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Title: Stagewise optimisation of extracellular enzyme extraction
Author: Barton, M.
Awarding Body: University of Wales Swansea
Current Institution: Swansea University
Date of Award: 1998
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The extraction of lipase from fermentation broth using membrane separations was investigated. Lipase was produced from Pseudomonas fluorescens by aerobic fermentation with a corn oil substrate. Fermentation broths of up to 50 1 volume were microfiltered using ceramic and polypropylene crossflow modules. The permeation of lipase was found to be highly dependent on pH. The separation characteristics of the fermentation broths were further investigated on a small scale, using dead end and cross flow microfiltration. Hydrophilic membranes (ceramic and glassfibre) showed an optimum pH for lipase permeation centred around pH 6.8, with rejection coefficients for lipase falling from near unity to R=0.5. This pH peak was not at the enzyme iso-electric point (pH 4.5). Filtration using hydrophobic membranes (polypropylene and polysulphone) showed no such pH dependence, but these had high rejection coefficients across a range of pH values (R = 0.9-1.0 at pH values between 4.8 and 8.5). Rejection coefficients were lower in the absence of corn oil than when corn oil was present for all the membranes used. When protein solutions (BSA and lipase) were microfiltered more protein was adsorbed onto the surface of the hydrophobic than hydrophilic membranes. More lipase adsorbed when corn oil was present. Separation characteristics were due to lipase/corn oil/membrane interactions. Proteins other than lipase were found to permeate preferentially to the lipase at values away from the optimum pH value for lipase permeation. A process was proposed and tested in which the fermentation broth was harvested using a two stage microfiltration process. The fermentation broth was microfiltered at a pH where lipase would not permeate, to increase lipase concentration and specific activity. The pH was then adjusted to allow the concentrated lipase solution to permeate. This gave good overall yields (60% of original activity) and an increase in the specific activity of the lipase- 60 U/mg total protein for the two stage microfiltration from a feed of 9 U/mg protein. Conventional microfiltration gave no such increase in specific activity.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available