Lee, Yew Choon; (1991) The process scale release of intracellular enzymes from filamentous microorganisms. Doctoral thesis (Ph.D.), University College London.

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Abstract

The impact of fermentation and processing conditions on the disruption of a strain of Aspergillus niger for the release of intracellular enzymes has been examined. High pressure homogenisers were used, and the objective was to seek the optimisation of the fermentation, harvesting and disruption stages to maximise the production of intracellular enzymes. One major variable studied was the effect of cell morphology on enzyme release. The control work centred on filamentous and highly entangled or "clumped" microorganisms and here, the release of intracellular protein and enzymes was shown to be highly pressure dependent, and a weak function of the number of passes (after the first pass) through the homogeniser. Complete release of protein and glucose-6-phosphate dehydrogenase (G6PDH), a freely soluble cytoplasmic enzyme was achieved after 2 or 3 passes at 80 MPa. Glucose oxidase (G.O.), a peroxisomally (membrane bound organelle) located cytoplasmic enzyme follows similar kinetics but was only released fully at 100 MPa. The morphology was changed to pelleted forms of up to 1.8 mm diameter by a decrease in spore inoculum concentration. This change in morphology from the "clumped" form did not alter the shapes of the disruption curves although there was a trend towards greater rate of release with increasing pellet size: e.g. 30-40 % more protein was released for pellets of 1.8 mm diameter at 60 MPa for a single pass, compared to the filamentous "clumped" morphology. "Junlon", a poly aery lie resin, incorporated in the growth medium, led to a more freely filamentous morphology and a 20% increase in G6PDH release. However, filamentous growth arising from a reduction in agitation speed did not result in any change in the rate of release. The effect of fermentation broth conditions on protein and enzyme release was also studied, the most important result being the effect of a change in the resuspending buffer for disruption from Tris HCl at pH 7.5 to an acetate buffer at pH 5.2. This led to the precipitation of 90% of the released protein, but not any of the glucose oxidase, and hence a considerable increase in the specific activity of the released enzyme. A mechanism of disruption for filamentous microorganism is postulated. During the first pass, there could be disentanglement of the mycelia resulting in disruption. At subsequent passes, the disruption kinetics appears to follow first order kinetics. This two stage process adequately describes the release of protein, G6PDH or G.O., the model equation being: (R1/ Rm) = k1.pa (model for N=l) where is the amount of product released at the first pass (N=l); R1 is the maximum product released; k1 is a rate constant; P is pressure and "a" is the pressure exponent. For subsequent passes (N > 1) the disruption can be described by a first order rate equation: log10 [(Rm - R1) / (Rm - R)] = k2. pb.(N - 1) (model for N > 1) where R is the product released at N number of passes; k2 is another rate constant, and "b" another pressure exponent. These two models are applicable to all the various morphologies, thus indicating a similar mechanism of disruption. It is the rate constants and pressure exponents which change with the different morphologies. The implications of this study on further purification and process development are discussed.

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