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Thermostable glycosidases and glycosynthases as biocatalysts in green chemistry applications

  • Tania Pozzo
Publiceringsår: 2012
Språk: Engelska
Sidor: 166
Dokumenttyp: Doktorsavhandling


The constant need to develop environmental friendly processes using renewable raw materials and to reduce harmful waste production motivates the present investigation focusing on biocatalyst development for enzymatic synthesis, a biotechnological approach in the field of green chemistry.
At first, structural functional experiments were made for a β-glucosidase from T. neapolitana (TnBgl3B). As a result the first three-domain thermostable member of a glycoside hydrolase (GH) from family 3 was reported, giving insights on substrate specificity and some structural explanations for its use in synthesis of alkyl glucosides. Moreover, efficiency in hexyl glucoside synthesis was compared between two thermostable β-glucosidases from T. neapolitana, belonging to family 1 (GH1) and 3 (GH3), TnBgl1A and TnBgl3B, respectively. For this purpose, a novel direct screening method in 96-well format was developed, using glucose, a cheap substrate from renewables, together with hexanol for reverse hydrolysis in a two-phase system.
The GH1 β-glucosidase TnBgl1A was, in a following study, used as biocatalyst in hydrolysis reactions, connected to flavonoid extractions from onion waste using pressurized hot water, to obtain a uniform deglycosylated product. Structural homology models and mutagenesis around the aglycone in the active site, was in this case used to identify important residues that led to increased hydrolytic activity towards flavonoid 3-glucosides.
The second part of the project focused on thermostable glycosynthases, which were engineered and tested using oligosaccharide synthesis as main model reaction. Two methodologies were used to obtain synthesis products, one using fluorinated sugar as donor, and the second using an exogenous nucleophile. The product specificity was shown to be towards synthesis of β-1,3-linkages for both TnBgl1A and TnBgl3B.
Structural examination of theTnBgl1A glycosynthase, after docking with the substrate, showed flexibility at the active site with catalytic residues located on loops, a statement verified using molecular dynamics.
The construction of a glycosynthase from TnBgl3B required an extra mutation next to the nucleophile. A residue that stabilized the product at the +1 subsite was also identified, together with one more hydrophobic residue located on the loop from the second domain, suggested to be important for the accommodation of acceptor molecules.
A natural product (antioxidant), such as the flavonoid quercetin-3-glucoside was also used as acceptor molecule in glycosynthase reactions at high temperature (70° C), in an attempt to look at an alternative acceptor and to expand the glycosynthase application to selective glycosylation of antioxidants. This assay used the exo-glycosynthases from TnBgl1A and TnBgl3B plus a new endo-glycosynthase constructed of a cellulase belonging to GH12 from R. marinus (RmCel12A). The main product of the thermostable glycosynthases was quercetin-3,4’-diglucoside when quercetin-3-glucoside was used as acceptor. Hence the 4’-hydroxyl of the acceptor was selected in the transglycosylation reactions, when the molecule was fitted in the active site.
In conclusion, thermostable glycoside hydrolases and glycosynthases can be useful biocatalysts for synthesising chemicals from renewable resources, such as alkyl glucosides, oligosaccharides or flavonoids with modified glycosylation. Thanks to the structural and functional information, the biocatalysts can be developed to harbour new or altered activities relevant for use to process renewable resources, such as in the modification of flavonoids combined with extraction form onion waste. Structural data also aided the construction of new glycosynthases coming from less investigated families like GH3. New interesting applications in antioxidant stabilization were assayed using thermostable glycosynthase for the glucosylation of flavonoids.


Lecture Hall K:C, Center of Chemistry and Chemical Engineering, Sölvegatan 39, Lund University Faculty of Engineering.
  • Marco Moracci (Dr.)


  • Industrial Biotechnology
  • Green chemistry
  • 3D sturcture
  • Glycosyl hydrolases
  • Glycosynthases
  • Alkyl glucosides
  • Oligosaccharides
  • Flavonoids


  • Eva Nordberg Karlsson
  • ISBN: 978-91-89627-91-8

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