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Synthesis of xyloglucan oligo- and polysaccharides with glycosynthase technology
KTH, School of Biotechnology (BIO), Glycoscience. (Glycoscience)
2009 (English)Licentiate thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology , 2009. , 47 p.
Trita-BIO-Report, ISSN 1654-2312 ; 2009:6
Keyword [en]
glycosynthase, xyloglucan, xyloglucan endo-transglycosylase, retaining glycoside hydrolase, xyloglucanase, polysaccharide synthesis
National Category
Industrial Biotechnology Biocatalysis and Enzyme Technology Other Industrial Biotechnology Biomaterials Science
URN: urn:nbn:se:kth:diva-10178ISBN: 978-91-7415-279-1OAI: diva2:210037
2009-04-29, FD51, Roslagstullsbacken 21, Stockholm, AlbaNova Universitetscentrum, 10:00 (English)
Available from: 2009-05-14 Created: 2009-03-30 Last updated: 2010-10-13Bibliographically approved
List of papers
1. Glycosynthase activity of hybrid aspen xyloglucan endo-transglycosylase PttXET16-34 nucleophile mutants
Open this publication in new window or tab >>Glycosynthase activity of hybrid aspen xyloglucan endo-transglycosylase PttXET16-34 nucleophile mutants
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2007 (English)In: Organic and Biomolecular Chemistry, ISSN 1477-0520, Vol. 5, no 24Article in journal (Refereed) Published
Abstract [en]

Glycosynthases are active-site mutants of glycoside hydrolases that catalyse glycosyl transfer using suitable activated donor substrates without competing product hydrolysis ( S. M. Hancock, M. D. Vaughan and S. G. Withers, Curr. Opin. Chem. Biol., 2006, 10, 509-519). Site-directed mutagenesis of the catalytic nucleophile, Glu-85, of a Populus tremula x tremuloides xyloglucan endo-transglycosylase (PttXET16-34, EC into alanine, glycine, and serine yielded enzymes with glycosynthase activity. Product analysis indicated that PttXET16-34 E85A in particular was able to catalyse regio- and stereospecific homo- and hetero- condensations of alpha-xylogluco-oligosaccharyl fluoride donors XXXG alpha F andXLLG alpha F to produce xyloglucans with regular sidechain substitution patterns. This substrate promiscuity contrasts that of the Humicola insolens Ce17B E197A glycosynthase, which was not able to polymerise the di-galactosylated substrate XLLG alpha F. The production of the PttXET16-34 E85A xyloglucosynthase thus expands the repertoire of glycosynthases to include those capable of synthesising structurally homogenenous xyloglucans

National Category
Organic Chemistry
urn:nbn:se:kth:diva-7554 (URN)10.1039/b714570e (DOI)000251274700012 ()2-s2.0-36749095084 (ScopusID)
QC 20100902Available from: 2007-10-22 Created: 2007-10-22 Last updated: 2010-10-13Bibliographically approved
2. Functional Characterization of Xyloglucan Glycosynthases from GH7, GH12, and GH16 Scaffolds
Open this publication in new window or tab >>Functional Characterization of Xyloglucan Glycosynthases from GH7, GH12, and GH16 Scaffolds
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2009 (English)In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 10, no 7, 1782-1788 p.Article in journal (Refereed) 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.

National Category
Industrial Biotechnology
urn:nbn:se:kth:diva-24295 (URN)10.1021/bm900215p (DOI)000268139300015 ()2-s2.0-67650459915 (ScopusID)
QC 20100902Available from: 2010-08-31 Created: 2010-08-31 Last updated: 2010-10-13Bibliographically approved

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