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Functional studies of a membrane-anchored cellulase from poplar
KTH, School of Biotechnology (BIO), Wood Biotechnology.
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 [en]
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: urn:nbn:se:kth:diva-4520ISBN: 978-91-7178-790-3 (print)OAI: oai:DiVA.org:kth-4520DiVA: diva2:12662
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
List of papers
1. Molecular features of family GH9 cellulases in hybrid aspen and the filamentous fungus Phanerochaete chrysosporium
Open this publication in new window or tab >>Molecular features of family GH9 cellulases in hybrid aspen and the filamentous fungus Phanerochaete chrysosporium
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.

Keyword
POPULUS TREMULA, POPULUS TREMULOIDES, HYBRIDS, CELLULASE, PHANEROCHAETE CHRYSOSPORIUM, CELLULOSE
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-7586 (URN)
Note
QC 20100802Available from: 2007-11-07 Created: 2007-11-07 Last updated: 2010-08-02Bibliographically approved
2. Recombinant Expression and Enzymatic characterization of PttCel9A, a KOR homologue from Populus tremula x tremuloides
Open this publication in new window or tab >>Recombinant Expression and Enzymatic characterization of PttCel9A, a KOR homologue from Populus tremula x tremuloides
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2004 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 31, 10080-10089 p.Article in journal (Refereed) Published
Abstract [en]

PttCel9A is a membrane-bound, family 9 glycosyl hydrolase from Populus tremula x tremuloides that is upregulated during secondary cell wall synthesis. The catalytic domain of PttCel9A, Delta(1-105)PttCel9A, was purified, and its activity was compared to TfCel9A and TfCel9B from Thermobifida fusca. Since aromatic amino acids involved in substrate binding at subsites -4, -3, and -2 are missing in PttCel9A, the activity of TfCel9A mutant enzymes W256S, W209A, and W313G was also investigated. Delta(1-105)PttCel9A hydrolyzed a comparatively narrow range of polymeric substrates, and the preferred substrate was (carboxymethyl)cellulose 4M. Moreover, Delta(1-105)PttCel9A did not hydrolyze oligosaccharides shorter than cellopentaose, whereas TfCel9A and TfCel9B hydrolyzed cellotetraose and cellotriose, respectively. These data suggest that the preferred substrates of PttCel9A are long, low-substituted, soluble cellulosic polymers. At 30degreesC and pH 6.0, the k(cat) for cellohexaose of Delta(1-105)PttCel9A, TfCel9A, and TfCel9B were 0.023 +/- 0.001, 16.9 +/- 2.0, and 1.3 +/- 0.2, respectively. The catalytic efficiency (k(cat)/K-m) of TfCel9B was 39% of that of TfCel9A, whereas the catalytic efficiency of Delta(1-105)PttCel9A was 0.04% of that of TfCel9A. Removing tryptophan residues at subsites -4, -3, and -2 decreased the efficiency of cellohexaose hydrolysis by TfCel9A. Mutation of W313 to G had the most drastic effect, producing a mutant enzyme with 1% of the catalytic efficiency of TfCel9A. The apparent narrow substrate range and catalytic efficiency of PttCel9A are correlated with a lack of aromatic amino acids in the substrate binding cleft and may be necessary to prevent excessive hydrolysis of cell wall polysaccharides during cell wall formation.

Keyword
CULTURED POPLAR CELLS; IN-GEL DIGESTION; CELLULOSE SYNTHESIS; THERMOMONOSPORA-FUSCA; ARABIDOPSIS-THALIANA; AGROBACTERIUM-TUMEFACIENS; CRYSTAL-STRUCTURE; ENDO-1, 4-BETA-GLUCANASE; PROTEINS; KORRIGAN
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-7587 (URN)10.1021/bi049453x (DOI)000223121200016 ()2-s2.0-3543110792 (Scopus ID)
Note
QC 20100802 QC 20110927Available from: 2007-11-07 Created: 2007-11-07 Last updated: 2017-12-14Bibliographically approved
3. Comparative NMR analysis of cello-oligosaccharide hydrolysis by family GH9 endoglucanases from Thermobifida fusca and Populus tremula x tremuloides Mich
Open this publication in new window or tab >>Comparative NMR analysis of cello-oligosaccharide hydrolysis by family GH9 endoglucanases from Thermobifida fusca and Populus tremula x tremuloides Mich
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(English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995Article in journal (Refereed) Submitted
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-7588 (URN)
Note
QC 20100802Available from: 2007-11-07 Created: 2007-11-07 Last updated: 2017-12-14Bibliographically approved
4. Expression of aspen homologue of KORRIGAN1, PttCel9A1, affects cellulose crystallinity in secondary growth
Open this publication in new window or tab >>Expression of aspen homologue of KORRIGAN1, PttCel9A1, affects cellulose crystallinity in secondary growth
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(English)Manuscript (preprint) (Other academic)
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-7589 (URN)
Note
QC 20100802Available from: 2007-11-07 Created: 2007-11-07 Last updated: 2010-08-02Bibliographically approved

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