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Novel Retaining Glycoside Hydrolases : Potential candidates for transglycosylation and hydrolysis

Gulshan Kazi, Zubaida LU (2020)
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... (More)
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. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Prof. Moracci, Marco, University of Naples Federico II, Italy.
organization
publishing date
type
Thesis
publication status
published
subject
keywords
glycoside hydrolase, transglucosylation, alkyl glycoside, cyclodextrin glucanotransferase, Rhodothermus marinus, xylanases, hydrolysis, beta-glucosidases
publisher
Division of Biotechnology, Lund University
defense location
Lecture hall KC:B, Kemicentrum, Naturvetarvägen 14, Faculty of Engineering LTH, Lund University, Lund. Join via Zoom: https://lu-se.zoom.us/s/61949547440
defense date
2020-09-09 14:15:00
ISBN
978-91-7422-730-7
978-91-7422-731-4
language
English
LU publication?
yes
id
9014dcb3-7ef0-4153-86bc-7a27fac94d12
date added to LUP
2020-03-05 16:40:33
date last changed
2025-04-04 14:12:05
@phdthesis{9014dcb3-7ef0-4153-86bc-7a27fac94d12,
  abstract     = {{Our society is moving towards renewable resources, where biomass, rich in<br/>carbohydrates, is producing chemicals and fuel. However, there are several<br/>limitations when it comes to valorisation of the carbohydrates from renewable<br/>biomass. One major hurdle is the over-functional nature of carbohydrates, making them difficult to process by conventional chemistry. Carbohydrate active<br/>enzymes can help overcome this limitation by providing excellent tools to utilise<br/>renewable feedstocks, supplying competitive alternatives to the traditional<br/>chemical process.<br/>The enzymatic toolbox is the green alternative when it comes to synthesis of<br/>glycoconjugates and to make the transition towards bioeconomy, use of these<br/>tools is an essential step. In nature, glycosylation is executed mostly by<br/>glycosyltransferases. However, they are not ideal for industrial applications due<br/>to their need to use expensive activated donors. Whereas, transglycosylases<br/>(classified under glycoside hydrolase families: GHs) do not need any activated<br/>donor, making them perfect candidates. The only limitation with them is that<br/>there are not many that have been characterised. Transglycosylases are classified<br/>in the same families as their hydrolysing counterparts, and are closely related in<br/>sequence and structure, making it difficult to select them based on sequence<br/>similarities. A typical exception from this is the cyclodextrin glucanotransferases<br/>(CGTases) which belongs to GH13.<br/>This thesis investigates the transglycosylation activity of cyclodextrin<br/>glucanotransferases, for expanding the utilisation of transglycosylases. The focus<br/>of the work was on the elongation of the carbohydrate part of alkyl glycosides. A<br/>novel cyclodextrin glucanotransferase (<i>Csp</i>CGT13) from <i>Carboxydocella</i> sp.<br/>was characterised and compared with available commercial enzymes to evaluate<br/>the applicability in alkyl glycoside modification. The novel enzyme showed<br/>significant coupling activity with γ-cyclodextrin as the donor, however it was not<br/>as efficient as the commercial CGTases. Later, the coupling activity was<br/>improved by protein engineering and bioinformatic analysis, making it a<br/>competitive candidate for alkyl glycosides modification.<br/>The majority of the enzymes in the GH-families are hydrolases and are widely<br/>available. Using glycoside hydrolases in synthesis requires reduced hydrolytic<br/>activity. In this thesis, oligosaccharide synthesis was studied by using glycosides<br/>hydrolases. Significantly reduced hydrolysis was achieved for an <i>endo</i>-xylanase<br/>from the thermophilic bacterium <i>Rhodothermus</i> <i>marinus </i>DSM 4252T through<br/>protein engineering. The enzyme variants displayed enhanced transglycosylation<br/>activity.<br/>In addition, novel candidates from the enzymatic toolbox from another strain of<br/>this marine thermophilic bacterium were also investigated in this thesis, aiming<br/>to gain more insight into the hydrolytic mechanism used for saccharification<br/>processes. Six novel <i>exo</i>-hydrolases from a single GH family (GH3) originating<br/>from R. marinus DSM 4253 were characterised. The study showed these enzymes<br/>to have broad substrate specificities and activities at moderately high temperature. Also, more information was obtained regarding their structural<br/>features and genomic distributions, providing more knowledge to tailor the<br/>enzymes for industrial applications.}},
  author       = {{Gulshan Kazi, Zubaida}},
  isbn         = {{978-91-7422-730-7}},
  keywords     = {{glycoside hydrolase; transglucosylation; alkyl glycoside; cyclodextrin glucanotransferase; Rhodothermus marinus; xylanases; hydrolysis; beta-glucosidases}},
  language     = {{eng}},
  publisher    = {{Division of Biotechnology, Lund University}},
  school       = {{Lund University}},
  title        = {{Novel Retaining Glycoside Hydrolases : Potential candidates for transglycosylation and hydrolysis}},
  url          = {{https://lup.lub.lu.se/search/files/83129517/Ara_KZG_Kappa_med_omslag.pdf}},
  year         = {{2020}},
}