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Genetic engineering of the Trichoderma reesei endoglucanase I (Cel7B) for enhanced partitioning in aqueous two-phase systems containing thermoseparating ethylene oxide-propylene oxide copolymers

Collén, Anna LU ; Ward, Michael; Tjerneld, Folke LU and Stålbrand, Henrik LU (2001) In Journal of Biotechnology 87(2). p.179-191
Abstract
Endoglucanases (endo-1,4---glucan-4-glucanohydrolase, EC 3.2.1.4) are industrially important enzymes. In this study endoglucanase I (EGI or Cel7B) of the filamentous fungi Trichoderma reesei has been genetically engineered to investigate the influence of tryptophan rich peptide extensions (tags) on partitioning in an aqueous two-phase model system. EGI is a two-domain enzyme and is composed of a N-terminal catalytic domain and a C-terminal cellulose binding domain, separated by a linker. The aim was to find an optimal tag and fusion position, which further could be utilised for large scale extractions. Peptide tags of different length and composition were attached at various localisations of EGI. The fusion proteins were expressed from T.... (More)
Endoglucanases (endo-1,4---glucan-4-glucanohydrolase, EC 3.2.1.4) are industrially important enzymes. In this study endoglucanase I (EGI or Cel7B) of the filamentous fungi Trichoderma reesei has been genetically engineered to investigate the influence of tryptophan rich peptide extensions (tags) on partitioning in an aqueous two-phase model system. EGI is a two-domain enzyme and is composed of a N-terminal catalytic domain and a C-terminal cellulose binding domain, separated by a linker. The aim was to find an optimal tag and fusion position, which further could be utilised for large scale extractions. Peptide tags of different length and composition were attached at various localisations of EGI. The fusion proteins were expressed from T. reesei with the use of the gpdA promoter from Aspergillus nidulans. Variations in secreted levels between the engineered proteins were obtained. The partitioning of EGI in an aqueous two-phase system composed of a thermoseparating ethylene oxide–propylene oxide random copolymer (EO50PO50) and dextran, could be significantly improved by relatively minor genetic engineering. The (Trp-Pro)4 tag added after a short stretch of the linker, containing five proline residues, gave in the highest partition coefficient of 12.8. The yield in the top phase was 94%. The specific activity was 83% of the specific activity of unmodified EGI on soluble substrate. The efficiency of a tag fused to a protein is shown by the tag efficiency factor (TEF). A hypothetical TEF of 1.0 would indicate full tag exposure and optimal contribution to the protein partitioning by the fused tag. The location of the fusion point after the sequence of five proline residues in the linker of EGI is the most beneficial in two-phase separation. The highest TEF (0.97) was obtained with the (Trp-Pro)2 tag at this position, indicating full exposure and intactness of the tag. However, the peptide tag composed of (Trp-Pro)4 improved the partition properties the most but had lower TEF in comparison to (Trp-Pro)2 (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Separation, Aqueous two-phase system, Gene fusion, Trichoderma reesei, Endoglucanase I, Tryptophan
in
Journal of Biotechnology
volume
87
issue
2
pages
179 - 191
publisher
Elsevier
external identifiers
  • scopus:0035805435
ISSN
1873-4863
DOI
10.1016/S0168-1656(01)00241-3
language
English
LU publication?
yes
id
5b86f3b0-2bbf-4ac7-9faf-11bdecd83562 (old id 124942)
date added to LUP
2007-07-04 15:38:33
date last changed
2018-10-03 10:39:03
@article{5b86f3b0-2bbf-4ac7-9faf-11bdecd83562,
  abstract     = {Endoglucanases (endo-1,4---glucan-4-glucanohydrolase, EC 3.2.1.4) are industrially important enzymes. In this study endoglucanase I (EGI or Cel7B) of the filamentous fungi Trichoderma reesei has been genetically engineered to investigate the influence of tryptophan rich peptide extensions (tags) on partitioning in an aqueous two-phase model system. EGI is a two-domain enzyme and is composed of a N-terminal catalytic domain and a C-terminal cellulose binding domain, separated by a linker. The aim was to find an optimal tag and fusion position, which further could be utilised for large scale extractions. Peptide tags of different length and composition were attached at various localisations of EGI. The fusion proteins were expressed from T. reesei with the use of the gpdA promoter from Aspergillus nidulans. Variations in secreted levels between the engineered proteins were obtained. The partitioning of EGI in an aqueous two-phase system composed of a thermoseparating ethylene oxide–propylene oxide random copolymer (EO50PO50) and dextran, could be significantly improved by relatively minor genetic engineering. The (Trp-Pro)4 tag added after a short stretch of the linker, containing five proline residues, gave in the highest partition coefficient of 12.8. The yield in the top phase was 94%. The specific activity was 83% of the specific activity of unmodified EGI on soluble substrate. The efficiency of a tag fused to a protein is shown by the tag efficiency factor (TEF). A hypothetical TEF of 1.0 would indicate full tag exposure and optimal contribution to the protein partitioning by the fused tag. The location of the fusion point after the sequence of five proline residues in the linker of EGI is the most beneficial in two-phase separation. The highest TEF (0.97) was obtained with the (Trp-Pro)2 tag at this position, indicating full exposure and intactness of the tag. However, the peptide tag composed of (Trp-Pro)4 improved the partition properties the most but had lower TEF in comparison to (Trp-Pro)2},
  author       = {Collén, Anna and Ward, Michael and Tjerneld, Folke and Stålbrand, Henrik},
  issn         = {1873-4863},
  keyword      = {Separation,Aqueous two-phase system,Gene fusion,Trichoderma reesei,Endoglucanase I,Tryptophan},
  language     = {eng},
  number       = {2},
  pages        = {179--191},
  publisher    = {Elsevier},
  series       = {Journal of Biotechnology},
  title        = {Genetic engineering of the Trichoderma reesei endoglucanase I (Cel7B) for enhanced partitioning in aqueous two-phase systems containing thermoseparating ethylene oxide-propylene oxide copolymers},
  url          = {http://dx.doi.org/10.1016/S0168-1656(01)00241-3},
  volume       = {87},
  year         = {2001},
}