Browsing by Author "Mocke, Leanie"

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    Kinetic characterisation and mathematical modelling of the coenzyme A biosynthesis salvage pathway in Mycobacterium tuberculosis
    (Stellenbosch : Stellenbosch University, ) Mocke, Leanie; Mocke, Leanie; Snoep, Jacob Leendert; Strauss, Erick; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Mycobacterium tuberculosis, the bacterium responsible for tuberculosis (TB), is becoming increasingly resistant to the current arsenal of anti-TB drugs, lead- ing to an increase in multi-drug resistant disease cases and death, particularly in developing countries. There is an urgent need to discover new anti-TB drugs that target pathways different from the current treatments. A possible target is the biosynthesis salvage pathway of Coenzyme A (CoA). By constructing, validating and analysis of a mechanistic mathematical model for this pathway, based on the enzyme kinetic characteristics, a rational approach is used for drug target identification in the pathway. A detailed kinetic model for the three enzyme pathway was constructed and validated. The workflow for the construction uses classic initial rate kinetics as a first step, followed by progress curve analysis for the individual enzymes and final modelling of intermediate dynamics for the complete reconstituted pathway. For initial rate kinetics, the enzymes of interest were firstly expressed and purified and parameters experimentally determined to describe each step with a parameterised rate equation. The in silico model was built by linking the three steps with ordinary differential equations. Model construction is fol- lowed by validation by comparing in vitro fluxes with model simulated fluxes. After construction and validation, the model was deemed able to describe the system behaviour. With the reconstituted pathway, different metabolic control analysis sim- ulations were evaluated to determine how control is distributed within the pathway. Experimental and model predictions of the reconstituted salvage pathway were used for metabolic control analysis. Reconstitutions were made with enzyme ratios chosen to reflect in vivo enzyme ratios. It was observed that not the PanK which is usually indicated as “the rate-limiting-step” but rather DPCK seems to have the highest control.
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    Kinetic modelling of wine fermentations : why does yeast prefer glucose to fructose
    (Stellenbosch : Stellenbosch University, 2013-03) Mocke, Leanie; Snoep, Jacky L.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: In the present-day competitive global market, wine industries are constantly aiming to improve the wine-making process,including the role of yeast. The most commonly used wine yeast is Saccharomyces cerevisiae, which is able to produce high quality wines, but problem fermentations do sometimes arise. The occurrence of stuck and sluggish fermentations pose a serious problem leading to loss of productivity and quality. Although the precise mechanism leading to stuck fermentations is unknown, they are often correlated with high fructose to glucose ratios in the wine-must. S. cerevisiae is a glucophylic yeast, indicating its preference for consuming glucose over fructose. Both these hexose sugars are present in unfermented wine must, mostly in equal concentrations. As fermentation progresses, glucose is consumed at a faster rate than fructose, leading to an increase in the fructose to glucose ratio. Yeast are left with the undesirable fructose at the later stages of fermentation, when the environmental stresses on the yeast can lead to stuck or sluggish fermentation. This residual fructose can lead to undesirable sweetness, as fructose is about twice as sweet as glucose. Even with the extensive research into yeast metabolism, there is as yet no definitive explanation as to why yeasts ferment glucose faster than fructose. This study aimed to investigate the mechanism responsible for the faster consumption of glucose over fructose of a commercially used wine yeast strain S. cerevisiae VIN 13. The first two steps of sugar metabolism, uptake and phosphorylation, were investigated as the possible sites of discrepancy in fermentation rates. Enzyme rates and affinities for both glucose and fructose as substrates for the relevant enzymes were experimentally determined. These kinetic parameter values were used to improve an existing model of yeast glycolytic pathway to model wine fermentations. The feasibility of constructing and validating a kinetic model of wine fermentations were investigated, by comparing model predicted fluxes with experimentally determined fluxes. Another aspect of this study was an investigation into the effect of hexose sugar type on fermentation profiles. Wine fermentations were done with only one hexose sugar as carbon source to determine if it has an effect on the flux through metabolism. This work succeeded in the construction of a kinetic model that distinguished between glucose and fructose as carbon source. The glucose was consumed faster than fructose, with control lying in the hexose transport step. It was also established that fermentation prfiles of fermentations with only one sugar was the same for both one sugar type fermentations. Fermentation with either glucose or fructose as the sole carbohydrate source had the same specfic production and consumption rates as normal fermentations with both sugars. Construction of detailed kinetic models can aid in the metabolic and cellular engineering of novel yeast strains. By identifying the importance of hexose transport, and thus the glucophilic character of the yeast, in flux control, yeast transporters can be targeted for strain improvement. This may in turn lead to more effective fermentation practices for controlling problem fermentations, or to the development of novel strains that utilizes fructose in the same manner as glucose, and in so doing lower the risk of stuck or sluggish wine fermentation.