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
Heterologous expression, characterization and applications of carbohydrate active enzymes and binding modules
KTH, School of Biotechnology (BIO), Wood Biotechnology.
2006 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Wood and wood products are of great economical and environmental importance, both in Sweden and globally. Biotechnology can be used both for achieving raw material of improved quality and for industrial processes such as biobleaching. Despite the enormous amount of carbon that is fixed as wood, the knowledge about the enzymes involved in the biosynthesis, re-organization and degradation of plant cell walls is relatively limited. In order to exploit enzymes more efficiently or to develop new biotechnological processes, it is crucial to gain a better understanding of the function and mechanism of the enzymes. This work has aimed to increase the knowledge about some of the enzymes putatively involved in the wood forming processes in Populus. Xyloglucan endotransglycosylases and a putative xylanase represent transglycosylating and hydrolytic enzymes, respectively. Carbohydrate binding modules represent non-catalytic modules, which bind to the substrate.

Among 24 genes encoding for putative xyloglucan endotransglycosylases or xyloglucan endohydrolases that were identified in the Populus EST database, two were chosen for further studies (PttXTH16-34 and PttXTH16-35). The corresponding proteins, PttXET16-34 and PttXET16-35, were expressed in P. pastoris, purified and biochemically characterized. The importance of the N-glycans was investigated by comparing the recombinant wild-type proteins with their deglycosylated counterparts. In order to obtain the large amounts of PttXET16-34 that were needed for crystallization and development of biotechnological applications, the conditions for the large-scale production of PttXET16-34 in a fermenter were optimized.

In microorganisms, endo-(1,4)-β-xylanases are important members of the xylan degrading machinery. These enzymes are also present in plants where they might fulfill a similar, but probably more restrictive function. One putative endo-(1,4)-β-xylanase, denoted PttXYN10A, was identified in the hybrid aspen EST library. Sequence analysis shows that this protein contains three putative carbohydrate-binding modules (CBM) from family 22 in addition to the catalytic module from GH10. Heterologous expression and reverse genetics were applied in order to elucidate the function of the catalytic module as well as the binding modules of PttXYN10A.

Just as in microorganisms, some of the carbohydrate active enzymes from plants have one or more CBM attached to the catalytic module. So far, a very limited number of plant CBMs has been biochemically characterized. A detailed bio-informatic analysis of the CBM family 43 revealed interesting modularity patterns. In addition, one CBM43 (CBM43PttGH17_84) from a putative Populus b-(1,3)-glucanase was expressed in E. coli and shown to bind to laminarin (β-(1,3)-glucan), mixed-linked β-(1,3)(1,4)-glucans and crystalline cellulose. Due to their high specificity for different carbohydrates, CBMs can be used as probes for the analysis of plant materials. Generally, they are more specific than both staining techniques and carbohydrate-binding antibodies. We have used cellulose- and mannan binding modules from microorganisms as tools for the analysis of intact fibers as well as processed pulps.

Place, publisher, year, edition, pages
Stockholm: KTH , 2006. , 69 p.
Keyword [en]
Populus, xyloglucan endotransglycosylase, carbohydrate binding modules, endo-(1, 4)-b-xylanase, Escherichia coli, Pichia pastoris, N-glycosylation, fiber analysis
National Category
Industrial Biotechnology Wood Science
Identifiers
URN: urn:nbn:se:kth:diva-3950ISBN: 91-7178-349-0 (print)OAI: oai:DiVA.org:kth-3950DiVA: diva2:10160
Public defence
2006-05-24, FR4, Albanova University Center, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20100903Available from: 2006-05-11 Created: 2006-05-11 Last updated: 2011-11-23Bibliographically approved
List of papers
1. Enzymatic properties of native and deglycosylated hybrid aspen (Populus tremula x tremuloides) xyloglucan endotransglycosylase 16A expressed in Pichia pastoris
Open this publication in new window or tab >>Enzymatic properties of native and deglycosylated hybrid aspen (Populus tremula x tremuloides) xyloglucan endotransglycosylase 16A expressed in Pichia pastoris
Show others...
2005 (English)In: Biochemical Journal, ISSN 0264-6021, Vol. 390, 105-113 p.Article in journal (Refereed) Published
Abstract [en]

The cDNA encoding a xyloglucan endotransglycosylase, PttXET16A, from hybrid aspen (Populus tremula x tremuloides) has been isolated from an expressed sequence tag library and expressed in the methylotrophic yeast Pichia pastoris. Sequence analysis indicated a high degree of similarity with other proteins in the XTH (xyloglucan transglycosylase/hydrolase) gene subfamily of GH16 (glycoside hydrolase family 16). In addition to the conserved GH16 catalytic sequence motif, PttXET16A contains a conserved N-glycosylation site situated proximal to the predicted catalytic residues. MS analysis indicated that the recombinant PttXET16A expressed in P. pastoris is heterogeneous due to the presence of variable N-glycosylation and incomplete cleavage of the a-factor secretion signal peptide. Removal of the N-glycan by endoglycosidase H treatment did not influence the catalytic activity significantly. Similarly, site-directed mutagenesis of Asn(93) to serine to remove the N-glycosylation site resulted in an enzyme which was comparable with the wild-type enzyme in specific activity and thermal stability but had clearly reduced solubility. Hydrolytic activity was detected neither in wild-type PttXET16A before or after enzymatic deglycosylation nor in PttXET16A N93S (Asn(93) -> Ser) mutant.

Keyword
glycoside hydrolase family 16 (GH16), hybrid aspen (Populus tremula x tremuloides), Pichia pastoris, protein glycosylation, protein MS, xyloglucan endotransglycosylase (XET)
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-5704 (URN)10.1042/BJ20041749 (DOI)000231492800012 ()2-s2.0-23944474570 (Scopus ID)
Note
QC 20100903Available from: 2006-05-11 Created: 2006-05-11 Last updated: 2010-09-03Bibliographically approved
2. Enzymatic characterization of a recombinant xyloglucan endotransglycosylase PttXET16-35 from Populus tremula x tremuloides
Open this publication in new window or tab >>Enzymatic characterization of a recombinant xyloglucan endotransglycosylase PttXET16-35 from Populus tremula x tremuloides
Show others...
(English)Manuscript (Other academic)
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-5705 (URN)
Note
QC 20100624Available from: 2006-05-11 Created: 2006-05-11 Last updated: 2010-09-03Bibliographically approved
3. Production of poplar xyloglucan endotransglycosylase using the methylotrophic yeast Pichia pastoris
Open this publication in new window or tab >>Production of poplar xyloglucan endotransglycosylase using the methylotrophic yeast Pichia pastoris
Show others...
2005 (English)In: Applied Biochemistry and Biotechnology, ISSN 0273-2289, Vol. 126, 61-77 p.Article in journal (Refereed) Published
Abstract [en]

The gene XET16A encoding the enzyme xyloglucan endotransglycosylase (XET) from hybrid aspen (Populus tremula x tremuloides Mich) was transformed into Pichia pastoris GS115 and the enzyme was secreted to the medium. The influence of process conditions on the XET production, activity, and proteolytic degradation were examined. Inactivation of XET occurred in the foam, but could be decreased significantly by using an efficient antifoam. Rich medium (yeast extract plus peptone) was needed for product accumulation, but not for growth. The proteolytic degradation of the enzyme in the medium was substantially decreased by also adding yeast extract and peptone to the glycerol medium before induction with methanol. Decreasing the fermentation pH from 5.0 to 4.0 further reduced the proteolysis. The specific activity was further improved by production at 15 degrees C instead of 22 degrees C. In this way a XET production of 54 mg/L active enzyme could be achieved in the process with a specific activity of 18 Unit/mg protein after a downstream process including centrifugation, micro- and ultrafiltration, and ion exchange chromatography.

Keyword
Pichia pastoris, xyloglucan endotransglycosylase, foaming, proteolysis
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-5706 (URN)10.1007/s12010-005-0006-4 (DOI)000230687500006 ()2-s2.0-22344448657 (Scopus ID)
Note
QC 20100903Available from: 2006-05-11 Created: 2006-05-11 Last updated: 2010-09-03Bibliographically approved
4. Characterization of a CBM43 module from hybrid aspen (Populus tremula x tremuloides): specificity of polysaccharide interaction and bioinformatic analysis
Open this publication in new window or tab >>Characterization of a CBM43 module from hybrid aspen (Populus tremula x tremuloides): specificity of polysaccharide interaction and bioinformatic analysis
Show others...
(English)Manuscript (Other academic)
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-5707 (URN)
Note
QC 20100903Available from: 2006-05-11 Created: 2006-05-11 Last updated: 2010-09-03Bibliographically approved
5. Mapping of crystalline cellulose and mannan on the surfaces of wood tissues and pulp fibers using carbohydrate binding modules
Open this publication in new window or tab >>Mapping of crystalline cellulose and mannan on the surfaces of wood tissues and pulp fibers using carbohydrate binding modules
Show others...
2007 (English)In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 8, no 1, 91-97 p.Article in journal (Refereed) Published
Abstract [en]

Carbohydrate binding modules (CBMs) are noncatalytic substrate binding domains of many enzymes involved in carbohydrate metabolism. Here we used fluorescent labeled recombinant CBMs specific for crystalline cellulose (CBM1(HjCel7A)) and mannans (CBM27(TmMan5) and CBM35(CjMan5C)) to analyze the complex surfaces of wood tissues and pulp fibers. The crystalline cellulose CBM1(HjCel7A) was found as a reliable marker of both bacterially produced and plant G-layer cellulose, and labeling of spruce pulp fibers with CBM1(HjCel7A) revealed a signal that increased with degree of fiber damage. The mannan-specific CBM27(TmMan5) and CBM35(CjMan5C) CBMs were found to be more specific reagents than a monoclonal antibody specific for (1 -> 4)-beta-mannan/galacto-(1 -> 4)-beta-mannan for mapping carbohydrates on native substrates. We have developed a quantitative fluorometric method for analysis of crystalline cellulose accumulation on fiber surfaces and shown a quantitative difference in crystalline cellulose binding sites in differently processed pulp fibers. Our results indicated that CBMs provide useful, novel tools for monitoring changes in carbohydrate content of nonuniform substrate surfaces, for example, during wood or pulping processes and possibly fiber biosynthesis.

Keyword
plant-cell walls, trichoderma-reesei, tension-wood, cellobiohydrolase-i, domain, family, identification, recognition, architecture, degradation
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-5708 (URN)10.1021/bm060632z (DOI)000243337600013 ()2-s2.0-33846461294 (Scopus ID)
Note
QC 20100903Available from: 2006-05-11 Created: 2006-05-11 Last updated: 2010-09-03Bibliographically approved

Open Access in DiVA

fulltext(1703 kB)2816 downloads
File information
File name FULLTEXT01.pdfFile size 1703 kBChecksum MD5
4b9d9d22e65918aa5e33de4df1e9053326c03151016a4a2e81498d2d9daa1d487705ff73
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Kallas, Åsa
By organisation
Wood Biotechnology
Industrial BiotechnologyWood Science

Search outside of DiVA

GoogleGoogle Scholar
Total: 2816 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 1027 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