Browsing by Author "Jordaan, Justin"
Now showing 1 - 4 of 4
Results Per Page
Sort Options
- ItemRestrictedImmobilisation and characterisation of biocatalytic co-factor recycling enzymes, glucose dehydrogenase and NADH oxidase, on aldehyde functional ReSyn™ polymer microspheres(Elsevier, 2012) Twala, Busisiwe V; Sewell, Trevor B; Jordaan, JustinThe use of enzymes in industrial applications is limited by their instability, cost and difficulty in their recovery and re-use. Immobilisation is a technique which has been shown to alleviate these limitations in biocatalysis. Here we describe the immobilisation of two biocatalytically relevant co-factor recycling enzymes, glucose dehydrogenase (GDH) and NADH oxidase (NOD) on aldehyde functional ReSyn™ polymer microspheres with varying functional group densities. The successful immobilisation of the enzymes on this new high capacity microsphere technology resulted in the maintenance of activity of ∼40% for GDH and a maximum of 15.4% for NOD. The microsphere variant with highest functional group density of ∼3500 μmol g−1 displayed the highest specific activity for the immobilisation of both enzymes at 33.22 U mg−1 and 6.75 U mg−1 for GDH and NOD with respective loading capacities of 51% (0.51 mg mg−1) and 129% (1.29 mg mg−1). The immobilised GDH further displayed improved activity in the acidic pH range. Both enzymes displayed improved pH and thermal stability with the most pronounced thermal stability for GDH displayed on ReSyn™ A during temperature incubation at 65 °C with a 13.59 fold increase, and NOD with a 2.25-fold improvement at 45 °C on the same microsphere variant. An important finding is the suitability of the microspheres for stabilisation of the multimeric protein GDH.
- ItemRestrictedImmobilisation and characterisation of biocatalytic co-factor recycling enzymes, glucose dehydrogenase and NADH oxidase, on aldehyde functional ReSyn™ polymer microspheres.(Elsevier, 2012) Twalaa, Busisiwe V; Sewell, Trevor B; Jordaan, JustinThe use of enzymes in industrial applications is limited by their instability, cost and difficulty in their recovery and re-use. Immobilisation is a technique which has been shown to alleviate these limitations in biocatalysis. Here we describe the immobilisation of two biocatalytically relevant co-factor recycling enzymes, glucose dehydrogenase (GDH) and NADH oxidase (NOD) on aldehyde functional ReSynTM polymer microspheres with varying functional group densities. The successful immobilisation ofthe enzymes on this new high capacity microsphere technology resulted in the maintenance of activity of ∼40% for GDH and a maximum of 15.4% for NOD. The microsphere variant with highest functional group density of ∼3500 mol g−1 displayed the highest specific activity for the immobilisation of both enzymes at 33.22 U mg−1 and 6.75 U mg−1 for GDH and NOD with respective loading capacities of 51% (0.51 mg mg−1) and 129% (1.29 mg mg−1). The immobilised GDH further displayed improved activity in the acidic pH range. Both enzymes displayed improved pH and thermal stability with the most pronounced thermal stability for GDH displayed on ReSynTM A during temperature incubation at 65 ◦C with a 13.59 fold increase, and NOD with a 2.25-fold improvement at 45 ◦C on the same microsphere variant. An important finding is the suitability of the microspheres for stabilisation of the multimeric protein GDH.
- ItemOpen AccessPreparation and evaluation of polymer microspheres for enhanced lateral flow immunoassay: the case study for malaria(2015) Hobbs, Henriëtte Renée; Blackburn, Jonathan; Jordaan, JustinWe proposed that the development of a new high capacity polymer microsphere technology, termed ReSyn, could be developed as viable detection reagents for lateral flow technology. This body of work outlines the development of this new high capacity polymer microsphere technology for suitability to flow on lateral flow membranes, and highly specific biomarker detection for immunoassay development. Proof-of-concept was achieved using hCG (pregnancy biomarker) and validated for detection of pLDH and HRP2 as biomarkers of malaria. The sensitivity, stability and multiplex capability of these microspheres were further explored and compared against the current ‘gold' standard detection agent for lateral flow, colloidal gold. Malaria was selected as a suitable target for evaluation of the microsphere technology since it is considered to be a global epidemic that can benefit greatly from improved point-of-care diagnostics. Malaria affects almost half of the world's population and is responsible for causing approximately 655 000 deaths per annum in 2010, with 90% of these deaths occurring in Africa and 85% of these deaths occurring in children under 5 years of age (del Prado et al., 2014; Kokwaro, 2009; White et al., 2014; WHO, 2009). Febrile disease diagnosis at point-of-care is often based on symptomatic diagnosis rather than on the use of validated diagnostic technologies, and is considered one of the major contributing factors for the high morbidity and mortality rate of malaria (Chandler et al., 2008; Kain et al., 1998; Kokwaro, 2009). Improved diagnostic technologies, allowing for sensitive and accurate diagnosis at the point-of-care, could assist alleviating these problems through the improved management of disease (Bell et al., 2006). Lateral flow rapid diagnostic tests are the preferred method for point-of-care diagnostics in resource constrained areas but have several limitations including sensitivity and stability in resource constrained settings (Bell et al., 2006). Improvements in detection agents are seen as a viable approach to improving these features of diagnostic assays. The results of this study show that the polymer microspheres provide improved stability to immobilised antibodies, with potential for translation into improved stability for diagnostic assays in tropical malaria endemic regions. The polymer microspheres offered high specificity and comparable visual sensitivity to the market leader colloidal gold and is therefore considered as alternate detector agents in lateral flow assays. Additionally, the microspheres can be dyed various colours (red and blue in this study), allowing for specific and sensitive multiplex detection of multiple analytes in a single sample. This increases the versatility of the microspheres for lateral flow diagnostic application, and improves the interpretation of lateral flow diagnostic technology at the point-of-care.
- ItemOpen AccessPreparation of a self-contained NADH co-factor recycling particle system(2010) Twala, Busisiwe V; Sewell, Bryan Trevor; Jordaan, JustinOxidoreductases 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.