance removal of 25 %. For 1 g chitosan these values were 130 % flow increase and 57 % resistance removal. The flow rate was optimised in relation to the chitosan amount, biomass (cell) amount and pressure. The maximum flow rate was obtained at a pressure of 40 kPa, using the minimum amount of cells (0.4 g) and a maximum amount of chitosan (1.6 g) / Thesis (M.Sc.)--North-West University, Potchefstroom Campus, 2004.

Biocatalysis and enzyme technology represent significant research topics of contemporary biotechnology. The immobilisation of these catalysts on or in static supports serves the purpose of transforming the catalyst into a particle that can be handled through effortless mechanical operations, while the entrapment within a membrane or capsule leads to the restraint of the enzyme to a distinct space. This confinement leads to a catalyst with a superior stability, and cell durability under reaction conditions. Epoxide hydrolase is a widely available co-factor independent enzyme, which is known to have remarkable chemio-, regio- and stereoselectivity for a wide range of substrates. Recently it was found that certain yeasts, including Rhodosporidium toruloides, contain this enzyme and are able to enantioselectively catalyse certain hydrolysis reactions. The objective of this project was four-sided: a) to immobilise Rhodospridium toruloides in an optimised immobilisation matrix (calcium alginate beads), for the kinetic resolution of 1.2- epoxyoctane in order to obtain an optically pure epoxide and its corresponding vicinal diol, b) to determine the effect of immobilisation on activity as well as stability of the enzyme and gain better understanding of the parameters that influence enzyme activity in a support, c) to determine the effect of formulation parameters on some of the bead characteristics and, d) to gain some insight in the distribution of epoxide and diol in the water and bead phases and the formulation parameters that have an effect thereon. Rhodospridium toruloides was immobilised in calcium alginate beads consisting of different combinations of alginate and CaCl2 concentrations. Best results were obtained with a combination of 0,5 % (m/v) alginate and 0,2 M CaC12. The immobilised cells exhibited lower initial activity. but more than 40 times the residual activity of that of the free cells after a 12-hour storage period. Both the immobilised and free cells exhibited an increase in reaction rate (V) with an increase in substrate concentration. An increase in the alginate concentration lead to the formation of smaller beads, but a decrease in enzume activity, while an increase in the CaCl2 solution concentration had no effect on bead diameter or enzyme activity. Epoxide diffused preferentially into the beads (± 96 %), and the diol into the water phase, which leads to the natural separation of the epoxide and the diol. The CaCl2 concentration affected epoxide diffusion with no effect on diol diffusion, which opens up the possibility to regulate the diffusion of epoxide into the beads. Although only a very small fraction of the epoxide inside the beads could be extracted, the alginate proved to be chirally selective for the (R)-epoxide, improving the reaction efficiency by increasing the % ee, of the epoxide extracted from the beads between 26 % and 43 %. The possibility to develop a system where the product is formed, purified and concentrated in a one-step reaction by extracting the product from the bead phase was clearly demonstrated. / Thesis (M.Sc. (Pharm.) (Pharmaceutical Chemistry))--Potchefstroom University for Christian Higher Education, 2003.

Thesis (M.Sc. (Pharm.) (Pharmaceutical Chemistry))--Potchefstroom University for Christian Higher Education, 2003.

Biocatalysis and enzyme technology represent significant research topics of contemporary biotechnology. The immobilisation of these catalysts on or in static supports serves the purpose of transforming the catalyst into a particle that can be handled through effortless mechanical operations, while the entrapment within a membrane or capsule leads to the restraint of the enzyme to a distinct space. This confinement leads to a catalyst with a superior stability, and cell durability under reaction conditions. Epoxide hydrolase is a widely available co-factor independent enzyme, which is known to have remarkable chemio-, regio- and stereoselectivity for a wide range of substrates. Recently it was found that certain yeasts, including Rhodosporidium toruloides, contain this enzyme and are able to enantioselectively catalyse certain hydrolysis reactions. The objective of this project was four-sided: a) to immobilise Rhodospridium toruloides in an optimised immobilisation matrix (calcium alginate beads), for the kinetic resolution of 1.2- epoxyoctane in order to obtain an optically pure epoxide and its corresponding vicinal diol, b) to determine the effect of immobilisation on activity as well as stability of the enzyme and gain better understanding of the parameters that influence enzyme activity in a support, c) to determine the effect of formulation parameters on some of the bead characteristics and, d) to gain some insight in the distribution of epoxide and diol in the water and bead phases and the formulation parameters that have an effect thereon. Rhodospridium toruloides was immobilised in calcium alginate beads consisting of different combinations of alginate and CaCl2 concentrations. Best results were obtained with a combination of 0,5 % (m/v) alginate and 0,2 M CaC12. The immobilised cells exhibited lower initial activity. but more than 40 times the residual activity of that of the free cells after a 12-hour storage period. Both the immobilised and free cells exhibited an increase in reaction rate (V) with an increase in substrate concentration. An increase in the alginate concentration lead to the formation of smaller beads, but a decrease in enzume activity, while an increase in the CaCl2 solution concentration had no effect on bead diameter or enzyme activity. Epoxide diffused preferentially into the beads (± 96 %), and the diol into the water phase, which leads to the natural separation of the epoxide and the diol. The CaCl2 concentration affected epoxide diffusion with no effect on diol diffusion, which opens up the possibility to regulate the diffusion of epoxide into the beads. Although only a very small fraction of the epoxide inside the beads could be extracted, the alginate proved to be chirally selective for the (R)-epoxide, improving the reaction efficiency by increasing the % ee, of the epoxide extracted from the beads between 26 % and 43 %. The possibility to develop a system where the product is formed, purified and concentrated in a one-step reaction by extracting the product from the bead phase was clearly demonstrated. / Thesis (M.Sc. (Pharm.) (Pharmaceutical Chemistry))--Potchefstroom University for Christian Higher Education, 2003.