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Functional Characterization of Xyloglucan Glycosynthases from GH7, GH12, and GH16 Scaffolds
KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
Vise andre og tillknytning
2009 (engelsk)Inngår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 10, nr 7, s. 1782-1788Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Glycosynthases, hydrolytically inactive mutant glycosidases that catalyze glycosylation reactions using glycosyl fluoride donor substrates, are emerging as useful tools for the synthesis of large, complex polysaccharides [Faijes, M.; Planas, A. Carbohydr. Res. 2007, 342, 1581-1594]. Guided by wild-type xyloglucanase activity, we have produced and characterized new glycosynthases for the synthesis of xyloglucan oligo- and polysaccharides, based on family GH7, GH12, and GH16 scaffolds. The Humicola insolens GH7 glycosynthase, HiCel7B E197S, is capable of synthesizing nongalactosylated, XXXG-based homoxyloglucan up to Mw 60000 [G = Glcβ(1→4); X = Xylα(1→6)Glcβ(1→4); L = Galβ(1→2)Xylα(1→6)Glcβ(1→4)], which is among the largest products so far obtained with glycosynthase technology. Novel glycosynthases based on the GH16 xyloglucan hydrolase from Tropaeolum majus (nasturtium), TmNXG1, are capable of synthesizing XLLG-based xyloglucan oligosaccharides at rates feasible for preparative synthesis, thus providing an essential expansion of product range. Finally, a new glycosynthase based on the recently characterized GH12 xyloglucanase from Bacillus licheniformis, BlXG12 E155A, can perform the condensation of xyloglucosyl fluorides, albeit at poor rates. Altogether, the high catalytic efficiency demonstrated by HiCel7B E197S and the extended product range provided by TmNXG1 E94A are key achievements toward a robust and versatile method for the preparative synthesis of homogeneous xyloglucans with regular substitution patterns not available in nature. Such compounds enable in vitro experimental studies regarding the role of particular structural elements for xyloglucan properties and its interaction with cellulose.

sted, utgiver, år, opplag, sider
2009. Vol. 10, nr 7, s. 1782-1788
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-24295DOI: 10.1021/bm900215pISI: 000268139300015Scopus ID: 2-s2.0-67650459915OAI: oai:DiVA.org:kth-24295DiVA, id: diva2:346376
Merknad
QC 20100902Tilgjengelig fra: 2010-08-31 Laget: 2010-08-31 Sist oppdatert: 2017-12-12bibliografisk kontrollert
Inngår i avhandling
1. On the engineering of proteins: methods and applications for carbohydrate-active enzymes
Åpne denne publikasjonen i ny fane eller vindu >>On the engineering of proteins: methods and applications for carbohydrate-active enzymes
2010 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Stockholm: KTH, 2010. s. xii, 74
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2010:14
Emneord
enzyme engineering, rational design, directed evolution, DNA shuffling, glycosynthase, xyloglucan, xyloglucan endo-transglycosylase, retaining glycoside hydrolase, xyloglucanase, carbohydrate binding module, polysaccharide synthesis
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-24296 (URN)978-91-7415-709-3 (ISBN)
Disputas
2010-09-22, FD5, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 10:15 (engelsk)
Opponent
Veileder
Merknad
QC 20100902Tilgjengelig fra: 2010-09-02 Laget: 2010-08-31 Sist oppdatert: 2010-09-02bibliografisk kontrollert
2. Synthesis of xyloglucan oligo- and polysaccharides with glycosynthase technology
Åpne denne publikasjonen i ny fane eller vindu >>Synthesis of xyloglucan oligo- and polysaccharides with glycosynthase technology
2009 (engelsk)Licentiatavhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glycans 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 glycans such as cellulose.

Xyloglucan is widely used in bulk quantities in the food, textile and paper making industries. With an increasing interest in technically more advanced applications of xyloglucan, such as novel biocomposites, there is a need to understand and control the properties and interactions of xyloglucan with other compounds, to decipher the relationship between xyloglucan structure and function, and in particular the effect of different branching patterns. However, due to the structural heterogeneity of the polysaccharide as obtained from natural sources, relevant studies have not been possible to perform in practise. This fact has stimulated an interest in synthetic methods to obtain xyloglucan mimics and analogs with well-defined structure and decoration patterns.

Glycosynthases are hydrolytically inactive mutant glycosidases that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Since its first conception in 1998, the technology is emerging as a useful tool in the synthesis of large, complex polysaccharides. This thesis presents the generation and characterisation of glycosynthases based on xyloglucanase scaffolds for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns.

sted, utgiver, år, opplag, sider
Stockholm: KTH Royal Institute of Technology, 2009. s. 47
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2009:6
Emneord
glycosynthase, xyloglucan, xyloglucan endo-transglycosylase, retaining glycoside hydrolase, xyloglucanase, polysaccharide synthesis
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-10178 (URN)978-91-7415-279-1 (ISBN)
Presentation
2009-04-29, FD51, Roslagstullsbacken 21, Stockholm, AlbaNova Universitetscentrum, 10:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2009-05-14 Laget: 2009-03-30 Sist oppdatert: 2010-10-13bibliografisk kontrollert

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