Bioprospecting for amylases, cellulases and xylanases from ericoid associated fungi, their production and characterisation for the bio-economy
- Authors: Adeoyo, Olusegun Richard
- Date: 2018
- Subjects: Mycorrhizal fungi , Hydrolases , Ericaceae South Africa , Ericaceae Molecular aspects
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/64327 , vital:28533
- Description: South Africa is one of the most productive areas for ericaceous plants with about 850 identified species in the Cape Floral Region. The Albany Centre of Endemism where all fungi used in this study were isolated from, falls within this region. Ericaceous plants interact with some fungi via an association called the ericoid mycorrhizal (ERM) association. All fungi used in this study were isolated from roots of six ericaceous plants; Erica cerinthoides, Erica demissa, Erica chamissonis, Erica glumiflora, Erica caffra and Erica nemorosa. Fungal enzymes are known to play a significant role in the food, brewing, detergent, pharmaceutical and biofuel industries. The enzyme industry is among the major sectors of the world, and additional novel sources are being explored from time to time. This study focussed on amylases (amyloglucosidase, AMG), cellulases (endoglucanase) and xylanases (endo-1,4-P-xylanase) production from ERM fungal isolates. Out of the fifty-one (51), fungal isolates screened, ChemRU330 (Leohumicola sp.), EdRU083 and EdRU002 were among the fungi that had the highest activities of all the enzymes. They were tested for the ability to produce amylases and cellulases under different pH and nutritional conditions that included: carbon sources, nitrogen sources and metal ions, at an optimum temperature of 28°C in a modified Melin-Norkrans (MMN) liquid medium. Cellulase specific activity of 3.99, 2.18 and 4.31 (U/mg protein) for isolates EdRU083, EdRU002 and ChemRU330, respectively, was produced at an optimal pH of 5.0. For amylase, ChemRU330 had the highest specific activity of 1.11 U/mg protein while EdRU083 and EdRU02 had a specific activity of 0.80 and 0.92 U/mg protein, respectively, at the same pH with corresponding biomass yield of 113, 125 and 97 mg/50 ml, respectively. Increased enzyme activities and improved mycelial biomass production were obtained in the presence of supplements such as potassium, sodium, glucose, maltose, cellobiose, tryptone and peptone, while NaFe-EDTA and cobalt inhibited enzyme activity. ChemRU330 was selected to determine the consistency and amount of amylase, cellulase and xylanase formed after several in vitro subculturing events. AMG and endo-1,4-P-xylanase were found to have the most consistent production throughout the study period. The AMG was stable at 45oC (pH 5.0), retaining approximately 65% activity over a period of 24 h. The molecular mass of AMG and endo-1,4-P-xylanase were estimated to be 101 kDa and 72 kDa, respectively. The Km and kcat were 0.38 mg/ml and 70 s-1, respectively, using soluble starch (AMG). For endo-1,4-P-xylanase, the Km and Vmax were 0.93 mg/ml and 8.54 U/ml, respectively, using beechwood xylan (endo-1,4-P-xylanase) as substrate. Additionally, crude extracts of five root endophytes with unique morphological characteristics were screened for antibacterial properties and was followed by determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). L. incrustata (ChemRU330) and Chaetomium sp. extracts exhibited varying degrees of inhibition against two Gram-positive and Gram-negative bacteria. The crude extract of L. incrustata was the most effective which was found to inhibit Staphylococcus aureus (MIC: 1 mg/ml), Bacillus subtilis (MIC: 2 mg/ml) and Proteus vulgaris (MIC: 16 mg/ml). The L. incrustata displayed potential for antibacterial production and could be considered as an additional source of new antimicrobial agents in drug and food preservation. Also, the three isolates used for enzyme production were identified to genus and species levels, i.e., Leohumicola incrustata (ChemRU330), Leohumicola sp. (EdRU083) and Oidiodendron sp. (EdRU002) using both ITS and Cox1 DNA regions. The molecular analysis results indicated that these ERM mycorrhizal fungi were similar to those successfully described by some researchers in South Africa and Australia. Therefore, this study opens new opportunities for exploring ERM fungal biomolecules for the bio-economy. The promising physicochemical properties, starch and xylan hydrolysis end- products, and being non-pathogenic make AMG and endo-1,4-P-xylanase potential candidates for future applications as additives in the food industry for the production of glucose, glucose syrups, high-fructose corn syrups, and as well as the production of bioethanol. Finally, the findings of this study revealed that it is possible to produce hydrolytic enzymes from ERM fungi in vitro using chemically defined media. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Authors: Adeoyo, Olusegun Richard
- Date: 2018
- Subjects: Mycorrhizal fungi , Hydrolases , Ericaceae South Africa , Ericaceae Molecular aspects
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/64327 , vital:28533
- Description: South Africa is one of the most productive areas for ericaceous plants with about 850 identified species in the Cape Floral Region. The Albany Centre of Endemism where all fungi used in this study were isolated from, falls within this region. Ericaceous plants interact with some fungi via an association called the ericoid mycorrhizal (ERM) association. All fungi used in this study were isolated from roots of six ericaceous plants; Erica cerinthoides, Erica demissa, Erica chamissonis, Erica glumiflora, Erica caffra and Erica nemorosa. Fungal enzymes are known to play a significant role in the food, brewing, detergent, pharmaceutical and biofuel industries. The enzyme industry is among the major sectors of the world, and additional novel sources are being explored from time to time. This study focussed on amylases (amyloglucosidase, AMG), cellulases (endoglucanase) and xylanases (endo-1,4-P-xylanase) production from ERM fungal isolates. Out of the fifty-one (51), fungal isolates screened, ChemRU330 (Leohumicola sp.), EdRU083 and EdRU002 were among the fungi that had the highest activities of all the enzymes. They were tested for the ability to produce amylases and cellulases under different pH and nutritional conditions that included: carbon sources, nitrogen sources and metal ions, at an optimum temperature of 28°C in a modified Melin-Norkrans (MMN) liquid medium. Cellulase specific activity of 3.99, 2.18 and 4.31 (U/mg protein) for isolates EdRU083, EdRU002 and ChemRU330, respectively, was produced at an optimal pH of 5.0. For amylase, ChemRU330 had the highest specific activity of 1.11 U/mg protein while EdRU083 and EdRU02 had a specific activity of 0.80 and 0.92 U/mg protein, respectively, at the same pH with corresponding biomass yield of 113, 125 and 97 mg/50 ml, respectively. Increased enzyme activities and improved mycelial biomass production were obtained in the presence of supplements such as potassium, sodium, glucose, maltose, cellobiose, tryptone and peptone, while NaFe-EDTA and cobalt inhibited enzyme activity. ChemRU330 was selected to determine the consistency and amount of amylase, cellulase and xylanase formed after several in vitro subculturing events. AMG and endo-1,4-P-xylanase were found to have the most consistent production throughout the study period. The AMG was stable at 45oC (pH 5.0), retaining approximately 65% activity over a period of 24 h. The molecular mass of AMG and endo-1,4-P-xylanase were estimated to be 101 kDa and 72 kDa, respectively. The Km and kcat were 0.38 mg/ml and 70 s-1, respectively, using soluble starch (AMG). For endo-1,4-P-xylanase, the Km and Vmax were 0.93 mg/ml and 8.54 U/ml, respectively, using beechwood xylan (endo-1,4-P-xylanase) as substrate. Additionally, crude extracts of five root endophytes with unique morphological characteristics were screened for antibacterial properties and was followed by determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). L. incrustata (ChemRU330) and Chaetomium sp. extracts exhibited varying degrees of inhibition against two Gram-positive and Gram-negative bacteria. The crude extract of L. incrustata was the most effective which was found to inhibit Staphylococcus aureus (MIC: 1 mg/ml), Bacillus subtilis (MIC: 2 mg/ml) and Proteus vulgaris (MIC: 16 mg/ml). The L. incrustata displayed potential for antibacterial production and could be considered as an additional source of new antimicrobial agents in drug and food preservation. Also, the three isolates used for enzyme production were identified to genus and species levels, i.e., Leohumicola incrustata (ChemRU330), Leohumicola sp. (EdRU083) and Oidiodendron sp. (EdRU002) using both ITS and Cox1 DNA regions. The molecular analysis results indicated that these ERM mycorrhizal fungi were similar to those successfully described by some researchers in South Africa and Australia. Therefore, this study opens new opportunities for exploring ERM fungal biomolecules for the bio-economy. The promising physicochemical properties, starch and xylan hydrolysis end- products, and being non-pathogenic make AMG and endo-1,4-P-xylanase potential candidates for future applications as additives in the food industry for the production of glucose, glucose syrups, high-fructose corn syrups, and as well as the production of bioethanol. Finally, the findings of this study revealed that it is possible to produce hydrolytic enzymes from ERM fungi in vitro using chemically defined media. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
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:
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