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Thesis submitted in partial fulfilment of the requirements for the degree Doctor of Technology: Biomedical Technology In the Faculty of Health and Wellness Sciences At the Cape Peninsula University of Technology 2014 / Peroxidases are ubiquitous catalysts that oxidise a wide variety of organic and inorganic compounds employing peroxide as the electron acceptor. They are an important class of oxidative enzymes which are found in nature, where they perform diverse physiological functions. Apart from the white rot fungi, actinomycetes are the only other known source of extracellular peroxidases. In this study, the production of extracellular peroxidase in wild type actinomycete strains was investigated, for the purpose of large-scale production and finding suitable applications. The adjustment of environmental parameters (medium components, pH, temperature and inducers) to optimise extracellular peroxidase production in five different strains was carried out. Five Streptomyces strains isolated from various natural habitats were initially selected for optimisation of their peroxidase production. Streptomyces sp. strain BSII#1 and Streptomyces sp. strain GSIII#1 exhibited the highest peroxidase activities (1.30±0.04 U ml-1 and 0.757±0.01 U ml-1, respectively) in a complex production medium at 37°C and pH 8.0 in both cases. Maximum enzyme production for Streptomyces strain BSII#1 was obtained in the presence of 0.1 mM veratryl alcohol or pyrogallol, while 0.1 mM guaiacol induced the highest peroxidase production in Streptomyces sp. strain GSIII#1. As the highest peroxidase producer, Streptomyces sp. strain BSII#1 was selected for further studies. The strain was first characterised by a polyphasic approach, and was shown to belong to the genus Streptomyces using various chemotaxonomic, genotypic and phenotypic tests. Production of peroxidase was scaled up to larger volumes in different bioreactor formats. The airlift configuration was optimal for peroxidase production, with Streptomyces sp. strain BSII#1 achieving maximum production (4.76±0.46 U ml-1) in the 3 l culture volume within 60 hrs of incubation. A protocol for the purification of the peroxidase was developed, which involved sequential steps of acid and acetone precipitation, as well as ultrafiltration. A purification factor of at least 46-fold was achieved using this method and the protein was further analysed by LC-MS. The protein was shown to be a 46 kDa protein, and further biochemical characterisation showed that the peroxidase had a narrower spectrum of substrates as compared to reports on other peroxidases derived from actinomycetes. With 2,4-dichlorophenol as the substrate, the Km and Vmax for this enzyme were 0.893 mM and 1.081 μmol min-1, respectively. The purified peroxidase was also capable of catalysing coupling reactions between several phenolic monomer pairs. Overall, the peroxidase from Streptomyces sp. strain BSII#1 could feasibly be produced in larger scales and there remains further room to investigate other potential applications for this enzyme.

Pore forming toxins are known for their ability to efficiently form transmembrane pores which eventually leads to cell lysis. PFTs have potential applications in devel-oping novel drug and gene delivery strategies. Although structural aspects of many pore forming toxins have been studied, very little is known about the dynamics and subsequent rupture mechanisms. In the first part of the thesis, a combined experimental and modeling study to understand the lytic action of Cytolysin A (ClyA) toxins on red blood cells (RBCs) has been presented. Lysis experiments are carried out on a 1% suspension of RBCs for different initial toxin concentrations ranging from 100 – 500 ng/ml and the extent of lysis is monitored spectrophotometrically. Using a mean field approach, we propose a non – equilibrium adsorption-reaction model to quantify the rate of pore formation on the cell surface. By analysing the model in a pre-lysis regime, the number of pores per RBC to initiate rupture was found to lie between 400 and 800. The time constants for pore formation are estimated to lie between 1-25 s and monomer conformation time scales were found to be 2-4 times greater than the oligomerization times. Using this model, we are able to predict the extent of cell lysis as a function of the initial toxin concentration. Various kinetic models for oligomerization mechanism have been explored. Irreversible sequential kinetic model has the best agreement with the available experimental data. Subsequent to the mean field approach, a population balance model was also formulated. The mechanics of cell rupture due to pore formation is poorly understood. Efforts to address this issue are concerned with understanding the changes in the membrane mechanical properties such as the modulus and tension in the presence of pores. The second part of the thesis is concerned with using atomic force microscopy to measure the mechanical properties of cells. We explore the possibility of employing tapping mode AFM (TM-AFM) to obtain the elastic modulus of soft samples. The dynamics of TM-AFM is modelled to predict the elastic modulus of soft samples, and predict optimal cantilever stiffness for soft biological samples. From experiments using TM-AFM on Nylon-6,6 the elastic modulus is predicted to lie between 2 and 5 GPa. For materials having elastic moduli in the range of 1– 20 GPa, the cantilever stiffness from simulations is found to lie in the range of 1 – 50 N/m. For soft biological samples, whose elastic moduli are in the range of 10-1000 kPa, a narrower range of cantilever stiffness (0.1 – 0.6 N/m), should be used.