Rijksuniversiteit Groningen
The evolution of cyclodextrin glucanotransferases, model enzymes of
glycoside hydrolase family 13
Datum: 04 september 2009
Promotie: R. Kelly, 14.45 uur, Academiegebouw, Broerstraat 5,
Groningen
Proefschrift: The evolution of cyclodextrin glucanotransferases, model
enzymes of glycoside hydrolase family 13
Promotor(s): prof.dr. L. Dijkhuizen
Faculteit: Wiskunde en Natuurwetenschappen
Reageerbuis-evolutie
Ronan Kelly onderzocht het ontstaan en de evolutionaire geschiedenis
van de GH13-enzymfamilie. Hij maakte daarbij gebruikt van "Darwinian"
evolutie methoden die bekend staan onder de naam "directed evolution",
dat wil zeggen: evolutie op de laboratoriumtafel. De in het
proefschrift beschreven voorbeelden en methoden vormen goede
uitgangspunten voor het verder vernieuwen en verbeteren van enzymen
van belang in de zetmeelbewerkende industrie.
Ronan Kelly (Ierland, 1980, officieel heet hij Ronan O'Ceallaigh)
studeerde aan het University College Dublin. Het onderzoek werd
uitgevoerd bij de afdeling Microbial Physiology. Kelly werkt nu als
postdoc hij het National Institute of Bioprocessing Research and
Training, University College Dublin (Ierland).
The scope of this thesis is to develop a greater understanding of the
structural factors responsible for the evolutionary diversification of
reaction and product specificity amongst GH13 family members, using
cyclodextrin glucanotransferases as model enzymes.
Chapter 1 provides an introduction to the a -amylase family of enzymes
and the directed evolution strategies frequently applied in the
modulation of specific properties of these enzymes.
In Chapter 2 the comparison of product profiles and amino acid
sequences of CGTases from mesophilic, thermophilic and
hyperthermophilic organisms enzymes revealed that specific
incorporation and / or substitution of residues at the substrate
binding sites, during the evolutionary progression of CGTases resulted
in diversification of cyclodextrin product specificity.
Chapter 3 investigates the evolutionary input required to efficiently
change T. thermosulfurigenes CGTase reaction specificities and
compares the effectiveness of laboratory evolution techniques applied.
Conversion of a CGTase into an a -amylase-like mutant enzyme suggests
that GH13 members may have diversified by introduction of a limited
number of mutations from a common ancestor.
Chapter 4 reports on the reduction of the hydrolytic side reaction of
the enzyme by directed evolution whilst maintaining the cyclization
activity. The best mutant obtained is located on the outer region of
the active site and lowered the hydrolytic activity up to 15-fold with
retention of cyclization activity.
Chapter 5 investigates the the considerable cost incurred to native
enzyme function and stability by evolving B. circulans CGTase enzyme
toward insensitivity to the protein's small molecule inhibitor,
acarbose.
Laatst gewijzigd: 25 augustus 2009 10:25
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