Browsing by Author "Anane, Emmanuel"

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    Comparison of constitutive and inducible β-fructofuranosidase production by recombinant Pichia pastoris in fed-batch culture using defined and semi-defined media
    (Elsevier, 2016) Anane, Emmanuel; Van Rensburg, Eugene; Gorgens, Johann F.
    ENGLISH ABSTRACT: Short-chain fructooligosaccharides produced from sucrose by transfructosylation using β-fructofuranosidase (FFase), an industrially important enzyme, finds application in pre-biotics, sweeteners and confectionary products. Using recombinant Pichia pastoris, the influence of replacing the commonly-used Invitrogen® medium with a semi-defined medium for FFase production under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) and alcohol oxidase (AOX) promoters was investigated. Replacing the trace metals (PTM1) solution with yeast extract resulted in a 54.3% decrease in FFase volumetric activity under control of the AOX promoter, suggesting a distinct requirement for trace metals for recombinant protein synthesis during methanol induction, given that the biomass yield on methanol decreased by only 10%. The same medium adjustment had no effect on enzyme production under GAP promoter control, although AOX promoter control resulted in double the FFase volumetric activity compared to glycerol-fed cultures. Decreasing basal salts by half did not affect the cultures, but alleviated precipitation during sterilisation. Optimisation of the glycerol feed rate and dissolved oxygen tension in DO-stat fed-batch fermentations using the semi-defined medium resulted in 17% increase in volutmetric activity of FFase expressed under the GAP promoter. This study highlighted the influence of carbon source and trace metals on heterologous protein production by P. pastoris using constitutive and inducible promoters.
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    Process optimisation and scale-up of industrial enzymes production
    (Stellenbosch : Stellenbosch University, 2013-12) Anane, Emmanuel; Gorgens, Johann F.; Stellenbosch University. Faculty of Engineering. Dept. of Process Engineering.
    ENGLISH ABSTRACT: Industrial enzymes offer excellent prospects for the development of ‘green’ processes and high quality products with a diminished negative impact on the environment. This study endeavours to develop fed-batch process methods to improve the production of two industrially relevant enzymes in dedicated yeast systems, namely Saccharomyces cerevisiae and Pichia pastoris, at laboratory and pilot scale. This goal was achieved by specifically focussing on key bioprocessing parameters, namely the substrate feed rate during fed-batch fermentation, fermentation process conditions including the dissolved oxygen tension (DOT), growth medium improvement and scale-up effects during enzyme production. In the first system, the glucose feed rate was used to optimise the specific growth rate of recombinant Saccharomyces cerevisiae for the production of α-glucuronidase. In the second system, a semi-defined growth medium was developed, and both the substrate feed rate and DOT were optimised in the production of β-fructofuranosidase (FFase) by Pichia pastoris. These two systems serve to demonstrate the potential for optimisation of fed-batch cultures to maximise production of industrial enzymes by engineered yeasts.