Thesis (MSc (Chemistry and Polymer Science))--University of Stellenbosch, 2006. / This work focuses on the biocatalytic production of coenzyme A (CoA) analogues
with different tether lengths in its pantetheine moiety, and on analogues where the
cysteamine moiety has been replaced with a range of other amines. An attempt
was made to develop a simple biocatalytic method for the optimum production of
such CoA analogues by chemo-enzymatic means.
Pantothenic acid ethyl thioesters with different tether lengths were first synthesized
as substrates of the CoA biosynthetic enzymes, CoaA, CoaD and CoaE. The
acceptability of these compounds as substrates for the pantothenate kinase
(CoaA) from prokaryotic and eukaryotic organisms was investigated through
kinetic studies. These substrates were subsequently exposed to CoaA, CoaD and
CoaE to produce various general CoA synthons (ethyl pre-CoAs). Finally
aminolysis of these ethyl pre-CoAs by cysteamine and homocysteamine gave the
various CoA analogues of different tether lengths in their pantetheine moiety. The
identical production of a second type of CoA synthon (phenyl pre-CoA) from
pantothenic acid phenyl thioesters was also investigated as a means to increase
reactivity of the thioester substrates. Aminolysis of the phenyl pre-CoA produced
the corresponding CoA derivative, but reactivity was lower than expected.
A second strategy was also developed where the pantothenic acid phenyl
thioesters were first aminolyzed, resulting in various pantothenamide
intermediates. Aminolysis was attempted with thiol-bearing amines such as
cysteamine and homocysteamine as well as with amines without sulfhydryl
functionalities. These pantothenamide intermediates were then used in the
biosynthesis of the corresponding CoA analogues by addition of CoaA, CoaD and
CoaE.
The ideal method of CoA analogue production will utilize a continuous bioreactor
system in which these analogues can be prepared on large scale. However, to
construct a bioreactor the enzymes involved need to be immobilized on a matrix in order to transform substrate to product. The enzymes CoaA, CoaD and CoaE can
be immobilized on cellulose via a cellulose binding domain (CBD) affinity tag.
Various types of CBDs were investigated and used in the construction of suitable
expression vectors. Optimum expression conditions to obtain soluble CBD-fused
enzymes were developed.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/2911 |
| Date | 03 1900 |
| Creators | Van Wyk, Marianne |
| Contributors | Strauss, Erick, University of Stellenbosch. Faculty of Science. Dept. of Chemistry and Polymer Science. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 2741201 bytes, application/pdf |
| Rights | University of Stellenbosch |
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