Item – Theses Canada

​
OCLC number
751632137
Link(s) to full text
LAC copy
LAC copy
Author
Clouthier, Christopher Michael,1973-
Title
Design and development of Baeyer-Villiger monooxygenases as reagents in organic synthesis.
Degree
Ph. D. -- University of New Brunswick, 2008
Publisher
Ottawa : Library and Archives Canada = Bibliothèque et Archives Canada, [2011]
Description
3 microfiches
Notes
Includes bibliographical references.
Abstract
<?Pub Inc> Directed evolution has been found to be a very useful tool for the targeted improvement of enzymatic properties. The application of directed evolution for the improvement of cyc1ohexanone monooxygenase (CHMO) selectivity for a specific substrate target (4-hydroxycyc1ohexanone) identified a number of mutant CHMO variants possessing enhanced enantioselectivity. Four of the best performing mutants of CHMO were investigated as catalysts for the oxidation of a variety of4-substituted and 4, 4-disubstituted cyc1ohexanones. Overall several excellent catalyst matches (mutant/substrate) were identified. In a number of cases a mutant containing a single amino acid exchange (Phe432Ser) was found to be the most robust and selective enzyme catalyst. In several cases the Phe432Ser mutant was found to outperform wild type CHMO (WT-CHMO) in terms of conversion, efficiency and selectivity. Based on the lessons learned from the directed evolutionary studies on CHMO, a rational design approach was used to improve the selectivity of another monooxygenase enzyme, cyc1opentanone monooxygenase (CPMO). Through the application of a novel mutagenesis strategy, Complete Active Site Saturation Test (CAST), several successful mutants with enhanced enantioselectivities were identified. For example, the mutant catalyzed oxidation of 4-methylcyc1ohexanone gave the corresponding lactone with 92% enantiomeric excess (ee) compared to 46% ee achieved with wild type CPMO. This methodology provided a relatively inexpensive and rapid way to obtain mutant enzymes with the desired characteristics. Six of the mutant CPMO variants obtained through the application of the CAST methodology were investigated further for the oxidation of a series of cyclohexanone substrates substituted at the 4-position. Several mutant enzymes with improved enantioselectivities were identified. In general, the CPMO mutant variants involving mutation at the Phe156Gly157 position (library B) were found to be the most robust and selective of the CPMO clones. The successful improvement of CPMO illustrates how a family of cyclohexanone substrates may be used to explore the putative active sites of selected Baeyer-Villiger monooxygenases (BYMOs) and can aid in potential identification of mutational "hot-spots" within an active site. To explore the effects of reaction conditions on the enzyme catalyzed oxidation of selected 4-substituted cyclohexanones, biooxidations using WT-CHMO, WT-CPMO, and selected CHMO and CPMO mutants under both growing and non-growing cell conditions were studied. The surprising result of this study was that several substrates that were known to give negative results (non-acceptance) under growing conditions afforded excellent conversions in the transformations under non-growing conditions.
ISBN
9780494637913
0494637919