Production, purification, and characterisation of proteases from an ericoid mycorrhizal fungus, Oidiodendron maius
- Authors: Manyumwa, Colleen Varaidzo
- Date: 2018
- Subjects: Ascomycetes , Mycorrhizal fungi , Ericaceae , Proteolytic enzymes , Silver Recycling
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/62833 , vital:28298
- Description: The aim of this study was to produce, purify and characterise proteases from the ericoid mycorrhizal fungus, Oidiodendron maius (CafRU082b/KP119480), as well as to explore their potential application in the recovery of silver from X-ray film. Firstly, the growth of the ericoid mycorrhizal fungus, Oidiodendron maius (CafRU082b), was studied, and its ability to produce proteolytic enzymes was investigated. O. maius proved to grow well in the dark, submerged in Modified Melin Norkran’s liquid medium at a pH of 5 and at 25°C. Pure cultures of the fungus were maintained on Potato Dextrose Agar (PDA). The fungus grew on PDA plates containing different substrates including haemoglobin, casein, gelatin as well as azocasein. Zones of clearance, however, were only observed on plates containing gelatin after treatment with mercuric chloride, HgCl2. Proteases were successfully produced after 14 days when gelatin was incorporated into the growth medium. After production of the proteases, purification and characterisation of the enzymes was performed. Purification of the enzymes was performed by acetone precipitation followed by ultrafiltration with 50 kDa and 30 kDa cut off membrane filters. A final purification fold of approximately 37.6 was achieved. Unusual yields of above 100% were observed after each purification step with the final yield achieved being 196% with a final specific activity of 2707 U/mg. SDS-PAGE revealed a protease band of 35 kDa which was also visible on the zymogram at approximately 36 kDa. The zymogram showed clear hydrolysis bands against a blue background after staining with Coomassie Brilliant Blue. Physico-chemical characterisation of the protease revealed its pH optimum to be pH 3.0 and its temperature optimum 68°C. Another peak was observed on the pH profile at pH 7.0. The protease exhibited high thermostability at temperatures 37°C, 80°C as well as 100°C with the enzyme retaining close to 50% of its initial activity after 4 h of exposure to all three temperatures. All ions tested for their effects on the proteases, except Ca2+, enhanced protease activity. Ca2+ did not exhibit any significant effect on the enzyme’s activity while Zn2+ had the highest effect, enhancing enzyme activity by 305%. The proteases, however, were not significantly inhibited by EDTA, a metal chelating agent and a known metalloprotease inhibitor. The enzyme was classified as an aspartic protease due to complete inhibition by 25 μM of pepstatin A, coupled to its low pH optimum of 3.0. Addition of trans-Epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), a cysteine protease inhibitor, and 2-mercaptoethanol increased protease activity. The proteases exhibited a narrow substrate specificity towards gelatin and no other substrate. Substrate kinetics values were plotted on a Michaelis-Menten Graph and showed that the enzyme had a Vmax of 55.25 U/ml and a Km of 2.7 mg/ml gelatin. A low Km indicated that the protease had a high affinity for gelatin. Silver recovery studies from X-ray film revealed the proteases’ capability to remove silver from X-ray film, leaving the film intact. The recovery of silver was perceived visually, by film observation, as well as by scan electron microscopy (SEM) images, where clearance of the film was observed after incubation with the enzyme. Energy dispersive X-ray spectroscopy (EDS) profiles also confirmed removal of silver from the film, with a Ag peak showing on the profile of the film before treatment with the proteases and no peak after treatment. The crude protease sample was, however, catalytically more efficient compared to the partially purified sample. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Authors: Manyumwa, Colleen Varaidzo
- Date: 2018
- Subjects: Ascomycetes , Mycorrhizal fungi , Ericaceae , Proteolytic enzymes , Silver Recycling
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/62833 , vital:28298
- Description: The aim of this study was to produce, purify and characterise proteases from the ericoid mycorrhizal fungus, Oidiodendron maius (CafRU082b/KP119480), as well as to explore their potential application in the recovery of silver from X-ray film. Firstly, the growth of the ericoid mycorrhizal fungus, Oidiodendron maius (CafRU082b), was studied, and its ability to produce proteolytic enzymes was investigated. O. maius proved to grow well in the dark, submerged in Modified Melin Norkran’s liquid medium at a pH of 5 and at 25°C. Pure cultures of the fungus were maintained on Potato Dextrose Agar (PDA). The fungus grew on PDA plates containing different substrates including haemoglobin, casein, gelatin as well as azocasein. Zones of clearance, however, were only observed on plates containing gelatin after treatment with mercuric chloride, HgCl2. Proteases were successfully produced after 14 days when gelatin was incorporated into the growth medium. After production of the proteases, purification and characterisation of the enzymes was performed. Purification of the enzymes was performed by acetone precipitation followed by ultrafiltration with 50 kDa and 30 kDa cut off membrane filters. A final purification fold of approximately 37.6 was achieved. Unusual yields of above 100% were observed after each purification step with the final yield achieved being 196% with a final specific activity of 2707 U/mg. SDS-PAGE revealed a protease band of 35 kDa which was also visible on the zymogram at approximately 36 kDa. The zymogram showed clear hydrolysis bands against a blue background after staining with Coomassie Brilliant Blue. Physico-chemical characterisation of the protease revealed its pH optimum to be pH 3.0 and its temperature optimum 68°C. Another peak was observed on the pH profile at pH 7.0. The protease exhibited high thermostability at temperatures 37°C, 80°C as well as 100°C with the enzyme retaining close to 50% of its initial activity after 4 h of exposure to all three temperatures. All ions tested for their effects on the proteases, except Ca2+, enhanced protease activity. Ca2+ did not exhibit any significant effect on the enzyme’s activity while Zn2+ had the highest effect, enhancing enzyme activity by 305%. The proteases, however, were not significantly inhibited by EDTA, a metal chelating agent and a known metalloprotease inhibitor. The enzyme was classified as an aspartic protease due to complete inhibition by 25 μM of pepstatin A, coupled to its low pH optimum of 3.0. Addition of trans-Epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), a cysteine protease inhibitor, and 2-mercaptoethanol increased protease activity. The proteases exhibited a narrow substrate specificity towards gelatin and no other substrate. Substrate kinetics values were plotted on a Michaelis-Menten Graph and showed that the enzyme had a Vmax of 55.25 U/ml and a Km of 2.7 mg/ml gelatin. A low Km indicated that the protease had a high affinity for gelatin. Silver recovery studies from X-ray film revealed the proteases’ capability to remove silver from X-ray film, leaving the film intact. The recovery of silver was perceived visually, by film observation, as well as by scan electron microscopy (SEM) images, where clearance of the film was observed after incubation with the enzyme. Energy dispersive X-ray spectroscopy (EDS) profiles also confirmed removal of silver from the film, with a Ag peak showing on the profile of the film before treatment with the proteases and no peak after treatment. The crude protease sample was, however, catalytically more efficient compared to the partially purified sample. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
Assessing the potential role of microorganisms in the production of seedlings for the restoration of Albany Thicket
- Authors: Mpama, Nelisa
- Date: 2017
- Subjects: Vesicular-arbuscular mycorrhizas , Rhizobacteria , Restoration ecology South Africa Albany , Microorganisms
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/64555 , vital:28558
- Description: The role of microorganisms in restoration of the Albany Thicket has not been well documented, although the benefits to plants of these various interactions has been well documented. Microorganisms are chief ecological engineers and assist in resolving environmental problems and act to restore degraded ecosystem function by forming mutual relationships with the roots of the plants. The aim of this study was to assess the potential of microorganisms for the improved biomass production of selected woody and succulent seedlings used in mesic thicket restoration. Three tree species were selected for propagation in this study namely; Mystroxylon aethiopicum Scutia myrtina and Aloe ferox. Soil samples were collected from a degraded and intact thicket site from Bathurst, South Africa. Soils were evaluated for number of arbuscular mycorrhizal (AM) spores, mycorrhizal infectivity potential and nutrient availability both before and after seedling propagation. Pasteurized soil from the degraded site was used in a pot trial. Ten replicates seedling for plant species were planted and subjected to four treatments which included inoculation with AM fungi and the rhizobacterium, Enterobacter sp., alone and in combination; the fourth treatment was an un-inouclated control. Plant growth parameters were recorded at regular intervals where appropriate and seedlings were harvested after 24 weeks for biomass measurements and AM colonisation assessments. Although generally low (< 1 spore per gram) the density of AM fungal spores was significantly higher in soils from the intact site when compared with soils from the degraded site. The mycorrhizal potential of the soils was however not significantly different. Mystroxylon aethiopicum seedling shoot height, canopy diameter and shoot biomass showed a significant increase when inoculated with AM fungi while S. myrtina seedlings showed increased shoot height when inoculated with both AM fungi and Enterobacter sp. Aloe ferox seedlings did not respond to microbial inoculation. The concentration of soil P and Na increased in treatments with Enterobacter sp. alone and in combination with AM fungi. Mystroxylon aethiopicum and S. myrtina seedlings showed a dependency on microbial inoculants indicating the importance of inoculation in the nursery before planting out into the field. Overall AM fungal inoculants applied to seedlings can be used to compensate for nutrient deficiency in soils. Although the Enterobacter isolate used was known to have various plant growth promoting capabilities. It is recommended that other rhizobacterial isolates be investigated. , Thesis (MSc) -- Faculty of Science, Environmental Science, 2017
- Full Text:
- Authors: Mpama, Nelisa
- Date: 2017
- Subjects: Vesicular-arbuscular mycorrhizas , Rhizobacteria , Restoration ecology South Africa Albany , Microorganisms
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/64555 , vital:28558
- Description: The role of microorganisms in restoration of the Albany Thicket has not been well documented, although the benefits to plants of these various interactions has been well documented. Microorganisms are chief ecological engineers and assist in resolving environmental problems and act to restore degraded ecosystem function by forming mutual relationships with the roots of the plants. The aim of this study was to assess the potential of microorganisms for the improved biomass production of selected woody and succulent seedlings used in mesic thicket restoration. Three tree species were selected for propagation in this study namely; Mystroxylon aethiopicum Scutia myrtina and Aloe ferox. Soil samples were collected from a degraded and intact thicket site from Bathurst, South Africa. Soils were evaluated for number of arbuscular mycorrhizal (AM) spores, mycorrhizal infectivity potential and nutrient availability both before and after seedling propagation. Pasteurized soil from the degraded site was used in a pot trial. Ten replicates seedling for plant species were planted and subjected to four treatments which included inoculation with AM fungi and the rhizobacterium, Enterobacter sp., alone and in combination; the fourth treatment was an un-inouclated control. Plant growth parameters were recorded at regular intervals where appropriate and seedlings were harvested after 24 weeks for biomass measurements and AM colonisation assessments. Although generally low (< 1 spore per gram) the density of AM fungal spores was significantly higher in soils from the intact site when compared with soils from the degraded site. The mycorrhizal potential of the soils was however not significantly different. Mystroxylon aethiopicum seedling shoot height, canopy diameter and shoot biomass showed a significant increase when inoculated with AM fungi while S. myrtina seedlings showed increased shoot height when inoculated with both AM fungi and Enterobacter sp. Aloe ferox seedlings did not respond to microbial inoculation. The concentration of soil P and Na increased in treatments with Enterobacter sp. alone and in combination with AM fungi. Mystroxylon aethiopicum and S. myrtina seedlings showed a dependency on microbial inoculants indicating the importance of inoculation in the nursery before planting out into the field. Overall AM fungal inoculants applied to seedlings can be used to compensate for nutrient deficiency in soils. Although the Enterobacter isolate used was known to have various plant growth promoting capabilities. It is recommended that other rhizobacterial isolates be investigated. , Thesis (MSc) -- Faculty of Science, Environmental Science, 2017
- Full Text:
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