Document Detail


Relation between cell disruption conditions, cell debris particle size, and inclusion body release.
MedLine Citation:
PMID:  15449302     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The efficiency of physical separation of inclusion bodies from cell debris is related to cell debris size and inclusion body release and both factors should be taken into account when designing a process. In this work, cell disruption by enzymatic treatment with lysozyme and cellulase, by homogenization, and by homogenization with ammonia pretreatment is discussed. These disruption methods are compared on the basis of inclusion body release, operating costs, and cell debris particle size. The latter was measured with cumulative sedimentation analysis in combination with membrane-associated protein quantification by SDS-PAGE and a spectrophotometric peptidoglycan quantification method. Comparison of the results obtained with these two cell debris quantification methods shows that enzymatic treatment yields cell debris particles with varying chemical composition, while this is not the case with the other disruption methods that were investigated. Furthermore, the experiments show that ammonia pretreatment with homogenization increases inclusion body release compared to homogenization without pretreatment and that this pretreatment may be used to control the cell debris size to some extent. The enzymatic disruption process gives a higher product release than homogenization with or without ammonia pretreatment at lower operating costs, but it also yields a much smaller cell debris size than the other disruption process. This is unfavorable for centrifugal inclusion body purification in this case, where cell debris is the component going to the sediment and the inclusion body is the floating component. Nevertheless, calculations show that centrifugal separation of inclusion bodies from the enzymatically treated cells gives a high inclusion body yield and purity.
Authors:
Pim Van Hee; Anton P J Middelberg; Rob G J M Van Der Lans; Luuk A M Van Der Wielen
Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Biotechnology and bioengineering     Volume:  88     ISSN:  0006-3592     ISO Abbreviation:  Biotechnol. Bioeng.     Publication Date:  2004 Oct 
Date Detail:
Created Date:  2004-09-30     Completed Date:  2005-03-01     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  7502021     Medline TA:  Biotechnol Bioeng     Country:  United States    
Other Details:
Languages:  eng     Pagination:  100-10     Citation Subset:  IM    
Affiliation:
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
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MeSH Terms
Descriptor/Qualifier:
Biotechnology / methods*
Cell Fractionation*
Cell Separation / methods*
Centrifugation
Flocculation
Inclusion Bodies*
Particle Size
Pseudomonas putida / cytology*,  genetics

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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