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Production and engineering of a xyloglucan endo-transglycosylase from Populus tremula x tremuloides
KTH, School of Biotechnology (BIO).
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 [en]
xyloglucan endo-transglycosylase, retaining glycoside hydrolase, glycosynthase, Pichia pastoris, fed-batch fermentation, expanded-bed adsorption
National Category
Plant Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-4511ISBN: 978-91-71-78-776-7 OAI: oai:DiVA.org:kth-4511DiVA: diva2:12618
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
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
Show others...
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

National Category
Organic Chemistry
Identifiers
urn:nbn:se:kth:diva-7554 (URN)10.1039/b714570e (DOI)000251274700012 ()2-s2.0-36749095084 (Scopus ID)
Note
QC 20100902Available from: 2007-10-22 Created: 2007-10-22 Last updated: 2010-10-13Bibliographically approved
2. Improvements of Pichia pastoris fermentation technique for production of recombinant proteins
Open this publication in new window or tab >>Improvements of Pichia pastoris fermentation technique for production of recombinant proteins
(English)Manuscript (Other academic)
National Category
Plant Biotechnology
Identifiers
urn:nbn:se:kth:diva-7555 (URN)
Note
QC 20101108Available from: 2007-10-22 Created: 2007-10-22 Last updated: 2010-11-08Bibliographically approved

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