Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Molecular features of family GH9 cellulases in hybrid aspen and the filamentous fungus Phanerochaete chrysosporium
KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
2003 (English)In: Journal of applied glycoscience, ISSN 1344-7882, Vol. 50, no 2, 253-256 p.Article in journal (Refereed) Published
Abstract [en]

Most plant cellulases belong to the glycosyl hydrolase family 9, which is both enzymatically and structurally well characterized in microbes. The microbial enzymes include both randomly acting endoglucanases active on soluble and amorphous substrates and processive endoglucanases, which can also degrade crystalline cellulose. The corresponding plant enzymes have been difficult to purify and express in heterologous hosts and thus their modes of action have remained obscure. Here we have taken a molecular modelling approach and describe some structural features characteristic for soluble and membrane-bound family 9 cellulases in plants and filamentous fungi.

Place, publisher, year, edition, pages
2003. Vol. 50, no 2, 253-256 p.
Keyword [en]
POPULUS TREMULA, POPULUS TREMULOIDES, HYBRIDS, CELLULASE, PHANEROCHAETE CHRYSOSPORIUM, CELLULOSE
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-7586OAI: oai:DiVA.org:kth-7586DiVA: diva2:12658
Note
QC 20100802Available from: 2007-11-07 Created: 2007-11-07 Last updated: 2010-08-02Bibliographically approved
In thesis
1. Functional studies of a membrane-anchored cellulase from poplar
Open this publication in new window or tab >>Functional studies of a membrane-anchored cellulase from poplar
2007 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Cellulose in particular and wood in general are valuable biomaterials for humanity, and cellulose is now also in the spotlight as a starting material for the production of biofuel. Understanding the processes of wood formation and cellulose biosynthesis could therefore be rewarding, and genomics and proteomics approaches have been initiated to learn more about wood biology. For example, the genome of the tree Populus trichocarpa has been completed during 2006. A single-gene approach then has to follow, to elucidate specific patterns and enzymatic details.

This thesis depicts how a gene encoding a membrane-anchored cellulase was isolated from Populus tremula x tremuloides Mich, how the corresponding protein was expressed in heterologous hosts, purified and characterized by substrate analysis using different techniques. The in vivo function and modularity of the membrane-anchored cellulase was also addressed using overexpression and complementation analysis in Arabidopsis thaliana.

Among 9 genes found in the Populus EST database, encoding enzymes from glycosyl hydrolase family 9, two were expressed in the cambial tissue, and the membrane-anchored cellulase, PttCel9A1, was the most abundant transcript. PttCel9A1 was expressed in Pichia pastoris, and purified by affinity chromatography and ion exchange chromatography. The low yield of recombinant protein from shake flask experiments was improved by scaling up in the fermentor. PttCel9A1 was however highly heterogenous, both mannosylated and phosphorylated, which made the protein unsuitable for crystallization experiments and 3D X-ray structure determination. Instead, a homology model using a well-characterized, homologous bacterial enzyme was built. From the homology model, interesting point mutations in the active site cleft that would highlight the functional differences of the two proteins could be identified. The real-time cleavage patterns of cello-oligosaccharides by mutant bacterial enzymes, the wildtype bacterial enzyme and PttCel9A1 were studied by 1H NMR spectroscopy, and compared with results from HPAEC-PAD analysis. The inverting stereochemistry for the hydrolysis reaction of the membrane-anchored poplar cellulase was also determined by 1H NMR spectroscopy, and it was concluded that transglycosylation in vivo is not a possible scenario.

The preferred in vitro polymeric substrates for PttCel9A1 were shown to be long, low-substituted cellulose derivatives, and the endo-1,4--glucanase activity was not extended to branched or mixed linkage substrates to detectable levels. This result indicates an in vivo function in the hydrolysis of “amorphous” regions of cellulose, either during polymerization or crystallization of cellulose. In addition, overexpressing PttCel9A1 in A. thaliana, demonstrated a correlation with decreased crystallinity of cellulose. The significance of the different putative modules of PttCel9A1 was investigated by the construction of hybrid proteins, that were introduced into a knock-out mutant of A. thaliana, and the potential complementation of the phenotype was examined. A type B plant cellulase catalytic domain could not substitute for a type A plant cellulase catalytic domain, although localization and interaction motifs were added to the N- and C-terminus.

Place, publisher, year, edition, pages
Stockholm: KTH, 2007. [7], 61 p.
Series
Trita-ARK. Akademisk avhandling, ISSN 1402-7461
Keyword
Populus, cellulose biosynthesis, endo-1, 4-b-glucanase, cellulase, Pichia pastoris, Arabidopsis thaliana, NMR spectroscopy, cleavage pattern, stereochemistry, transglycosylation, complementation, hybrid proteins, overexpression, substrate analysis, overglycosylation
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-4520 (URN)978-91-7178-790-3 (ISBN)
Public defence
2007-11-23, FB42, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20100802Available from: 2007-11-07 Created: 2007-11-07 Last updated: 2010-08-02Bibliographically approved

Open Access in DiVA

No full text

Search in DiVA

By author/editor
Rudsander, UllaDenman, StuartTeeri, Tuula
By organisation
Albanova VinnExcellence Center for Protein Technology, ProNova
Industrial Biotechnology

Search outside of DiVA

GoogleGoogle Scholar

urn-nbn

Altmetric score

urn-nbn
Total: 145 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf