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The evolution of cyclodextrin glucanotransferases, model enzymes of glycoside hydrolase family 13

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 α -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 α -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:15 september 2017 15:39

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