Preparation of a self-contained NADH co-factor recycling particle system

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dc.contributor.advisorSewell, Bryan Trevoren_ZA
dc.contributor.advisorJordaan, Justinen_ZA
dc.contributor.authorTwala, Busisiwe Ven_ZA
dc.date.accessioned2014-07-30T17:42:12Z
dc.date.available2014-07-30T17:42:12Z
dc.date.issued2010en_ZA
dc.descriptionIncludes abstract.
dc.descriptionIncludes bibliographical references (leaves 101-116).
dc.description.abstractOxidoreductases are interesting enzymes with potential applications in a number of different industries such as the textile, food and feed, chemical and biomedical industries. Oxidoreductases require the use of co-factors. These small molecules are relatively expensive and are required in stoichiometric amounts for their enzymatic reaction; this negatively impacts the economic viability of their potential applications. Several methods have been developed to counteract this problem, the most preferred of which is the enzymatic co-factor recycling method. A few methods for the co-immobilisation of enzymes and co-factors have been developed. These systems are of interest as they offer the advantages of recycling the enzymes together with the co-factor, thereby enabling re-use. The immobilisation of enzymes also provides a platform for improving their stability, activity, specificity and selectivity. Since glucose dehydrogenase (GDH) and NADH oxidase, are industrially relevant co-factor recycling enzymes for NAD(P)H and NAD+ respectively, characterisation of their immobilisation is of interest. The current work describes the use of the proprietary particle technology, termed ReSyn™, for the construction of a self-contained co-factor recycling system. The research included the optimisation of immobilisation for the individual enzymes, followed by the co-immobilisation with subsequent co-factor entrapment. The immobilised enzymes displayed improved thermal and pH stability compared to the non-immobilised enzymes. Immobilised GDH also displayed increased activity over the acidic range when compared to free GDH. The system was shown to be capable of recycling NADH/NAD+ up to at least 142 times with a specific activity of 10.18 U.mg¯1. The system was recovered and recycled with a 77% activity efficiency indicating recovery of the system and reusability. Preparation of a functional self-contained co-factor recycling system was demonstrated consisting of the biological components NADH oxidase and glucose dehydrogenase, immobilised on a polyethylenimine support with entrapped cofactor. This serves as proof-of-principle for the construction of derivative systems that could be used for the development of applications such as efficient biosynthesis, novel biosensors, diagnostic and therapeutic systems.en_ZA
dc.identifier.apacitationTwala, B. V. (2010). <i>Preparation of a self-contained NADH co-factor recycling particle system</i>. (Thesis). University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology. Retrieved from http://hdl.handle.net/11427/4343en_ZA
dc.identifier.chicagocitationTwala, Busisiwe V. <i>"Preparation of a self-contained NADH co-factor recycling particle system."</i> Thesis., University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 2010. http://hdl.handle.net/11427/4343en_ZA
dc.identifier.citationTwala, B. 2010. Preparation of a self-contained NADH co-factor recycling particle system. University of Cape Town.en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Twala, Busisiwe V AB - Oxidoreductases are interesting enzymes with potential applications in a number of different industries such as the textile, food and feed, chemical and biomedical industries. Oxidoreductases require the use of co-factors. These small molecules are relatively expensive and are required in stoichiometric amounts for their enzymatic reaction; this negatively impacts the economic viability of their potential applications. Several methods have been developed to counteract this problem, the most preferred of which is the enzymatic co-factor recycling method. A few methods for the co-immobilisation of enzymes and co-factors have been developed. These systems are of interest as they offer the advantages of recycling the enzymes together with the co-factor, thereby enabling re-use. The immobilisation of enzymes also provides a platform for improving their stability, activity, specificity and selectivity. Since glucose dehydrogenase (GDH) and NADH oxidase, are industrially relevant co-factor recycling enzymes for NAD(P)H and NAD+ respectively, characterisation of their immobilisation is of interest. The current work describes the use of the proprietary particle technology, termed ReSyn™, for the construction of a self-contained co-factor recycling system. The research included the optimisation of immobilisation for the individual enzymes, followed by the co-immobilisation with subsequent co-factor entrapment. The immobilised enzymes displayed improved thermal and pH stability compared to the non-immobilised enzymes. Immobilised GDH also displayed increased activity over the acidic range when compared to free GDH. The system was shown to be capable of recycling NADH/NAD+ up to at least 142 times with a specific activity of 10.18 U.mg¯1. The system was recovered and recycled with a 77% activity efficiency indicating recovery of the system and reusability. Preparation of a functional self-contained co-factor recycling system was demonstrated consisting of the biological components NADH oxidase and glucose dehydrogenase, immobilised on a polyethylenimine support with entrapped cofactor. This serves as proof-of-principle for the construction of derivative systems that could be used for the development of applications such as efficient biosynthesis, novel biosensors, diagnostic and therapeutic systems. DA - 2010 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 2010 T1 - Preparation of a self-contained NADH co-factor recycling particle system TI - Preparation of a self-contained NADH co-factor recycling particle system UR - http://hdl.handle.net/11427/4343 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/4343
dc.identifier.vancouvercitationTwala BV. Preparation of a self-contained NADH co-factor recycling particle system. [Thesis]. University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 2010 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/4343en_ZA
dc.language.isoengen_ZA
dc.publisher.departmentDepartment of Molecular and Cell Biologyen_ZA
dc.publisher.facultyFaculty of Scienceen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.subject.otherCell Biologyen_ZA
dc.titlePreparation of a self-contained NADH co-factor recycling particle systemen_ZA
dc.typeMaster Thesis
dc.type.qualificationlevelMasters
dc.type.qualificationnameMScen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
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