Van Staden, Johannes.Finnie, Jeffrey Franklin.Mtshali, Ntombizamatshali Prudence.2013-12-062013-12-0620112011http://hdl.handle.net/10413/10173Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.Acid phosphatases (apase) are important hydrolytic enzymes that function in the acquisition, production; transport and recycling of inorganic phosphate (Pi), thus making a significant contribution towards nutrients dynamic of many ecological niches. The aim of this study was to characterize the apase enzyme found in the lichen Cladonia portentosa at the molecular level. The initial experiment entailed cloning the apase gene by PCR using degenerate primers designed from close relatives of C. portentosa from the Ascomycete family. The isolation of apase gene from Cladonia portentosa using PCR was not successful. Attempts were then made to purify the secreted apase and to determine its biochemical and molecular properties and to allow comparison with already characterized secreted phosphatases from other fungal sources existing in the NCBI database. It was anticipated that the partial sequence of the purified enzymes would provide a corresponding apase gene. The acid phosphatase enzyme was partial purified to 45 fold by a gel filtration with a yield of 18%. It gave a single, broad glycoprotein band on native PAGE and SDS-PAGE corresponding in size to 250 and 148 kDa, respectively. Under reducing conditions, the purified enzyme migrated as two bands of 116 and 32 kDa, indicating the heterodimer nature of this enzyme. Only one distinct band, (pI 6.4) was observed after electrofocusing. The optimum temperature for the enzyme was 65 °C where an optimal pH was detected at 2.5. The enzyme was inhibited by known acid phosphatase inhibitors (fluoride, molybdate, orthovanadate and tartrate) and the metals (Cu²⁺ and Zn²⁺). The purified enzyme demonstrated broad substrates selectivity and had a KM of 31.2±0.25 μM for phytic acid. Peptide analysis by Mass Spectrometry (MS) MALDI-TOF indicated the presence of two apase proteins. The amino sequences of purified apase/s from Cladonia portentosa were FLAETNPAPFGH, AVGLGYVEELLAR and AQGLGYVQEVLAR. Comparing the amino acids of the sequenced protein with that of already known proteins confirmed the enzyme to be a secreted histidine acid phosphatase, resembling other acid phosphatases and phytase from several filamentous fungi with respect to amino acid composition. To investigate the effect of phosphorus on C. portentosa apase, the mycelium was grown under different concentrations of Pi [0.05, 1.0, 3.0, 10 and 100 mM (KH₂PO₄)]. The aim was to localize the apase enzyme and to screen for the occurrence of the gene coding for the acid phosphatase enzyme. A treatment of 3.0 mM Pi induced high levels of apase compared to all other treatments. In addition, cultures of C. portentosa were grown in axenic cultures to study the effect of pH and Pi versus menadione on the production of acid phosphatase and mycelia growth. A culture media of pH 4.8 and 6.0 resulted in higher apase secretion than when compared with pH 2.5 medium. The presence of 2.0 μM menadione marginally increased levels of the apase compared to the control treatment. Apase was further localized cytochemically using fluorescent substrate-enzyme-labelled fluorescence (ELF-97) which forms a fluorescent crystalline precipitate at the site of phosphate activity. Fluorescent microscope revealed that the enzyme was present in all treatments, irrespective of Pi concentration, however, the fluorescence signals were intense in low Pi concentrations (0.05 and 1.0 and 3.0 mM Pi). Ultrastructure localization using live mycelium under confocal microscopy using Vector blue III substrate revealed that the enzyme was localized in the cytoplasm, cell membrane, vacuole and small organelles, presumed to be endosomes. Co-staining with FM4-64, confirmed the punctuate structure to be secretory vesicles or a vacuolar network. To investigate the effect of P starvation on C. portentosa at a molecular level, the effect of Pi on the gene expression profile was examined. The generation of a cDNA library from axenic grown mycelium treated with P provided a foundation for the identification and characterization of genes expressed in the P treated mycelium through expressed sequence tags (ESTs). Several genes were identified whose transcriptional profiles have been significantly changed by phosphorus treatment and menadione. They include genes required for signal transduction and vesicular transport, cell biosynthesis and protein metabolism and stress response. In conclusion, this study constitutes the first step towards understanding the molecular mechanism governing acid phosphatase in C. portentosa.enCladonia--Molecular aspects.Phosphates.Cytochemistry.Theses--Botany.Acid phosphatase.Thesis