Biocatalytic production of coenzyme A analogues using cellulose-based enzyme reactors

Rootman, Ilse (2008-04)

Thesis (MSc) -- University of Stellenbosch, 2008.

Thesis

ENGLISH ABSTRACT: This study focuses on the development of an immobilized enzyme-based reactor for the chemo-enzymatic preparation of coenzyme A (CoA) analogues. The concept is based on published research proving that CoA biosynthetic enzymes can be used to catalyze the transformation of chemically-synthesized pantetheine analogues to their related CoA analogues. The three CoA biosynthetic enzymes that are used to catalyze the transformation reaction are pantothenate kinase (CoaA), phosphopantetheine adenylyltransferase (CoaD) and dephospho-coenzyme A kinase (CoaE). In this study we discuss the construction of fusion proteins of these three biosynthetic enzymes from two different bacterial sources (Escherichia coli and Staphylococcus aureus) with a cellulose binding domain (CBD) in order to achieve their immobilization on cellulose. Out of the eight biosynthetic enzyme combinations that can possibly be made up by these six CBD-fusion proteins we identified two combinations of three proteins that were fully characterized for their ability to transform the natural substrate and a substrate analogue into CoA and a CoA analogue respectively. One of these combinations is subsequently used in the construction of batch- and column-based reactors that are successfully employed in the preparation of two CoA analogues. This demonstrates that multi-enzyme reactors based on CBD-fusion proteins embody a viable strategy for the production of CoA and its analogues.

AFRIKAANSE OPSOMMING: Hierdie studie fokus op die ontwikkeling van 'n ge·immobiliseerde ensiem-gebaseerde reaktor wat gebruik kan word om analoe van koensiem A (KoA) chemo-ensimaties te berei. Die konsep is gebaseer op bestaande kennis wat KoA biosintetiese ensieme gebruik om die transformasie van chemies-gesintetiseerde pantete"ien-analoe na hul verwante KoA-analoe te kataliseer. Die drie ensieme wat nodig is om die transformasie te kataliseer is pantotenaatkinase (CoaA), fosfopantete"ienadenielieltransferase (CoaD) en defosfo-koensiem A-kinase (CoaE). In hierdie studie bespreek ans die versmelting van hierdie drie KoA biosintetiese ensieme afkomstig van twee verskillende bakteriele bronne (Escherichia coli en Staphylococcus aureus) met 'n sellulose-bindingsdomein (SBD) om sodoende die ensieme se immobilisering op sellulose te bewerkstellig. Vanuit die agt biosintetiese ensiemkombinasies wat moontlik saamgestel kan word uit die ses SBDfusieprote"iene, identifiseer ans twee kombinasies van drie ensieme wat ten volle gekarakteriseer is vir hulle vermoe om die natuurlike substraat en 'n substraatanaloog om te skakel na KoA en 'n KoA analoog onderskeidelik. Een van hierdie kombinasies is vervolgens gebruik om lot- en kolomreaktors saam te stel wat suksesvol aangewend is om twee KoA analoe te berei. Hierdie bevinding demonstreer dat multiensiemreaktore wat op SBD-fusieprote"iene gebaseer is 'n lewensvatbare strategie is om KoA en sy analoe mee te produseer.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/49415
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