Immobilisation of an Aspergillus niger derived endo-1,4-β-mannanase, Man26A, for the production of prebiotic mannooligosaccharides from soybean meal
- Authors: Anderson, Amy Sage
- Date: 2024-10-11
- Subjects: Aspergillus niger , Soybean meal , Mannosidases , Oligosaccharides , Immobilized microorganisms
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/463897 , vital:76455
- Description: This study investigated the potential for antibiotic alternatives in the form of prebiotics produced from the enzymatic breakdown of soybean meal (SBM). This study first investigated the immobilisation of an endo-1,4-β-mannanase derived from Aspergillus niger on glutaraldehyde-activated chitosan nanoparticles (CTS) and glutaraldehyde-activated chitosan-coated magnetic Fe3O4 nanoparticles (MAGS-CTS) - which could be effectively used to hydrolyse the galactomannan contained in SBM in a recyclable manner. The mannooligosaccharides (MOS) produced from the enzymatic digestion of SBM were then analysed for their prebiotic and antimicrobial capabilities to determine whether the strategy employed was capable of promoting and inhibiting probiotic and pathogenic growth, respectively. An Aspergillus niger endo-1,4-β-mannanase, Man26A, was confirmed by FTIR (Fourier-transform infrared spectroscopy) and XRD (X-ray powder diffraction) to be immobilised onto CTS and MAGS-CTS by covalent bonding. The immobilisation (%) and activity yields (%) were 81.14% and 35.45%, and 55.75% and 21.17%, respectively. The biochemical properties (pH and temperature optima, and temperature stability) of both the free CTS and MAGS-CTS immobilised Man26A enzymes were evaluated, with the pH optima shifting to a lower pH range after immobilisation (pH 2.0 – 3.0 vs. 5.0), while the temperature optima and stabilities remaining unchanged (at 60°C). CTS and free enzymes exhibited identical thermal stabilities, maintaining 100% activity for the first 6 hours at 55°C, while MAGS-CTS showed an immediate drop in relative activity after the first 30 minutes of incubation. Recyclability analysis revealed that CTS could be effectively reused for six reaction cycles, while the MAGS-CTS immobilised enzyme could only be used once. Both enzymes could be efficiently stored at 4ºC, showing a relative residual activity of 73% after 120 hours of storage. Substrate kinetic analysis showed that the free enzyme had the highest catalytic capabilities in hydrolysing locust bean gum (LBG), with the CTS immobilised enzyme was the most efficient in hydrolysing SBM, the insoluble, complex substrate. Sugar residues produced from the hydrolysis of LBG illustrated the effective breakdown of galactomannan to mannobiose (M2), mannotriose (M3), mannotetrose (M4), and mannohexose (M6). SBM-produced sugars analysed via TLC and HPLC indicated that the MOS residues were most probably glucose, galactose, and galactomannans (GM2 and GM3). The SBM-produced sugars were then evaluated for their prebiotic effect, illustrating their successful utilisation as a carbon source by probiotic bacteria; Streptococcus thermophilus, Bacillus subtilis and Lactobacillus bulgaricus. Evaluation of the antimicrobial activities of the SBM-produced sugars digested by probiotics suggested that their metabolites had the potential to be used as an antibiotic alternative. This study therefore illustrated that an endo-1,4-β-mannanase derived from Aspergillus niger could be immobilised successfully, for use in a recyclable reaction to produce MOS products. This study also described the successful use of SBM-sugars as a prebiotic, indicating a successful alternative to antibiotic growth promoters (AGP) by illustrating their positive effect on inhibiting growth of pathogenic bacterial species. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology & Bioinformatics, 2024
- Full Text:
- Date Issued: 2024-10-11
- Authors: Anderson, Amy Sage
- Date: 2024-10-11
- Subjects: Aspergillus niger , Soybean meal , Mannosidases , Oligosaccharides , Immobilized microorganisms
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/463897 , vital:76455
- Description: This study investigated the potential for antibiotic alternatives in the form of prebiotics produced from the enzymatic breakdown of soybean meal (SBM). This study first investigated the immobilisation of an endo-1,4-β-mannanase derived from Aspergillus niger on glutaraldehyde-activated chitosan nanoparticles (CTS) and glutaraldehyde-activated chitosan-coated magnetic Fe3O4 nanoparticles (MAGS-CTS) - which could be effectively used to hydrolyse the galactomannan contained in SBM in a recyclable manner. The mannooligosaccharides (MOS) produced from the enzymatic digestion of SBM were then analysed for their prebiotic and antimicrobial capabilities to determine whether the strategy employed was capable of promoting and inhibiting probiotic and pathogenic growth, respectively. An Aspergillus niger endo-1,4-β-mannanase, Man26A, was confirmed by FTIR (Fourier-transform infrared spectroscopy) and XRD (X-ray powder diffraction) to be immobilised onto CTS and MAGS-CTS by covalent bonding. The immobilisation (%) and activity yields (%) were 81.14% and 35.45%, and 55.75% and 21.17%, respectively. The biochemical properties (pH and temperature optima, and temperature stability) of both the free CTS and MAGS-CTS immobilised Man26A enzymes were evaluated, with the pH optima shifting to a lower pH range after immobilisation (pH 2.0 – 3.0 vs. 5.0), while the temperature optima and stabilities remaining unchanged (at 60°C). CTS and free enzymes exhibited identical thermal stabilities, maintaining 100% activity for the first 6 hours at 55°C, while MAGS-CTS showed an immediate drop in relative activity after the first 30 minutes of incubation. Recyclability analysis revealed that CTS could be effectively reused for six reaction cycles, while the MAGS-CTS immobilised enzyme could only be used once. Both enzymes could be efficiently stored at 4ºC, showing a relative residual activity of 73% after 120 hours of storage. Substrate kinetic analysis showed that the free enzyme had the highest catalytic capabilities in hydrolysing locust bean gum (LBG), with the CTS immobilised enzyme was the most efficient in hydrolysing SBM, the insoluble, complex substrate. Sugar residues produced from the hydrolysis of LBG illustrated the effective breakdown of galactomannan to mannobiose (M2), mannotriose (M3), mannotetrose (M4), and mannohexose (M6). SBM-produced sugars analysed via TLC and HPLC indicated that the MOS residues were most probably glucose, galactose, and galactomannans (GM2 and GM3). The SBM-produced sugars were then evaluated for their prebiotic effect, illustrating their successful utilisation as a carbon source by probiotic bacteria; Streptococcus thermophilus, Bacillus subtilis and Lactobacillus bulgaricus. Evaluation of the antimicrobial activities of the SBM-produced sugars digested by probiotics suggested that their metabolites had the potential to be used as an antibiotic alternative. This study therefore illustrated that an endo-1,4-β-mannanase derived from Aspergillus niger could be immobilised successfully, for use in a recyclable reaction to produce MOS products. This study also described the successful use of SBM-sugars as a prebiotic, indicating a successful alternative to antibiotic growth promoters (AGP) by illustrating their positive effect on inhibiting growth of pathogenic bacterial species. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology & Bioinformatics, 2024
- Full Text:
- Date Issued: 2024-10-11
Production of mannooligosaccharides from pineapple pulp and pine sawdust using Aspergillus niger derived Man26A and determination of their prebiotic effect
- Authors: Hlalukana, Nosipho Pretty
- Date: 2022-10-14
- Subjects: Oligosaccharides , Prebiotics , Lignocellulose , Mannans
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362853 , vital:65368
- Description: Lignocellulosic biomass is the most abundant source of renewable biomass on earth. Lignocellulosic biomass consists of cellulose, hemicelluloses and lignin. These can be used as a source of renewable fuel as well as other value-added products . Mannans are part of the hemicellulose fraction of lignocellulosic biomass and are the major hemicellulosic polysaccharide fraction in softwoods, where they are found as galactoglucomannans and as glucomannans. Mannans are also found in hardwoods in the form of glucomannans. Mannans can be enzymatically hydrolysed using endo-mannanases to produce of short chain mannooligosaccharides (MOS). MOS have received significant attention for their prebiotic properties, as they promote the growth of probiotic bacteria, which have positively affects on gut health. This study focused on the production of prebiotic MOS from lignocellulosic biomass waste (LBW) and an evaluation of the prebiotic potential of the produced MOS. An Aspergillus niger derived endo-mannanase, Man26A, was fractionated and biochemically analysed. Purified Man26A had a fold purification of 1.25 and a yield of 41.1%. SDS-PAGE analysis of the enzyme revealed that it had a molecular weight of 46 kDa. The pH and temperature optima of Man26A were determined and the pH optimum was found to be pH 4.0 (but the enzyme displayed high activity over a broad acidic pH range, with up to 90% of the activity retained between pH 3.0 and 7.0). The temperature optimum was 50℃. The enzyme was shown to have the highest specific activity on locust bean gum (52.27 U/mg) and ivory nut mannan (57.25 U/mg), compared to guar gum (29.07 U/mg), which indicated that it was affected by the substitution pattern of the mannans. Man26A produced MOS of different diversity on model mannan substrates, where the MOS produced were mannobiose, mannotriose, and mannotetraose for ivory nut mannan, mannobiose, mannotriose, mannotetraose, and mannopentaose and MOS with a higher degree of polymerisation for locust bean gum, and mannobiose, mannotriose, mannotetraose, mannopentaose, and mannohexose and MOS with a higher degree of polymerisation for guar gum, as determined by thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Pretreatment and characterisation of pineapple pulp (PP) and pine sawdust (PSD) was conducted, and the impact of the pretreatment procedures was analysed using Megazyme sugar kits, thermogravimetric analysis (TGA), Fourier-transform infrared spectroscopy (FTIR), and microscopic analysis using scanning electron microscopy (SEM) and light microscopy. Compositional analysis of the carbohydrates present in both substrates revealed that they had a glucan content of 36.41 and 50.47% for untreated PP and PSD, respectively. Their respective mannan content was 6.74 and 11.59% and was deemed sufficient for the production of MOS via enzymatic hydrolysis. TGA analysis revealed that untreated and sodium chlorite-acetic acid delignified samples decomposed at approximately the same time, and had a negligible ash content at 600℃, while delignified plus phosphoric acid swollen substrates decomposed at a faster rate, but had a residual ash content at 600℃. FTIR analysis of the substrates revealed slight changes in the structures of untreated and pretreated samples. SEM analysis of PP and PSD showed a change in the morphology of the substrates with subsequent pretreatment steps. Histochemical analysis for lignin for PP and PSD showed successful delignification upon pretreatment. Untreated and sodium chlorite delignified PP and PSD released low amounts of reducing sugars compared to delignified + phosphoric acid swollen substrates. The delignified + phosphoric acid swollen substrates were used for further experiments. MOS produced from delignified and phosphoric acid swollen (Del + PAS) PP and PSD at 0.1 mg/ml enzyme loading and 80 mg/ml (8% (w/v)) substrate concentration, ran between mannose and mannobiose and between mannobiose and manotriose on TLC, with low concentrations of MOS running between mannotetraose and mannopentaose. HPLC analysis of the MOS revealed that Del + PAS PP produced mannose to mannohexose, while Del + PAS PSD produced mannose, mannobiose, and mannotetraose. The MOS were analysed using FTIR, to determine whether the MOS produced contained any acetyl groups, which were present for Del + PAS PSD at 1706 cm-1. The MOS were stable at different pHs, and at temperatures below 200℃. The MOS were also found to be stable in a simulated gastrointestinal environment, in the presence of bile salts and digestive enzymes. The prebiotic effect of the MOS derived from Del + PAS PP and PSD was evaluated. MOS had a proliferative effect on probiotic bacteria (Lactobacillus bulgaricus, Bacillus subtilis and Streptococcus thermophilus). The production of short chain fatty acids (SCFAs) was evaluated on TLC, where no SCFAs were observed on the plate. The effect of MOS on the adhesion ability of bacteria revealed that they do not positively influence the adhesion of probiotic bacteria. The antioxidant activities of 1 mg/ml MOS produced from both substrates were determined to be approximately 15% using the ABTS radical scavenging assay, compared to a radical scavenging activity of 45% for the 0.02 mg/ml gallic acid standard. This study demonstrated that biomass waste could be used to produce prebiotic MOS, which play a positive role in gut ecology and provide health benefits. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Hlalukana, Nosipho Pretty
- Date: 2022-10-14
- Subjects: Oligosaccharides , Prebiotics , Lignocellulose , Mannans
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362853 , vital:65368
- Description: Lignocellulosic biomass is the most abundant source of renewable biomass on earth. Lignocellulosic biomass consists of cellulose, hemicelluloses and lignin. These can be used as a source of renewable fuel as well as other value-added products . Mannans are part of the hemicellulose fraction of lignocellulosic biomass and are the major hemicellulosic polysaccharide fraction in softwoods, where they are found as galactoglucomannans and as glucomannans. Mannans are also found in hardwoods in the form of glucomannans. Mannans can be enzymatically hydrolysed using endo-mannanases to produce of short chain mannooligosaccharides (MOS). MOS have received significant attention for their prebiotic properties, as they promote the growth of probiotic bacteria, which have positively affects on gut health. This study focused on the production of prebiotic MOS from lignocellulosic biomass waste (LBW) and an evaluation of the prebiotic potential of the produced MOS. An Aspergillus niger derived endo-mannanase, Man26A, was fractionated and biochemically analysed. Purified Man26A had a fold purification of 1.25 and a yield of 41.1%. SDS-PAGE analysis of the enzyme revealed that it had a molecular weight of 46 kDa. The pH and temperature optima of Man26A were determined and the pH optimum was found to be pH 4.0 (but the enzyme displayed high activity over a broad acidic pH range, with up to 90% of the activity retained between pH 3.0 and 7.0). The temperature optimum was 50℃. The enzyme was shown to have the highest specific activity on locust bean gum (52.27 U/mg) and ivory nut mannan (57.25 U/mg), compared to guar gum (29.07 U/mg), which indicated that it was affected by the substitution pattern of the mannans. Man26A produced MOS of different diversity on model mannan substrates, where the MOS produced were mannobiose, mannotriose, and mannotetraose for ivory nut mannan, mannobiose, mannotriose, mannotetraose, and mannopentaose and MOS with a higher degree of polymerisation for locust bean gum, and mannobiose, mannotriose, mannotetraose, mannopentaose, and mannohexose and MOS with a higher degree of polymerisation for guar gum, as determined by thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Pretreatment and characterisation of pineapple pulp (PP) and pine sawdust (PSD) was conducted, and the impact of the pretreatment procedures was analysed using Megazyme sugar kits, thermogravimetric analysis (TGA), Fourier-transform infrared spectroscopy (FTIR), and microscopic analysis using scanning electron microscopy (SEM) and light microscopy. Compositional analysis of the carbohydrates present in both substrates revealed that they had a glucan content of 36.41 and 50.47% for untreated PP and PSD, respectively. Their respective mannan content was 6.74 and 11.59% and was deemed sufficient for the production of MOS via enzymatic hydrolysis. TGA analysis revealed that untreated and sodium chlorite-acetic acid delignified samples decomposed at approximately the same time, and had a negligible ash content at 600℃, while delignified plus phosphoric acid swollen substrates decomposed at a faster rate, but had a residual ash content at 600℃. FTIR analysis of the substrates revealed slight changes in the structures of untreated and pretreated samples. SEM analysis of PP and PSD showed a change in the morphology of the substrates with subsequent pretreatment steps. Histochemical analysis for lignin for PP and PSD showed successful delignification upon pretreatment. Untreated and sodium chlorite delignified PP and PSD released low amounts of reducing sugars compared to delignified + phosphoric acid swollen substrates. The delignified + phosphoric acid swollen substrates were used for further experiments. MOS produced from delignified and phosphoric acid swollen (Del + PAS) PP and PSD at 0.1 mg/ml enzyme loading and 80 mg/ml (8% (w/v)) substrate concentration, ran between mannose and mannobiose and between mannobiose and manotriose on TLC, with low concentrations of MOS running between mannotetraose and mannopentaose. HPLC analysis of the MOS revealed that Del + PAS PP produced mannose to mannohexose, while Del + PAS PSD produced mannose, mannobiose, and mannotetraose. The MOS were analysed using FTIR, to determine whether the MOS produced contained any acetyl groups, which were present for Del + PAS PSD at 1706 cm-1. The MOS were stable at different pHs, and at temperatures below 200℃. The MOS were also found to be stable in a simulated gastrointestinal environment, in the presence of bile salts and digestive enzymes. The prebiotic effect of the MOS derived from Del + PAS PP and PSD was evaluated. MOS had a proliferative effect on probiotic bacteria (Lactobacillus bulgaricus, Bacillus subtilis and Streptococcus thermophilus). The production of short chain fatty acids (SCFAs) was evaluated on TLC, where no SCFAs were observed on the plate. The effect of MOS on the adhesion ability of bacteria revealed that they do not positively influence the adhesion of probiotic bacteria. The antioxidant activities of 1 mg/ml MOS produced from both substrates were determined to be approximately 15% using the ABTS radical scavenging assay, compared to a radical scavenging activity of 45% for the 0.02 mg/ml gallic acid standard. This study demonstrated that biomass waste could be used to produce prebiotic MOS, which play a positive role in gut ecology and provide health benefits. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
An evaluation of synergistic interactions between feruloyl esterases and xylanases during the hydrolysis of various pre-treated agricultural residues
- Authors: Mkabayi, Lithalethu
- Date: 2021-04
- Subjects: Esterases , Xylanases , Hydrolysis , Agricultural wastes -- Recycling , Enzymes , Lignocellulose -- Biodegradation , Escherichia coli , Oligosaccharides , Hydroxycinnamic acids
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/178224 , vital:42922 , 10.21504/10962/178224
- Description: Agricultural residues are readily available and inexpensive renewable resources that can be used as raw materials for the production of value-added chemicals. The application of enzymes to facilitate the degradation of agricultural residues has long been considered the most environmentally friendly strategy for converting this material into good quality value-added chemicals. However, agricultural residues are typically lignocellulosic in composition and recalcitrant to enzymatic hydrolysis. Due to this recalcitrant nature, the complete degradation of biomass residues requires the synergistic action of a broad range of enzymes. The development and optimisation of synergistic enzyme cocktails is an effective approach for achieving high hydrolysis efficiency of lignocellulosic biomass. The aim of the current study was to evaluate the synergistic interactions between two termite metagenome-derived feruloyl esterases (FAE6 and FAE5) and endo-xylanases for the production of hydroxycinnamic acids and xylo-oligosaccharides (XOS) from model substrates, and untreated and pre-treated agricultural residues. Firstly, the two fae genes were heterologously expressed in Escherichia coli, and the recombinant enzymes were purified to homogeneity. The biochemical properties of the purified recombinant FAEs and xylanases (XT6 and Xyn11) were then assessed to determine the factors which influenced their activities and to select suitable operating conditions for synergy studies. An optimal protein loading ratio of xylanases to FAEs required to maximise the release of both reducing sugar and ferulic acid (FA) was established using 0.5% (w/v) insoluble wheat arabinoxylan (a model substrate). The enzyme combination of 66% xylanase and 33% FAE (on a protein loading basis) produced the highest amounts of reducing sugars and FA. The enzyme combination of XT6 (GH10 xylanase) and FAE5 or FAE6 liberated the highest amount of FA while a combination of Xyn11 (GH11 xylanase) and FAE5 or FAE6 produced the highest reducing sugar content. The synergistic interactions which were established between the xylanases and FAEs were further investigated using agricultural residues (corn cobs, rice straw and sugarcane bagasse). The three substrates were subjected to hydrothermal and dilute acid pre-treatment prior to synergy studies. It is generally known that, during pre-treatment, many compounds can be produced which may influence enzymatic hydrolysis. The effects of these by-products were assessed and it was found that lignin and its degradation products were the most inhibitory to the FAEs. The optimised enzyme cocktail was then applied to 1% (w/v) of untreated and pre-treated substrates for the efficient production of XOS and hydroxycinnamic acids. A significant improvement in xylanase substrate degradation was observed, especially with the combination of 66% Xyn11 and 33% FAE6 which displayed an improvement in reducing sugars of approximately 1.9-fold and 3.4-fold for hydrothermal and acid pre-treated corn cobs (compared to when Xyn11 was used alone), respectively. The study demonstrated that pre-treatment substantially enhanced the enzymatic hydrolysis of corn cobs and rice straw. Analysis of the hydrolysate product profiles revealed that the optimised enzyme cocktail displayed great potential for releasing XOS with a low degree of polymerisation. In conclusion, this study provided significant insights into the mechanism of synergistic interactions between xylanases and metagenome-derived FAEs during the hydrolysis of various substrates. The study also demonstrated that optimised enzyme cocktails combined with low severity pre-treatment can facilitate the potential use of xylan-rich lignocellulosic biomass for the production of valuable products in the future. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-04
- Authors: Mkabayi, Lithalethu
- Date: 2021-04
- Subjects: Esterases , Xylanases , Hydrolysis , Agricultural wastes -- Recycling , Enzymes , Lignocellulose -- Biodegradation , Escherichia coli , Oligosaccharides , Hydroxycinnamic acids
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/178224 , vital:42922 , 10.21504/10962/178224
- Description: Agricultural residues are readily available and inexpensive renewable resources that can be used as raw materials for the production of value-added chemicals. The application of enzymes to facilitate the degradation of agricultural residues has long been considered the most environmentally friendly strategy for converting this material into good quality value-added chemicals. However, agricultural residues are typically lignocellulosic in composition and recalcitrant to enzymatic hydrolysis. Due to this recalcitrant nature, the complete degradation of biomass residues requires the synergistic action of a broad range of enzymes. The development and optimisation of synergistic enzyme cocktails is an effective approach for achieving high hydrolysis efficiency of lignocellulosic biomass. The aim of the current study was to evaluate the synergistic interactions between two termite metagenome-derived feruloyl esterases (FAE6 and FAE5) and endo-xylanases for the production of hydroxycinnamic acids and xylo-oligosaccharides (XOS) from model substrates, and untreated and pre-treated agricultural residues. Firstly, the two fae genes were heterologously expressed in Escherichia coli, and the recombinant enzymes were purified to homogeneity. The biochemical properties of the purified recombinant FAEs and xylanases (XT6 and Xyn11) were then assessed to determine the factors which influenced their activities and to select suitable operating conditions for synergy studies. An optimal protein loading ratio of xylanases to FAEs required to maximise the release of both reducing sugar and ferulic acid (FA) was established using 0.5% (w/v) insoluble wheat arabinoxylan (a model substrate). The enzyme combination of 66% xylanase and 33% FAE (on a protein loading basis) produced the highest amounts of reducing sugars and FA. The enzyme combination of XT6 (GH10 xylanase) and FAE5 or FAE6 liberated the highest amount of FA while a combination of Xyn11 (GH11 xylanase) and FAE5 or FAE6 produced the highest reducing sugar content. The synergistic interactions which were established between the xylanases and FAEs were further investigated using agricultural residues (corn cobs, rice straw and sugarcane bagasse). The three substrates were subjected to hydrothermal and dilute acid pre-treatment prior to synergy studies. It is generally known that, during pre-treatment, many compounds can be produced which may influence enzymatic hydrolysis. The effects of these by-products were assessed and it was found that lignin and its degradation products were the most inhibitory to the FAEs. The optimised enzyme cocktail was then applied to 1% (w/v) of untreated and pre-treated substrates for the efficient production of XOS and hydroxycinnamic acids. A significant improvement in xylanase substrate degradation was observed, especially with the combination of 66% Xyn11 and 33% FAE6 which displayed an improvement in reducing sugars of approximately 1.9-fold and 3.4-fold for hydrothermal and acid pre-treated corn cobs (compared to when Xyn11 was used alone), respectively. The study demonstrated that pre-treatment substantially enhanced the enzymatic hydrolysis of corn cobs and rice straw. Analysis of the hydrolysate product profiles revealed that the optimised enzyme cocktail displayed great potential for releasing XOS with a low degree of polymerisation. In conclusion, this study provided significant insights into the mechanism of synergistic interactions between xylanases and metagenome-derived FAEs during the hydrolysis of various substrates. The study also demonstrated that optimised enzyme cocktails combined with low severity pre-treatment can facilitate the potential use of xylan-rich lignocellulosic biomass for the production of valuable products in the future. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-04
The effect of GH family affiliations of mannanolytic enzymes on their synergistic associations during the hydrolysis of mannan-containing substrates
- Authors: Malgas, Samkelo
- Date: 2015
- Subjects: Lignocellulose , Biomass energy , Ethanol as fuel , Polysaccharides , Sugar -- Inversion , Glycosidases , Galactoglucomannans , Oligosaccharides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4148 , http://hdl.handle.net/10962/d1017909
- Full Text:
- Date Issued: 2015
- Authors: Malgas, Samkelo
- Date: 2015
- Subjects: Lignocellulose , Biomass energy , Ethanol as fuel , Polysaccharides , Sugar -- Inversion , Glycosidases , Galactoglucomannans , Oligosaccharides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4148 , http://hdl.handle.net/10962/d1017909
- Full Text:
- Date Issued: 2015
The synthesis of fructooligosaccharides by the fructofuranosidase FopAp from Aspergillus niger
- Pindura, Mitchell Kingsley Chido
- Authors: Pindura, Mitchell Kingsley Chido
- Date: 2012
- Subjects: Aspergillus niger , Oligosaccharides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4158 , http://hdl.handle.net/10962/d1018267
- Description: Fructooligosaccharides (FOS) are short-chain fructans with a terminal glucose moiety and are found naturally in many plant species. Besides their wide use as an alternative sweetener in food and beverage industry, FOS have shown great potential as neutraceuticals against diabetes, colon cancer and bowel disease. The uses of FOS are dependent on the degree of polymerisation that they exhibit. β-fructofuranosidase (FFase) and fructosyltransferase (FTase) enzymes are capable of synthesing FOS from carbohydrate raw materials such as chicory and sugar beet. The aim of this study was to investigate the synthesis of FOS of a pre-defined chain length, from sucrose, by the enzyme FopAp; a β-fructofuranosidase from Aspergillus niger. ATCC 20611. The crude enzyme FopAp was successfully purified, with a yield of 78.20 %, by ammonium sulphate precipitation and anion exchange chromatography. Two protein fractions, named FA and FB were shown to exhibit FFase activity. SDS PAGE analysis revealed two proteins with molecular weights of 112 kDa and 78 kDa, which were identified as a FFase and a hydrolase. Temperature and pH optima of 20 ºC and 9, respectively, were observed for the transfructosylation activity in the FFase. The purified FFase exhibited a half life of 1.5 hrs under optimal conditions. Substrate kinetic studies indicated a high hydrolytic activity at low sucrose concentrations, with Vmax and Km of 1.25 μmol/ml/min and 3.28 mM, respectively. Analysis by response surface methodology identified temperature and pH to be significant factors for the production of kestose and nystose, at a 95 % level of confidence. These findings were confirmed by neural networks constructed to identify optimal conditions of FOS synthesis.FOS synthesis was found to be optimal between pH 6 and pH 9 at 25 ºC. The factor of reaction time was found to be insignificant within the selected experimental constraints, for both FOS species. The findings of this investigation are very important as the foundations of a commercially viable synthetic process for the production of FOS.
- Full Text:
- Date Issued: 2012
- Authors: Pindura, Mitchell Kingsley Chido
- Date: 2012
- Subjects: Aspergillus niger , Oligosaccharides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4158 , http://hdl.handle.net/10962/d1018267
- Description: Fructooligosaccharides (FOS) are short-chain fructans with a terminal glucose moiety and are found naturally in many plant species. Besides their wide use as an alternative sweetener in food and beverage industry, FOS have shown great potential as neutraceuticals against diabetes, colon cancer and bowel disease. The uses of FOS are dependent on the degree of polymerisation that they exhibit. β-fructofuranosidase (FFase) and fructosyltransferase (FTase) enzymes are capable of synthesing FOS from carbohydrate raw materials such as chicory and sugar beet. The aim of this study was to investigate the synthesis of FOS of a pre-defined chain length, from sucrose, by the enzyme FopAp; a β-fructofuranosidase from Aspergillus niger. ATCC 20611. The crude enzyme FopAp was successfully purified, with a yield of 78.20 %, by ammonium sulphate precipitation and anion exchange chromatography. Two protein fractions, named FA and FB were shown to exhibit FFase activity. SDS PAGE analysis revealed two proteins with molecular weights of 112 kDa and 78 kDa, which were identified as a FFase and a hydrolase. Temperature and pH optima of 20 ºC and 9, respectively, were observed for the transfructosylation activity in the FFase. The purified FFase exhibited a half life of 1.5 hrs under optimal conditions. Substrate kinetic studies indicated a high hydrolytic activity at low sucrose concentrations, with Vmax and Km of 1.25 μmol/ml/min and 3.28 mM, respectively. Analysis by response surface methodology identified temperature and pH to be significant factors for the production of kestose and nystose, at a 95 % level of confidence. These findings were confirmed by neural networks constructed to identify optimal conditions of FOS synthesis.FOS synthesis was found to be optimal between pH 6 and pH 9 at 25 ºC. The factor of reaction time was found to be insignificant within the selected experimental constraints, for both FOS species. The findings of this investigation are very important as the foundations of a commercially viable synthetic process for the production of FOS.
- Full Text:
- Date Issued: 2012
Purification and characterization of fructosyltransferase for the synthesis of short-chain fructo-oligosaccharides and investigation into thier anti-carcinogenic properties
- Authors: Nemukula, Aluwani
- Date: 2009
- Subjects: Oligosaccharides , Polygalacturonase , Aspergillus , Fructose , Inulin , Cancer -- Prevention , Cancer -- Research , Carcinogens , High performance liquid chromatography
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3927 , http://hdl.handle.net/10962/d1003986 , Oligosaccharides , Polygalacturonase , Aspergillus , Fructose , Inulin , Cancer -- Prevention , Cancer -- Research , Carcinogens , High performance liquid chromatography
- Description: There is a growing attention in the synthesis of fructo-oligosaccharides (FOS) due to their excellent bio-functional and health-promoting properties. The current production processes are limited to chemical hydrolysis reactions of plant extracts, which are often associated with several drawbacks. In this study, fructosyltransferase (FTase) and polygalacturonase (PGase) activities, present in a commercial enzyme preparation (Pectinex® Ultra SP-L) sourced from Aspergillus aculeatus, have been separated and fully purified by anion-exchange and sizeexclusion chromatography. The FTase possesses fructosyl transfer activity for FOS synthesis and the PGase has pectin hydrolytic activity. Fructosyltransferase is a single-band protein with a molecular weight of 85 kDa, whereas PGase is a distinct protein of 40 kDa. The temperature and pH optima of FTase were 60 ºC and 6.0, with a half-life of 8 h; while that for PGase were 40 ºC and 6.0, respectively. FTase was slightly inhibited in the presence of Ni²⁺, Mg²⁺ and urea; but PGase was more susceptible to divalent ions such as Ca²⁺, Mg²⁺ and Mn²⁺. The kinetic parameters (Km and Vmax) of FTase for the hydrolysis of β-(2→1) linkages from sucrose were 752.3 mM and 120.5 μmol.min⁻¹.mL⁻¹, respectively; whereas the same parameters for pectin hydrolysis by PGase were 13.0 mg.mL⁻¹ and 263 μmol.min-1.mL⁻¹, respectively. The purified FTase was able to transfer fructosyl residues from sucrose, synthesizing the corresponding chains of FOS. PGase was relatively stable at 40 ºC (t½ > 3 h), depolymerizing the pectin backbone while releasing the inulins from within the chicory roots. Analysis of various mixtures of FOS by mass spectrometry, HPLC and ¹H-NMR was undertaken. Results indicated that MS with electrospray ionization and ¹H-NMR are capable of providing relative quantitative data of the FOS present in the mixtures. The pharmaceutical effects of various sc-FOS (0.5%, v/v) and SCFA (0.3%, v/v) on certain bacterial enzymes (β-glucuronidase, urease and β-glucosidase) associated with the formation of carcinogens were also studied. These enzyme activities were not directly influenced by the sc-FOS, but were found to be remarkably decreased by SCFA, pointing toward the prebiotic effect of FOS in intestinal microflora modulation.
- Full Text:
- Date Issued: 2009
- Authors: Nemukula, Aluwani
- Date: 2009
- Subjects: Oligosaccharides , Polygalacturonase , Aspergillus , Fructose , Inulin , Cancer -- Prevention , Cancer -- Research , Carcinogens , High performance liquid chromatography
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3927 , http://hdl.handle.net/10962/d1003986 , Oligosaccharides , Polygalacturonase , Aspergillus , Fructose , Inulin , Cancer -- Prevention , Cancer -- Research , Carcinogens , High performance liquid chromatography
- Description: There is a growing attention in the synthesis of fructo-oligosaccharides (FOS) due to their excellent bio-functional and health-promoting properties. The current production processes are limited to chemical hydrolysis reactions of plant extracts, which are often associated with several drawbacks. In this study, fructosyltransferase (FTase) and polygalacturonase (PGase) activities, present in a commercial enzyme preparation (Pectinex® Ultra SP-L) sourced from Aspergillus aculeatus, have been separated and fully purified by anion-exchange and sizeexclusion chromatography. The FTase possesses fructosyl transfer activity for FOS synthesis and the PGase has pectin hydrolytic activity. Fructosyltransferase is a single-band protein with a molecular weight of 85 kDa, whereas PGase is a distinct protein of 40 kDa. The temperature and pH optima of FTase were 60 ºC and 6.0, with a half-life of 8 h; while that for PGase were 40 ºC and 6.0, respectively. FTase was slightly inhibited in the presence of Ni²⁺, Mg²⁺ and urea; but PGase was more susceptible to divalent ions such as Ca²⁺, Mg²⁺ and Mn²⁺. The kinetic parameters (Km and Vmax) of FTase for the hydrolysis of β-(2→1) linkages from sucrose were 752.3 mM and 120.5 μmol.min⁻¹.mL⁻¹, respectively; whereas the same parameters for pectin hydrolysis by PGase were 13.0 mg.mL⁻¹ and 263 μmol.min-1.mL⁻¹, respectively. The purified FTase was able to transfer fructosyl residues from sucrose, synthesizing the corresponding chains of FOS. PGase was relatively stable at 40 ºC (t½ > 3 h), depolymerizing the pectin backbone while releasing the inulins from within the chicory roots. Analysis of various mixtures of FOS by mass spectrometry, HPLC and ¹H-NMR was undertaken. Results indicated that MS with electrospray ionization and ¹H-NMR are capable of providing relative quantitative data of the FOS present in the mixtures. The pharmaceutical effects of various sc-FOS (0.5%, v/v) and SCFA (0.3%, v/v) on certain bacterial enzymes (β-glucuronidase, urease and β-glucosidase) associated with the formation of carcinogens were also studied. These enzyme activities were not directly influenced by the sc-FOS, but were found to be remarkably decreased by SCFA, pointing toward the prebiotic effect of FOS in intestinal microflora modulation.
- Full Text:
- Date Issued: 2009
"Complexes of carbohydrates with magnesium-ions" : "the isolation of an oligosaccharide containing L-galactose from the polysaccharide of Aeodes orbitosa" : "horizontal cellulose colum chromatography of sugars"
- Authors: Van der Linde, Michael John
- Date: 1974
- Subjects: Magenesium , Ions , Carbohydrates , Oligosaccharides , Polysaccharides , Chromatographic analysis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4504 , http://hdl.handle.net/10962/d1013282
- Description: Sugar complexes with anions and cations are reviewed. Evidence is presented for the formation of a complex of stoichiometry 1:1 in aqueous solution between methyl-α-Dglucopyranoside and magnesium perchlorate. The complex may be detected by the method of continuous variations. Measurements of changes in the proton chemical shifts of the glucoside in dauterium oxide, indicate that the complex is probably formed between the vicinal hydroxyl groups at C-2 and C-3 of the pyranoside ring and the hydrated cation. At elevation temperatures there is evidence for the presence of a complex of stoichiometry 2:1. Experiments conducted on cellulose indicate the possible formation of cellulose - magnesium-ion complexes . These complexes provide an explanation for the "protective action" of mEgnesium compounds on the cellulose portion of pulp during alkali-oxygen bleaching.
- Full Text:
- Date Issued: 1974
- Authors: Van der Linde, Michael John
- Date: 1974
- Subjects: Magenesium , Ions , Carbohydrates , Oligosaccharides , Polysaccharides , Chromatographic analysis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4504 , http://hdl.handle.net/10962/d1013282
- Description: Sugar complexes with anions and cations are reviewed. Evidence is presented for the formation of a complex of stoichiometry 1:1 in aqueous solution between methyl-α-Dglucopyranoside and magnesium perchlorate. The complex may be detected by the method of continuous variations. Measurements of changes in the proton chemical shifts of the glucoside in dauterium oxide, indicate that the complex is probably formed between the vicinal hydroxyl groups at C-2 and C-3 of the pyranoside ring and the hydrated cation. At elevation temperatures there is evidence for the presence of a complex of stoichiometry 2:1. Experiments conducted on cellulose indicate the possible formation of cellulose - magnesium-ion complexes . These complexes provide an explanation for the "protective action" of mEgnesium compounds on the cellulose portion of pulp during alkali-oxygen bleaching.
- Full Text:
- Date Issued: 1974
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