Advanced

Improving selectivity in protein extraction by subtle interactions and environmental changes

Fernandes, Sheryl LU (2001)
Abstract
The challenges for efficient bioprocesses lie in developing and adapting further current technologies or in creating innovative separation technologies. The thesis is based on the studies on protein recovery from crude feedstocks by extraction in aqueous two-phase systems and detergent foam, respectively. The separation technologies have been adapted to suit individual proteins by utilizing different forms of molecular interactions and environmental changes in a very simple way. Interaction of the lipolytic enzyme, cutinase with it's substrate, butyrate resulted in sufficient conformational changes to change it's extraction behavior in a polyethylene glycol (PEG)/salt system. The ability of the polymer, polyvinylpyrrolidone (PVP) to... (More)
The challenges for efficient bioprocesses lie in developing and adapting further current technologies or in creating innovative separation technologies. The thesis is based on the studies on protein recovery from crude feedstocks by extraction in aqueous two-phase systems and detergent foam, respectively. The separation technologies have been adapted to suit individual proteins by utilizing different forms of molecular interactions and environmental changes in a very simple way. Interaction of the lipolytic enzyme, cutinase with it's substrate, butyrate resulted in sufficient conformational changes to change it's extraction behavior in a polyethylene glycol (PEG)/salt system. The ability of the polymer, polyvinylpyrrolidone (PVP) to complex reactive dyes and metal ions was used to set up a selective extraction process for the proteins (porcine Lactate dehydrogenase (LDH) and His6-LDH) binding these entities in a two-phase system employing PVP as one of the phase forming components. Extraction in aqueous two-phase systems was found to be an efficient separation technique for cold-active, highly labile enzyme molecules. Cold-active ß-galactosidase was purified with high yields using extraction as a primary separation sep. In case of cold-active ß-lactamase cloned in a mesophilic host, partial denaturation of the labile enzyme under conditions when the host proteins were still intact, allowed selective extraction in a PEG/salt system. Foam, generated by stirring of a non-ionic detergent Triton X-114 was used as a separation tool for isolation of cutinase mutants. The mutant with the hydrophobic fusion tag, (Trp-Pro)4 was recovered with high yields due to the stronger interaction with the detergent. Protein extraction into the foam was made more selective by integration of affinity interactions. The foam prepared from detergent modified with Cibacron blue could selectively extract hydrophilic LDH from muscle extract. The thermoseparating property of Triton X-114 further helped easy separation of the target enzyme from the bulk detergent. (Less)
Please use this url to cite or link to this publication:
author
opponent
  • Dr Jungbauer, Alois
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Difference Spectroscopy, Circular dichroism, Biotechnology, Bioteknik, Chemical technology and engineering, Kemiteknik och kemisk teknologi, Fluorescence spectroscopy, Metal ions, Cibacron blue, Polyvinylpyrrolidone, Polyethylene glycol, Triton X-114, His 6 tag, Cold-active ß-lactamase, Cold-active ß-galactosidase, Lactate dehydrogenase, Cutinase, Affinity Extraction, Conformational change, Partial Denaturation, Molecular interaction, Aqueous two-phase systems, Detergent foam
pages
200 pages
publisher
Department of Biotechnology, Lund University
defense location
Sal C, Kemicentrum
defense date
2001-12-17 10:15
external identifiers
  • Other:ISRN: LUTKDH/TKBT....01/1055--SE
ISBN
91-89627-03-2
language
English
LU publication?
yes
id
c5900631-48a7-4d60-a49b-841c978d2afa (old id 42225)
date added to LUP
2007-06-20 15:00:03
date last changed
2016-09-19 08:45:14
@misc{c5900631-48a7-4d60-a49b-841c978d2afa,
  abstract     = {The challenges for efficient bioprocesses lie in developing and adapting further current technologies or in creating innovative separation technologies. The thesis is based on the studies on protein recovery from crude feedstocks by extraction in aqueous two-phase systems and detergent foam, respectively. The separation technologies have been adapted to suit individual proteins by utilizing different forms of molecular interactions and environmental changes in a very simple way. Interaction of the lipolytic enzyme, cutinase with it's substrate, butyrate resulted in sufficient conformational changes to change it's extraction behavior in a polyethylene glycol (PEG)/salt system. The ability of the polymer, polyvinylpyrrolidone (PVP) to complex reactive dyes and metal ions was used to set up a selective extraction process for the proteins (porcine Lactate dehydrogenase (LDH) and His6-LDH) binding these entities in a two-phase system employing PVP as one of the phase forming components. Extraction in aqueous two-phase systems was found to be an efficient separation technique for cold-active, highly labile enzyme molecules. Cold-active ß-galactosidase was purified with high yields using extraction as a primary separation sep. In case of cold-active ß-lactamase cloned in a mesophilic host, partial denaturation of the labile enzyme under conditions when the host proteins were still intact, allowed selective extraction in a PEG/salt system. Foam, generated by stirring of a non-ionic detergent Triton X-114 was used as a separation tool for isolation of cutinase mutants. The mutant with the hydrophobic fusion tag, (Trp-Pro)4 was recovered with high yields due to the stronger interaction with the detergent. Protein extraction into the foam was made more selective by integration of affinity interactions. The foam prepared from detergent modified with Cibacron blue could selectively extract hydrophilic LDH from muscle extract. The thermoseparating property of Triton X-114 further helped easy separation of the target enzyme from the bulk detergent.},
  author       = {Fernandes, Sheryl},
  isbn         = {91-89627-03-2},
  keyword      = {Difference Spectroscopy,Circular dichroism,Biotechnology,Bioteknik,Chemical technology and engineering,Kemiteknik och kemisk teknologi,Fluorescence spectroscopy,Metal ions,Cibacron blue,Polyvinylpyrrolidone,Polyethylene glycol,Triton X-114,His 6 tag,Cold-active ß-lactamase,Cold-active ß-galactosidase,Lactate dehydrogenase,Cutinase,Affinity Extraction,Conformational change,Partial Denaturation,Molecular interaction,Aqueous two-phase systems,Detergent foam},
  language     = {eng},
  pages        = {200},
  publisher    = {ARRAY(0x85f9eb8)},
  title        = {Improving selectivity in protein extraction by subtle interactions and environmental changes},
  year         = {2001},
}