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

Master Thesis


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University of Cape Town

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¯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.

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Includes bibliographical references (leaves 101-116).