Browsing by Author "Prins, Alaric"
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Item Actinobacteria associated with two diverse soil environments and their multicopper oxidase diversity(University of the Western Cape, 2024) Prins, Alaric; McCullough, Bronwyn KirbyThe Cape Floristic Region (CFR) is a biodiverse region boasting unique plant diversity with a rich concentration of endemic plants. Aspalathus linearis (Rooibos) is an indigenous plant that grows in the Clanwilliam region of the Western Cape and is cultivated for its use as an herbal tea. Emerging peatlands in the CFR have gained increasing attention over recent years through research aiming to understand the microbial diversity associated with these environments. Little is known about the actinobacterial diversity of these regions, and as such, it is necessary to investigate the diversity of the actinobacteria associated with these environments, whilst simultaneously gaining knowledge on whether the associated actinobacteria may produce enzymes of biotechnological interest. Two CFR regions (the Rooibos environment – Clanwillian, and the Springfield emerging peatland environment – Agulhas) were explored through culture-based and genomic screening. Metabarcoding analyses using actinobacterial-specific 16S rRNA gene primers showed that the major taxa contributing to the Rooibos environment were members of the families Mycobacteriaceae, Pseudonocardiaceae, Frankiaceae and Geodermatophilaceae. Members of the families Mycobacteriaceaea, Pseudonocardiaceae, Acidimicrobiaceae and Nocardioiaceae was identified as the major taxa for the Springfield environment. Through selective isolation techniques, actinobacteria from rare (underrepresented) genera were isolated, including members of the genera Dactylosporangium, Actinokineospora, Curtobacterium, Modestobacter, Leifsonia and Actinomadura. The top strains, selected based on exhibiting extracellular multicopper oxidase (MCO) activity through culture-based screening, were subjected to whole genome sequence analysis. These rare genera are also vastly underrepresented among 3 400 bacterial MCO sequences found in the Laccase and Multicopper Oxidase Engineering Database (LccED).Item The degradation of the endocrine disrupting chemical, bisphenol-A : a comparative study between fungal and bacterial laccases(University of the Western Cape, 2015) Prins, Alaric; Le Roes-Hill, M..; Kirby, BronwynThe degradation of endocrine disrupting chemicals (EDCs) is a topic of high importance and one that research efforts are continually being focused on. These harmful chemicals are known to cause adverse health effects in humans and animals. In particular, bisphenol-A (BPA), a high volume chemical which is mainly used in the manufacturing of polycarbonate plastics and epoxy resins have been shown to be implicated in the development of a variety of health problems. In this study, the ability of two fungal laccases [Trametes versicolor (TvL) and Trametes pubescens (TpL)], and two bacterial laccases [Streptomyces coelicolor (SLAC), and a mutant of SLAC (SLAC- VN)] to degrade or remove BPA from solution was investigated. The commercial preparation of TvL was used for the purposes of this study, while TpL was produced from the native strain. T. pubescens was cultured in shake-flasks, the supernatant harvested and subjected to ammonium sulphate precipitation. SLAC and SLAC-VN were produced from recombinant strains using a standard protocol and the enzymes purified by size-exclusion chromatography. The presence of the laccases were confirmed by the 2,6-dimethoxyphenol assay and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).The removal or degradation of BPA from solution was determined for the free enzymes, as well as the enzymes in immobilised form. For immobilisation, the enzymes were encapsulated in sodium alginate beads and cross-linked to form cross- linked enzyme aggregates (CLEAs).High levels of BPA removal was exhibited by the fungal laccase, TpL (100% removal)and the bacterial mutant laccase, SLAC-VN (96%) in their free form. When all four laccases were encapsulated in sodium alginate beads, a number of changes to the characteristics of the enzymes were observed. Overall, the level of BPA removal was reduced for all enzymes as when compared to the free laccases, while SLAC-VN removed more BPA than either of the fungal laccases (59% for SLAC-VN versus 57% TvL and 54% for TpL). The encapsulation of the laccases in alginate beads also led to changes in the optimal temperature for BPA removal, with all encapsulated laccase being able to remove BPA optimally at 40°C. The immobilisation of the laccases in CLEA form had the most significant effect on the BPA removal ability of the laccases. The pH range for both fungal laccases was extended beyond the acidic range [for TpL, optimal removal occurred at pH 8.5 compared to pH 4.5 (free) and pH 6.0 (encapsulated)]. Most remarkable, however, was that the formation of CLEAs greatly enhanced the BPA removal ability of SLAC (60% removal compared to 25% when encapsulated).Item Draft Genome Sequence of Gordonia lacunae BS2T(American Society for Microbiology, 2017) Durrell, Kim; Prins, Alaric; Le Roes-Hill, MarilizeWe report here the draft genome sequence of the soil bacterium Gordonia lacunae BS2T ( DSM 45085T JCM 14873T NRRL B-24551T), isolated from an estuary in Plettenberg Bay, South Africa. Analysis of the draft genome revealed that more than 40% of the secondary metabolite biosynthetic genes encode new compounds.