PhD ceremony: R. Kelly, 14.45 uur, Academiegebouw, Broerstraat 5, Groningen
Thesis: The evolution of cyclodextrin glucanotransferases, model enzymes of glycoside hydrolase family 13
Promotor(s): prof. L. Dijkhuizen
Faculty: Mathematics and Natural Sciences
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
-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
-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.
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