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Affinity maturation generates greatly improved xyloglucan-specific carbohydrate binding modules
KTH, School of Biotechnology (BIO), Glycoscience.
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2009 (English)In: BMC Biotechnology, ISSN 1472-6750, Vol. 9Article in journal (Refereed) Published
Abstract [en]

Background: Molecular evolution of carbohydrate binding modules (CBM) is a new approach for the generation of glycan-specific molecular probes. To date, the possibility of performing affinity maturation on CBM has not been investigated. In this study we show that binding characteristics such as affinity can be improved for CBM generated from the CBM4-2 scaffold by using random mutagenesis in combination with phage display technology. Results: Two modified proteins with greatly improved affinity for xyloglucan, a key polysaccharide abundant in the plant kingdom crucial for providing plant support, were generated. Both improved modules differ from other existing xyloglucan probes by binding to galactose-decorated subunits of xyloglucan. The usefulness of the evolved binders was verified by staining of plant sections, where they performed better than the xyloglucan-binding module from which they had been derived. They discriminated non-fucosylated from fucosylated xyloglucan as shown by their ability to stain only the endosperm, rich in non-fucosylated xyloglucan, but not the integument rich in fucosylated xyloglucan, on tamarind seed sections. Conclusion: We conclude that affinity maturation of CBM selected from molecular libraries based on the CBM4-2 scaffold is possible and has the potential to generate new analytical tools for detection of plant carbohydrates.

Place, publisher, year, edition, pages
2009. Vol. 9
Keyword [en]
plant-cell walls, somatic hypermutation, protein stability, antibodies, selection, recognition, diversity, mechanism, evolution, display
URN: urn:nbn:se:kth:diva-19010DOI: 10.1186/1472-6750-9-92ISI: 000272337000001ScopusID: 2-s2.0-71049184894OAI: diva2:337057
QC 20100902Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2010-09-02Bibliographically approved
In thesis
1. On the engineering of proteins: methods and applications for carbohydrate-active enzymes
Open this publication in new window or tab >>On the engineering of proteins: methods and applications for carbohydrate-active enzymes
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis presents the application of different protein engineering methods on enzymes and non-catalytic proteins that act upon xyloglucans. Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glucans in the cell wall of plants. Their structure is complex with intricate branching patterns, which contribute to the physical properties of the polysaccharide including its binding to and interaction with other glucans such as cellulose.

One important group of xyloglucan-active enzymes is encoded by the GH16 XTH gene family in plants, including xyloglucan endo-transglycosylases (XET) and xyloglucan endo-hydrolases (XEH). The molecular determinants behind the different catalytic routes of these homologous enzymes are still not fully understood. By combining structural data and molecular dynamics (MD) simulations, interesting facts were revealed about enzyme-substrate interaction. Furthermore, a pilot study was performed using structure-guided recombination to generate a restricted library of XET/XEH chimeras.

Glycosynthases are hydrolytically inactive mutant glycoside hydrolases (GH) that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Different enzymes with xyloglucan hydrolase activity were engineered into glycosynthases, and characterised as tools for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns.

Carbohydrate-binding modules (CBM) are non-catalytic protein domains that bind to polysaccharidic substrates. An important technical application involves their use as molecular probes to detect and localise specific carbohydrates in vivo. The three-dimensional structure of an evolved xyloglucan binding module (XGBM) was solved by X-ray diffraction. Affinity-guided directed evolution of this first generation XGBM resulted in highly specific probes that were used to localise non-fucosylated xyloglucans in plant tissue sections.

Place, publisher, year, edition, pages
Stockholm: KTH, 2010. xii, 74 p.
Trita-BIO-Report, ISSN 1654-2312 ; 2010:14
enzyme engineering, rational design, directed evolution, DNA shuffling, glycosynthase, xyloglucan, xyloglucan endo-transglycosylase, retaining glycoside hydrolase, xyloglucanase, carbohydrate binding module, polysaccharide synthesis
National Category
Industrial Biotechnology
urn:nbn:se:kth:diva-24296 (URN)978-91-7415-709-3 (ISBN)
Public defence
2010-09-22, FD5, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 10:15 (English)
QC 20100902Available from: 2010-09-02 Created: 2010-08-31 Last updated: 2010-09-02Bibliographically approved

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