Process integration of cell disruption and fluidised bed adsorption of microbial enzymes: application to the retro-design of the purification of L-asparaginase

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http://eprints.org/relation/isVersionOf http://etheses.bham.ac.uk/id/document/167 http://eprints.org/relation/isVolatileVersionOf http://etheses.bham.ac.uk/id/document/167 http://eprints.org/relation/isIndexCodesVersionOf http://etheses.bham.ac.uk/id/document/167 archive 4031 disk0/00/00/00/71 2008-03-07 15:29:52 2019-01-23 09:51:28 2011-07-19 16:12:27 thesis show 0 Bierau Horst h.bierau@web.de Lyddiatt Andrew University of Birmingham Process integration of cell disruption and fluidised bed adsorption of microbial enzymes: application to the retro-design of the purification of L-asparaginase unpub TP 30sch_engi The practical feasibility and generic applicability of the direct integration in the same time frame of cell disruption by bead milling with the capture of intracellular products by fluidised bed adsorption has been demonstrated. Pilot-scale purification of the enzyme L-asparaginase from unclarified Erwinia chrysanthemi disruptates exploiting this novel approach yielded an interim product which rivalled or bettered that produced by the current commercial process employing discrete operations of alkaline lysis, centrifugal clarification and batch adsorption. In addition to improved yield and quality of product, the process time during primary stages of purification was greatly diminished. Two cation exchange adsorbents, CM HyperD LS (Biosepra/Life Technologies) and SP UpFront (custom made SP form of a prototype stainless steel/agarose matrix, UpFront Chromatography) were physically and biochemically evaluated for such direct product sequestration. Differences in performance with regard to product capacity and adsorption/desorption kinetics were demonstrated and are discussed with respect to the designof adsorbents for specific applications. In any purification of L-asparaginase (pI=8.6), product-debris interactions commonly diminish the recovery of available product. It was demonstrated herein, that immediate disruptate exposure to a fluidised bed adsorbent promoted concomitant reduction of product in the liquid phase, which clearly counter-acted the product-debris interactions to the benefit of overall product yield. 2001 graduation 2115137 eng author unchecked This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Centre for Applied Microbiology and Research public 182 University of Birmingham School of Chemical Engineering d_ph na 2001 graduation