Novel Retaining Glycoside Hydrolases: Potential candidates for transglycosylation and hydrolysis

Research output: ThesisDoctoral Thesis (compilation)

Abstract

Our society is moving towards renewable resources, where biomass, rich in
carbohydrates, is producing chemicals and fuel. However, there are several
limitations when it comes to valorisation of the carbohydrates from renewable
biomass. One major hurdle is the over-functional nature of carbohydrates, making them difficult to process by conventional chemistry. Carbohydrate active
enzymes can help overcome this limitation by providing excellent tools to utilise
renewable feedstocks, supplying competitive alternatives to the traditional
chemical process.
The enzymatic toolbox is the green alternative when it comes to synthesis of
glycoconjugates and to make the transition towards bioeconomy, use of these
tools is an essential step. In nature, glycosylation is executed mostly by
glycosyltransferases. However, they are not ideal for industrial applications due
to their need to use expensive activated donors. Whereas, transglycosylases
(classified under glycoside hydrolase families: GHs) do not need any activated
donor, making them perfect candidates. The only limitation with them is that
there are not many that have been characterised. Transglycosylases are classified
in the same families as their hydrolysing counterparts, and are closely related in
sequence and structure, making it difficult to select them based on sequence
similarities. A typical exception from this is the cyclodextrin glucanotransferases
(CGTases) which belongs to GH13.
This thesis investigates the transglycosylation activity of cyclodextrin
glucanotransferases, for expanding the utilisation of transglycosylases. The focus
of the work was on the elongation of the carbohydrate part of alkyl glycosides. A
novel cyclodextrin glucanotransferase (CspCGT13) from Carboxydocella sp.
was characterised and compared with available commercial enzymes to evaluate
the applicability in alkyl glycoside modification. The novel enzyme showed
significant coupling activity with γ-cyclodextrin as the donor, however it was not
as efficient as the commercial CGTases. Later, the coupling activity was
improved by protein engineering and bioinformatic analysis, making it a
competitive candidate for alkyl glycosides modification.
The majority of the enzymes in the GH-families are hydrolases and are widely
available. Using glycoside hydrolases in synthesis requires reduced hydrolytic
activity. In this thesis, oligosaccharide synthesis was studied by using glycosides
hydrolases. Significantly reduced hydrolysis was achieved for an endo-xylanase
from the thermophilic bacterium Rhodothermus marinus DSM 4252T through
protein engineering. The enzyme variants displayed enhanced transglycosylation
activity.
In addition, novel candidates from the enzymatic toolbox from another strain of
this marine thermophilic bacterium were also investigated in this thesis, aiming
to gain more insight into the hydrolytic mechanism used for saccharification
processes. Six novel exo-hydrolases from a single GH family (GH3) originating
from R. marinus DSM 4253 were characterised. The study showed these enzymes
to have broad substrate specificities and activities at moderately high temperature. Also, more information was obtained regarding their structural
features and genomic distributions, providing more knowledge to tailor the
enzymes for industrial applications.

Details

Authors
Organisations
Research areas and keywords

Subject classification (UKÄ) – MANDATORY

  • Biocatalysis and Enzyme Technology

Keywords

  • glycoside hydrolase, transglucosylation, alkyl glycoside, cyclodextrin glucanotransferase, Rhodothermus marinus, xylanases, hydrolysis, beta-glucosidases
Original languageEnglish
QualificationDoctor
Supervisors/Assistant supervisor
Award date2020 Sep 9
Publisher
  • Division of Biotechnology, Lund University
Print ISBNs978-91-7422-730-7
Electronic ISBNs978-91-7422-731-4
Publication statusPublished - 2020
Publication categoryResearch

Bibliographic note

Defence details Date: 2020-09-09 Time: 14:15 Place: Lecture hall KC:B, Kemicentrum, Naturvetarvägen 14, Faculty of Engineering LTH, Lund University, Lund. Join via Zoom: https://lu-se.zoom.us/s/61949547440 External reviewer(s) Name: Moracci, Marco Title: Prof. Affiliation: University of Naples Federico II, Italy. ---

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Research output: Contribution to journalArticle

Zubaida Gulshan Kazi, Pontus Lundemo, Olafur H Fridjonsson, Gudmundur O Hreggvidson, Patrick Adlercreutz & Eva Nordberg Karlsson, 2015, In: Glycobiology. 25, 5, p. 514-523

Research output: Contribution to journalArticle

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