Alcohol dehydrogenase mediated lactonization of 1,6-hexanediol

dc.contributor.advisorOpperman, D. J.en_ZA
dc.contributor.advisorSmit, M. S.en_ZA
dc.contributor.authorDithugoe, Choaro Daviden_ZA
dc.date.accessioned2018-08-06T08:18:56Z
dc.date.available2018-08-06T08:18:56Z
dc.date.issued2018en_ZA
dc.descriptionDissertation (M.Sc. (Microbiology and Biochemistry))--University of the Free State, 2018en_ZA
dc.description.abstractAlcohol dehydrogenases (ADHs) are oxidoreductases that catalyse the interconversion between alcohols and ketones. These are biotechnologically interesting enzymes due to their ability to produce optical active alcohols. Alternatively, lactonization of diols to corresponding lactones has been explored by researchers. The aim of this study was to screen ADHs for the lactonization of 1,6-hexanediol to Ɛ-caprolactone by evaluating a single ADHs enzyme system and a combinatorial ADHs enzyme system. Saccharomyces cerevisiae ADH 1 and ADH 6 together with Alcanivorax dieselolei B5 ADH 1 to ADH 3 genes were PCR amplified and subcloned into pET28b(+) expression vector. An additional 8 ADHs genes were synthesised and cloned into pET28b(+) by Genescript. The pET28b(+) ADHs were screened using cell-free extract (CFE), however, only four ADHs showed activity towards 1,6-hexanediol of which Aedes aegypti Farnesol dehydrogenase (AaSDR-1), a newly discovered ADH, showed activity towards 1,6-hexanediol. From the CFE reactions, an unknown side product was detected. Therefore, purified AaSDR-1 with Streptococcus mutant NADH oxidase 2 (SmNOX) for cofactor regeneration was used to study the reaction, however the same unknown side product also formed. Thereafter, it was speculated that tris buffer react with the products from 1,6-hexanediol. With NMR and GC-MS analysis, the product was identified to be a tricyclic-tris adduct. Different buffering systems, including sodium phosphate buffer, decreases the tricyclic-tris adduct. The AaSDR-1 and Equus caballus ADH (HLADH) were compared with low concentration of SmNOX. It was observed that at low SmNOX concentrations NADH regeneration is favoured, while, at high concentration NADPH regeneration is favoured. However, the single ADH enzyme system had limitation and a combinatorial ADH enzyme system was proposed to screen for the best primary and secondary ADH. From the combinatorial reaction, the combination of Thermus sp. ATN1 ADH (TADH)-AaSDR-1 gave the best activity. Unfortunately, during the single AaSDR-1 enzyme system and combinatorial TADH-AaSDR-1 system, a white-precipitate ‘plastic-like’ structure was formed over time. The precipitate could not be extracted and quantified. The precipitate could potentially be from 6-hydroxyhexanoic acid and therefore the Ɛ-caprolactone concentration might be underestimated. Chemical, photochemical regeneration and bi-convergent cascade systems were tested as alternatives to the SmNOX system. The FMN light driven reaction required high concentration of AaSDR-1 and also produced the white-precipitate ‘plastic-like’ structure. Although the bi-convergent system of NADPH-dependent Cyclohexanone monooxygenase (CHMO) and NADP+-dependent AaSDR-1 can produce Ɛ-caprolactone, the AaSDR-1 competes with the CHMO for substrate and cofactors resulting in low Ɛ-caprolactone yields.en_ZA
dc.description.sponsorshipNational Research Foundation (NRF)en_ZA
dc.identifier.urihttp://hdl.handle.net/11660/9056
dc.language.isoenen_ZA
dc.publisherUniversity of the Free Stateen_ZA
dc.rights.holderUniversity of the Free Stateen_ZA
dc.subject1,6-Hexanediolen_ZA
dc.subjectƐ-Caprolactoneen_ZA
dc.subjectAlcohol dehydrogenasesen_ZA
dc.subjectShort-chain dehydrogenases/reductasesen_ZA
dc.subjectMedium-chain dehydrogenasesen_ZA
dc.subjectLong-chain dehydrogenasesen_ZA
dc.subjectNAD(P)H oxidaseen_ZA
dc.subjectNMRen_ZA
dc.subjectCofactor regenerationen_ZA
dc.titleAlcohol dehydrogenase mediated lactonization of 1,6-hexanediolen_ZA
dc.typeDissertation
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