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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:cput/oai:localhost:20.500.11838/1525 |
| Date | January 2014 |
| Creators | Musengi, Amos |
| Publisher | Cape Peninsula University of Technology |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Rights | http://creativecommons.org/licenses/by-nc-sa/3.0/za/ |
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