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Glycosynthase activity of hybrid aspen xyloglucan endo-transglycosylase PttXET16-34 nucleophile mutants
KTH, School of Biotechnology (BIO), Glycoscience.
KTH, School of Biotechnology (BIO).
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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 2.4.1.207) 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

Place, publisher, year, edition, pages
2007. Vol. 5, no 24
National Category
Organic Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-7554DOI: 10.1039/b714570eISI: 000251274700012Scopus ID: 2-s2.0-36749095084OAI: oai:DiVA.org:kth-7554DiVA: diva2:12616
Note
QC 20100902Available from: 2007-10-22 Created: 2007-10-22 Last updated: 2010-10-13Bibliographically approved
In thesis
1. Production and engineering of a xyloglucan endo-transglycosylase from Populus tremula x tremuloides
Open this publication in new window or tab >>Production and engineering of a xyloglucan endo-transglycosylase from Populus tremula x tremuloides
2007 (English)Licentiate thesis, comprehensive summary (Other scientific)
Abstract [en]

The aim of this work was to develop a production process for the enzyme xyloglucan endo-transglycosylase from Populus tremula x tremuloides (PttXET16-34). The natural transglycosylating activity of this enzyme has previously been employed in a XET-Technology. This chemo enzymatic method is useful for biomimetic modification of cellulose surfaces and holds great potential for industrial applications. Thus, it requires that the XET-enzyme can be produced in larger scale.

This work also shows how the wildtype PttXET16-34 was modified into a glycosynthase. By mutation of the catalytic nucleophile into an alanine, glycine or serine residue, enzymes capable of synthesising defined xyloglucan fragments were obtained. These defined compounds are very valuable for further detailed studies of xyloglucan active-enzymes, but are also useful in molecular studies of the structurally important xyloglucan-cellulose interaction.

A heterologous production system for PttXET16-34 was previously developed in the methylotrophic yeast Pichia pastoris. A methanol-limited fed-batch process was also previously established, but the yield of active XET was low due to proteolysis problems and low productivity. Therefore, two alternative fed-batch techniques were investigated for the production of PttXET16-34: a temperature-limited fed-batch (TLFB) and an oxygen-limited high-pressure fed-batch (OLHPFB).

For the initial recovery of XET after the fermentation process, two different downstream processes were investigated: expanded bed adsorption (EBA) and cross-flow filtration (CFF).

Place, publisher, year, edition, pages
Stockholm: KTH, 2007. v, 43 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2007:8
Keyword
xyloglucan endo-transglycosylase, retaining glycoside hydrolase, glycosynthase, Pichia pastoris, fed-batch fermentation, expanded-bed adsorption
National Category
Plant Biotechnology
Identifiers
urn:nbn:se:kth:diva-4511 (URN)978-91-71-78-776-7 (ISBN)
Presentation
2007-11-09, FA32, AlbaNova, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 14:00
Supervisors
Note
QC 20101108Available from: 2007-10-22 Created: 2007-10-22 Last updated: 2011-11-23Bibliographically approved
2. 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
3. Synthesis of xyloglucan oligo- and polysaccharides with glycosynthase technology
Open this publication in new window or tab >>Synthesis of xyloglucan oligo- and polysaccharides with glycosynthase technology
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.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2009:6
Keyword
glycosynthase, xyloglucan, xyloglucan endo-transglycosylase, retaining glycoside hydrolase, xyloglucanase, polysaccharide synthesis
National Category
Industrial Biotechnology Biocatalysis and Enzyme Technology Other Industrial Biotechnology Biomaterials Science
Identifiers
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 (English)
Opponent
Supervisors
Available from: 2009-05-14 Created: 2009-03-30 Last updated: 2010-10-13Bibliographically approved

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