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Design of GH16 XET/XEH chimeric enzymes with SCHEMA: Manuscript
KTH, School of Biotechnology (BIO), Glycoscience. KTH, School of Biotechnology (BIO), Centres, Swedish Center for Biomimetic Fiber Engineering, BioMime.
KTH, School of Biotechnology (BIO), Glycoscience.
KTH, School of Biotechnology (BIO), Glycoscience.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

This manuscript contains experimental data obtained during a pilot study for the application of the SCHEMA method for structure-guided recombination on PttXET16-34 and TmNXG1, a model system for the evolution of different catalytic routes of GH16 XETs and XEHs.

A restricted library of PttXET16-34/TmNXG1 chimeras with high diversity and low calculated SCHEMA disruption was generated based on crossover points identified by the RASPP algorithm. Analysis of the library revealed a bias among certain regions to remain intact and recalcitrant to recombination, in particular the upper and lower β-sheet structures forming the part of the protein that binds the donor substrate. In contrast, sequence diversity was preferentially introduced at the N-terminus, the major part of the acceptor side of the protein, and most of the C-terminal extension characteristic to XET/XEH in the GH16 family. Finally, in order to test the predictive capacity of SCHEMA, six chimeras with low calculated disruption were chosen for subsequent cloning and expression in Pichia pastoris.

National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-24298OAI: oai:DiVA.org:kth-24298DiVA: diva2:346379
Note
QC 20100902Available from: 2010-08-31 Created: 2010-08-31 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.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2010:14
Keyword
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
Identifiers
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)
Opponent
Supervisors
Note
QC 20100902Available from: 2010-09-02 Created: 2010-08-31 Last updated: 2010-09-02Bibliographically approved

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