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
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
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