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
Expression, partial characterisation and utilization of a GH11 xylanase (Xyn2A) from Trichoderma viride as an additive in monogastric animal feeds
- Mzimkulu-Ncoyi, Nosabatha Happyness
- Authors: Mzimkulu-Ncoyi, Nosabatha Happyness
- Date: 2023-03-29
- Subjects: Feed additives , Xylanases , Trichoderma viride , Monogastric , Polysaccharides , Plant cell walls , Prebiotics
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/422409 , vital:71940
- Description: Endo-xylanases (shortly called xylanases) are a group of glycoside hydrolase enzymes that target β-D-1,4-linkages in the xylan backbone, leading to the production of xylooligosaccharides (XOS) of varying degree of polymerization (DP). Xylan is an indigestible non-starch polysaccharide present in monogastric animal feeds which in high amounts leads to increased digesta viscosity, slow movement of digesta in the intestines, malabsorption of nutrients among other challenges. The aim of this study was to investigate the effect of xylanase 2A (Xyn2A) from Trichoderma viride on broiler chicken feeds, particularly the hydrolysis of the xylan content, reduction of feed viscosity and the effect of produced XOS on eliciting the growth of gut associated probiotic bacteria. Xyn2AE was successfully induced with 0.8 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and produced in Escherichia coli BL21 (DE3) and Xyn2AC was expressed in tobacco mosaic plants. For the purification of Xyn2AE, an immobilized metal affinity chromatography (IMAC) column and diafiltration using a 3kDa cut-off Amicon filter membranes were used. Xyn2AE and Xyn2AC showed a xylanase active band at a relative weight of 21 kDa. Both enzymes showed high specificity towards soluble wheat arabinoxylan (WAX), with specific activities of 7.61 U/mg for Xyn2AE and 536.5 U/mg for Xyn2AC. Xyn2A kinetic parameters (Vmax and Km) were determined by Michaelis-Menten plots on soluble and insoluble WAX. The Vmax and Km values of Xyn2AC were 1003.01 U/mg and 9.25 mg/mL, 302.89 U/mg and 13.54 mg/mL, respectively. The Vmax and Km values of Xyn2AE for soluble and insoluble WAX were 20.45 U/mg and 12.95 mg/mL, and 8.31 U/mg and 13.15 mg/mL. Xyn2A enzymes displayed optimum activity at pH and temperature parameters of 5.0 and 50°C, respectively, and stability in temperatures ranging between 50 and 80°C and pH 4.0-9.0. Broiler chicken feeds were hydrolysed using Xyn2AE over a 24 h period and analysed using the dinitrosalicylic (DNS) assay, thin layer chromatography (TLC), viscometry and visualized using scanning electron microscope (SEM). The results showed a release of release of XOS xylotriose, xylopentose and xylohexose; enzyme’s ability to decrease the viscosity of the feeds and punched holes of feed surface, which was indicative of xylanase action. XOS produced during hydrolysis was used to study prebiotic effect on selected few bacteria and released short chain fatty acids (SCFAs) were measured. Additionally, SCFAs formation was detected in the presence of XOS as a carbon source for S. thermophilus and L. bulgaricus, whereas B. subtilis formed fewer organic acids in the presence of XOS. The results obtained from this study demonstrated that the supplementation of Xyn2A on broiler feeds has ii a positive effect in decreasing feed viscosity. Furthermore, the results of this investigation will assist the South African poultry farming sector to increase profitability in poultry farming and gain stability in the global trade as far as poultry feed is concerned. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-03-29
- Authors: Mzimkulu-Ncoyi, Nosabatha Happyness
- Date: 2023-03-29
- Subjects: Feed additives , Xylanases , Trichoderma viride , Monogastric , Polysaccharides , Plant cell walls , Prebiotics
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/422409 , vital:71940
- Description: Endo-xylanases (shortly called xylanases) are a group of glycoside hydrolase enzymes that target β-D-1,4-linkages in the xylan backbone, leading to the production of xylooligosaccharides (XOS) of varying degree of polymerization (DP). Xylan is an indigestible non-starch polysaccharide present in monogastric animal feeds which in high amounts leads to increased digesta viscosity, slow movement of digesta in the intestines, malabsorption of nutrients among other challenges. The aim of this study was to investigate the effect of xylanase 2A (Xyn2A) from Trichoderma viride on broiler chicken feeds, particularly the hydrolysis of the xylan content, reduction of feed viscosity and the effect of produced XOS on eliciting the growth of gut associated probiotic bacteria. Xyn2AE was successfully induced with 0.8 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and produced in Escherichia coli BL21 (DE3) and Xyn2AC was expressed in tobacco mosaic plants. For the purification of Xyn2AE, an immobilized metal affinity chromatography (IMAC) column and diafiltration using a 3kDa cut-off Amicon filter membranes were used. Xyn2AE and Xyn2AC showed a xylanase active band at a relative weight of 21 kDa. Both enzymes showed high specificity towards soluble wheat arabinoxylan (WAX), with specific activities of 7.61 U/mg for Xyn2AE and 536.5 U/mg for Xyn2AC. Xyn2A kinetic parameters (Vmax and Km) were determined by Michaelis-Menten plots on soluble and insoluble WAX. The Vmax and Km values of Xyn2AC were 1003.01 U/mg and 9.25 mg/mL, 302.89 U/mg and 13.54 mg/mL, respectively. The Vmax and Km values of Xyn2AE for soluble and insoluble WAX were 20.45 U/mg and 12.95 mg/mL, and 8.31 U/mg and 13.15 mg/mL. Xyn2A enzymes displayed optimum activity at pH and temperature parameters of 5.0 and 50°C, respectively, and stability in temperatures ranging between 50 and 80°C and pH 4.0-9.0. Broiler chicken feeds were hydrolysed using Xyn2AE over a 24 h period and analysed using the dinitrosalicylic (DNS) assay, thin layer chromatography (TLC), viscometry and visualized using scanning electron microscope (SEM). The results showed a release of release of XOS xylotriose, xylopentose and xylohexose; enzyme’s ability to decrease the viscosity of the feeds and punched holes of feed surface, which was indicative of xylanase action. XOS produced during hydrolysis was used to study prebiotic effect on selected few bacteria and released short chain fatty acids (SCFAs) were measured. Additionally, SCFAs formation was detected in the presence of XOS as a carbon source for S. thermophilus and L. bulgaricus, whereas B. subtilis formed fewer organic acids in the presence of XOS. The results obtained from this study demonstrated that the supplementation of Xyn2A on broiler feeds has ii a positive effect in decreasing feed viscosity. Furthermore, the results of this investigation will assist the South African poultry farming sector to increase profitability in poultry farming and gain stability in the global trade as far as poultry feed is concerned. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Date Issued: 2023-03-29
Fucoidans from South African brown seaweeds: establishing the link between their structure and biological properties (anti-diabetic and anti-cancer activities)
- Authors: Mabate, Blessing
- Date: 2022-10-14
- Subjects: Fucoidan , Diabetes Treatment , Cancer Treatment , Brown algae
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365677 , vital:65775 , DOI https://doi.org/10.21504/10962/365677
- Description: Type 2 diabetes mellitus (T2DM) and cancer are major non-communicable diseases causing a heavy morbidity-mortality and economic burden globally. The therapeutic efforts in managing these diseases are primarily chemotherapeutic and are associated with demerits, including side effects and toxicity, limiting the prescribed amounts. These dosage limits may cause drug resistance, another major challenge in maintaining quality global health. The pursuit of novel natural bioproducts is a reasonable strategy to add to the arsenal against T2DM and cancer. Fucoidans, sulphated fucose polysaccharides abundant in brown seaweeds, have recently become popular for their biological activities, including anti-diabetic and anti-cancer properties. However, endemic South African brown seaweeds have not been adequately explored. Therefore, this study sought to characterise fucoidans extracted from South African brown seaweeds and elucidate their structure to their biological activities. Also, this study highlighted carbohydrate and glucose metabolism as major target processes in the control efforts of T2DM and cancer using fucoidans. Harvested brown seaweeds were identified as Ecklonia radiata and Sargassum elegans. E. maxima was kindly donated by KelpX. The fucoidans were then extracted using hot water, EDTA assisted, and acid extraction protocols. The integrity of the extracted fucoidan was confirmed through structural analysis using FTIR, NMR and TGA. The fucoidan extracts were then chemically characterised to determine their carbohydrate and monosaccharide composition and sulphate content. The characterised fucoidans were profiled for inhibiting the major amylolytic enzymes, namely α-amylase and α-glucosidase. The mode of inhibition by fucoidans and synergy experiments with the commercial anti-diabetic drug acarbose were also investigated. Furthermore, the fucoidans were screened for potential anti-cancer activities on the human colorectal HCT116 cancer cell line. The cytotoxicity of fucoidans was quantified using the resazurin assay. The effect of fucoidan on HCT116 cell adhesion on the tissue culture plastic was also investigated using the crystal violet-based cell adhesion assay. In addition, cancer antimigration properties of fucoidans were also investigated using 2D wound healing and 3D spheroid-based assays. Furthermore, the long-term survival of HCT116 cells was investigated through the clonogenic assay after treatment with fucoidans. Lastly, glucose uptake and lactate export assays revealed the influence of fucoidan on glucose uptake and the glycolytic flux of HCT116 cells. Fucoidans were successfully extracted with a yield between 2.2% and 14.2% on a dry weight basis. EDTA extracts produced the highest yields than the water and the acid extracts. Ecklonia spp. fucoidans displayed the highest total carbohydrate content, with glucose and galactose being the major monosaccharides. S. elegans and commercial Fucus vesiculosus had lower carbohydrate contents but contained more sulphates than the Ecklonia spp. fucoidans. Furthermore, the extracted fucoidan contained little to no contaminants, including proteins, phenolics and uronic acids. In addition, the extracted fucoidans were determined to be >100 kDa through ultracentrifugation. Mass spectrometry also detected the most abundant peak for all fucoidans to be around 700 Da (m/z). Extracted fucoidans inhibited the activity of α-glucosidase more strongly than the commercial anti-diabetic agent acarbose but were inactive on α-amylase. Fucoidans were also shown to be mixed inhibitors of α-glucosidase. Compellingly, fucoidans synergistically inhibited α-glucosidase in combination with the anti-diabetic agent acarbose, highlighting prospects for combination therapy. Finally, fucoidans demonstrated some anti-proliferative characteristics on HCT116 cancer cells by inhibiting their ability to adhere to the tissue culture plate matrix. Furthermore, some fucoidan extracts inhibited the migration of HCT116 cancer cells from 3D spheroids. Some of our fucoidan extracts also inhibited HCT116 colony formation, demonstrating inhibition of long-term cell survival. The E. maxima water extract also inhibited glucose uptake by HCT116 cells, thereby influencing the glycolytic flux. In conclusion, biologically active fucoidans were successfully extracted from South African brown seaweeds. These fucoidans demonstrated anti-diabetic and anti-cancer properties, revealing their relevance as potential drugs for these diseases. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Mabate, Blessing
- Date: 2022-10-14
- Subjects: Fucoidan , Diabetes Treatment , Cancer Treatment , Brown algae
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365677 , vital:65775 , DOI https://doi.org/10.21504/10962/365677
- Description: Type 2 diabetes mellitus (T2DM) and cancer are major non-communicable diseases causing a heavy morbidity-mortality and economic burden globally. The therapeutic efforts in managing these diseases are primarily chemotherapeutic and are associated with demerits, including side effects and toxicity, limiting the prescribed amounts. These dosage limits may cause drug resistance, another major challenge in maintaining quality global health. The pursuit of novel natural bioproducts is a reasonable strategy to add to the arsenal against T2DM and cancer. Fucoidans, sulphated fucose polysaccharides abundant in brown seaweeds, have recently become popular for their biological activities, including anti-diabetic and anti-cancer properties. However, endemic South African brown seaweeds have not been adequately explored. Therefore, this study sought to characterise fucoidans extracted from South African brown seaweeds and elucidate their structure to their biological activities. Also, this study highlighted carbohydrate and glucose metabolism as major target processes in the control efforts of T2DM and cancer using fucoidans. Harvested brown seaweeds were identified as Ecklonia radiata and Sargassum elegans. E. maxima was kindly donated by KelpX. The fucoidans were then extracted using hot water, EDTA assisted, and acid extraction protocols. The integrity of the extracted fucoidan was confirmed through structural analysis using FTIR, NMR and TGA. The fucoidan extracts were then chemically characterised to determine their carbohydrate and monosaccharide composition and sulphate content. The characterised fucoidans were profiled for inhibiting the major amylolytic enzymes, namely α-amylase and α-glucosidase. The mode of inhibition by fucoidans and synergy experiments with the commercial anti-diabetic drug acarbose were also investigated. Furthermore, the fucoidans were screened for potential anti-cancer activities on the human colorectal HCT116 cancer cell line. The cytotoxicity of fucoidans was quantified using the resazurin assay. The effect of fucoidan on HCT116 cell adhesion on the tissue culture plastic was also investigated using the crystal violet-based cell adhesion assay. In addition, cancer antimigration properties of fucoidans were also investigated using 2D wound healing and 3D spheroid-based assays. Furthermore, the long-term survival of HCT116 cells was investigated through the clonogenic assay after treatment with fucoidans. Lastly, glucose uptake and lactate export assays revealed the influence of fucoidan on glucose uptake and the glycolytic flux of HCT116 cells. Fucoidans were successfully extracted with a yield between 2.2% and 14.2% on a dry weight basis. EDTA extracts produced the highest yields than the water and the acid extracts. Ecklonia spp. fucoidans displayed the highest total carbohydrate content, with glucose and galactose being the major monosaccharides. S. elegans and commercial Fucus vesiculosus had lower carbohydrate contents but contained more sulphates than the Ecklonia spp. fucoidans. Furthermore, the extracted fucoidan contained little to no contaminants, including proteins, phenolics and uronic acids. In addition, the extracted fucoidans were determined to be >100 kDa through ultracentrifugation. Mass spectrometry also detected the most abundant peak for all fucoidans to be around 700 Da (m/z). Extracted fucoidans inhibited the activity of α-glucosidase more strongly than the commercial anti-diabetic agent acarbose but were inactive on α-amylase. Fucoidans were also shown to be mixed inhibitors of α-glucosidase. Compellingly, fucoidans synergistically inhibited α-glucosidase in combination with the anti-diabetic agent acarbose, highlighting prospects for combination therapy. Finally, fucoidans demonstrated some anti-proliferative characteristics on HCT116 cancer cells by inhibiting their ability to adhere to the tissue culture plate matrix. Furthermore, some fucoidan extracts inhibited the migration of HCT116 cancer cells from 3D spheroids. Some of our fucoidan extracts also inhibited HCT116 colony formation, demonstrating inhibition of long-term cell survival. The E. maxima water extract also inhibited glucose uptake by HCT116 cells, thereby influencing the glycolytic flux. In conclusion, biologically active fucoidans were successfully extracted from South African brown seaweeds. These fucoidans demonstrated anti-diabetic and anti-cancer properties, revealing their relevance as potential drugs for these diseases. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
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
Cloning, expression, partial characterisation and application of a recombinant GH10 xylanase, XT6, from Geobacillus stearothermophilus T6 as an additive to chicken feeds
- Authors: Sithole, Tariro
- Date: 2022-04-06
- Subjects: Chicken feed industry , Chickens Feeding and feeds , Bacillus (Bacteria) , Xylanases , Polysaccharides , Geobacillus stearothermophilus
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/292693 , vital:57007
- Description: Monogastric animal farming has largely been sustained by feeding animals with grain feedstocks containing non-starch polysaccharides (NSPs) and anti-nutritive factors, which cause adverse effects, such as increased digesta viscosity and entrapment of nutrients, which leads to the inaccessibility of nutrients. These effects have been linked to a reduction in nutrient digestion and absorption, which results in a decreased feed conversion ratio, energy metabolism and animal growth. Monogastric animals do not produce enzymes that can hydrolyse these NSPs. The application of exogenous enzymes as supplements to animal feeds has been implemented to reduce viscosity and increase nutrient absorption in poultry and pigs over the past few decades. The aim of this study was to clone, express, partially characterise and apply a glycoside hydrolase (GH) family 10 xylanase (XT6), derived from Geobacillus stearothermophilus T6, as an additive to locally produced chicken feeds. The xt6 gene (1,236 bp) was subcloned and expressed in Escherichia coli DH5α and BL21(DE3) cells, respectively. Upon expression, XT6 had a molecular weight of 42 kDa and was partially purified by Ni-NTA chromatography and ultrafiltration. The purification step resulted in a yield of 66.7% with a 16.8-fold increase in purification. XT6 exhibited maximal activity when incubated at a pH and temperature of pH 6.0 and 70°C, respectively, with a high thermostability over a broad range of pH (2–9) and temperature (30–90 °C). The specific activities of XT6 on extracted soluble and insoluble wheat flour arabinoxylans were 110.9 U/mg and 63.98 U/mg, respectively. Kinetic data showed that XT6 displayed a higher catalytic activity and affinity (Vmax = 231.60 μmol/min/mg and KM = 2.759 mg/ml) for soluble wheat arabinoxylan, compared to insoluble wheat arabinoxylan (Vmax = 99.02 μmol/min/mg and KM = 5.058 mg/ml). High-performance liquid chromatography (HPLC) analysis showed that the enzyme hydrolysed wheat flour, arabinoxylan and chicken feeds, producing a range of xylooligosaccharides (XOS), with xylotetraose and xylopentaose being the predominant XOS species. Hydrolysis of both soluble and insoluble wheat flour arabinoxylans by XT6 led to a significant reduction in substrate viscosity. The effects of simulated gastrointestinal fluid contents, such as proteases, bile salts and mucins, on XT6 stability were also studied. Exposure of XT6 to pepsin did not significantly reduce its activity; however, the inhibitory effect of trypsin and mucin on XT6 was much greater. The presence of gut-derived bile salts had no iii | P a g e significant effect on XT6 activity. Finally, it was shown that the XOS produced from the hydrolysis of chicken feeds (starter and grower feeds) by XT6 significantly enhanced the growth of the probiotic bacteria B. subtilis, while there was no significant improvement in the growth of S. thermophilus and L. bulgaricus. In conclusion, the recombinantly produced XT6 demonstrated efficient hydrolysis of starter and grower feeds, and produced XOS that showed prebiotic activity on selected probiotic bacteria. In addition, the pH, temperature and simulated gastric juice content stability of XT6 renders it an attractive candidate as an additive for chicken feeds. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-04-06
- Authors: Sithole, Tariro
- Date: 2022-04-06
- Subjects: Chicken feed industry , Chickens Feeding and feeds , Bacillus (Bacteria) , Xylanases , Polysaccharides , Geobacillus stearothermophilus
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/292693 , vital:57007
- Description: Monogastric animal farming has largely been sustained by feeding animals with grain feedstocks containing non-starch polysaccharides (NSPs) and anti-nutritive factors, which cause adverse effects, such as increased digesta viscosity and entrapment of nutrients, which leads to the inaccessibility of nutrients. These effects have been linked to a reduction in nutrient digestion and absorption, which results in a decreased feed conversion ratio, energy metabolism and animal growth. Monogastric animals do not produce enzymes that can hydrolyse these NSPs. The application of exogenous enzymes as supplements to animal feeds has been implemented to reduce viscosity and increase nutrient absorption in poultry and pigs over the past few decades. The aim of this study was to clone, express, partially characterise and apply a glycoside hydrolase (GH) family 10 xylanase (XT6), derived from Geobacillus stearothermophilus T6, as an additive to locally produced chicken feeds. The xt6 gene (1,236 bp) was subcloned and expressed in Escherichia coli DH5α and BL21(DE3) cells, respectively. Upon expression, XT6 had a molecular weight of 42 kDa and was partially purified by Ni-NTA chromatography and ultrafiltration. The purification step resulted in a yield of 66.7% with a 16.8-fold increase in purification. XT6 exhibited maximal activity when incubated at a pH and temperature of pH 6.0 and 70°C, respectively, with a high thermostability over a broad range of pH (2–9) and temperature (30–90 °C). The specific activities of XT6 on extracted soluble and insoluble wheat flour arabinoxylans were 110.9 U/mg and 63.98 U/mg, respectively. Kinetic data showed that XT6 displayed a higher catalytic activity and affinity (Vmax = 231.60 μmol/min/mg and KM = 2.759 mg/ml) for soluble wheat arabinoxylan, compared to insoluble wheat arabinoxylan (Vmax = 99.02 μmol/min/mg and KM = 5.058 mg/ml). High-performance liquid chromatography (HPLC) analysis showed that the enzyme hydrolysed wheat flour, arabinoxylan and chicken feeds, producing a range of xylooligosaccharides (XOS), with xylotetraose and xylopentaose being the predominant XOS species. Hydrolysis of both soluble and insoluble wheat flour arabinoxylans by XT6 led to a significant reduction in substrate viscosity. The effects of simulated gastrointestinal fluid contents, such as proteases, bile salts and mucins, on XT6 stability were also studied. Exposure of XT6 to pepsin did not significantly reduce its activity; however, the inhibitory effect of trypsin and mucin on XT6 was much greater. The presence of gut-derived bile salts had no iii | P a g e significant effect on XT6 activity. Finally, it was shown that the XOS produced from the hydrolysis of chicken feeds (starter and grower feeds) by XT6 significantly enhanced the growth of the probiotic bacteria B. subtilis, while there was no significant improvement in the growth of S. thermophilus and L. bulgaricus. In conclusion, the recombinantly produced XT6 demonstrated efficient hydrolysis of starter and grower feeds, and produced XOS that showed prebiotic activity on selected probiotic bacteria. In addition, the pH, temperature and simulated gastric juice content stability of XT6 renders it an attractive candidate as an additive for chicken feeds. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-04-06
Biochemical characterization of the β-mannanase activity of Bacillus paralicheniformis SVD1
- Authors: Clarke, Matthew David
- Date: 2019
- Subjects: Mycobacterium avium paratuberculosis , Enzymes -- Biotechnology , Lignocellulose -- Biotechnology
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/67570 , vital:29112
- Description: Products produced via the enzymatic hydrolysis of lignocellulosic biomass, the most abundant renewable terrestrial source of carbon, can potentially replace a lot of the fuels and chemicals currently produced using non-renewable hydrocarbons. Mannan is a polysaccharide component of lignocellulose that is abundant in softwoods and legume seeds. Enzymatic hydrolysis of mannan by β-mannanases has various industrial applications, including use in biofuel and prebiotic mannooligosaccharide (MOS) production for the improvement of human and animal health. The industrial use of β-mannanases depends on their biochemical characteristics, such as their activity, stability and substrate specificity. Knowledge of their synergistic interactions with other enzymes is also useful for effective hydrolysis. Bacillus paralicheniformis SVD1 was used as a source for β-mannanases. The two mannanases of B. paralicheniformis SVD1 have not been biochemically characterized apart from minor characterization of crude β-mannanase activity. The protein sequences of the two β-mannanases, of glycosyl hydrolase family 5 and 26, have a 95% - 96% identity to the β-mannanases of B. licheniformis DSM13T (=ATCC14580T). These small protein sequence differences could lead to quite different biochemical characteristics. These mannanases were characterized as these enzymes may have industrially useful characteristics. To induce mannanase production, B. paralicheniformis SVD1 was cultured in broth containing the mannan substrate locust bean gum. Various growth curve parameters were measured over 72 h. Mannanase activity was the highest after 48 h of growth - this was the time at which mannanase activity was concentrated, using 3 kDa centrifugal filtration devices, for biochemical characterization of the crude activity. Zymography revealed that the crude concentrated mannanase fraction consisted of at least two mannanases with relative molecular weights (MWs) of 29.6 kDa and 33 kDa. This was smaller than expected – based on their theoretical molecular masses. Protease activity, which was detected in the broth, was probably the reason. There were two pH optima, pH 5.0 and pH 7.0, which also indicated the presence of two mannanases. The concentrated mannanase displayed characteristics that were expected of a B. paralicheniformis β-mannanase. The temperature optimum was 50°C and the activity loss was less than 7% at 50°C after 24 h. Substrate specificity assays revealed that there was predominantly mannanase activity present. Thin layer chromatography (TLC) analysis of mannan and MOS hydrolysis showed that mainly M2 and M3 MOS were produced; only MOS with a degree of polymerization of 4 or higher were hydrolyzed. Hydrolysis was minimal on mannoligosaccharides with galactose substituents. Activity and MOS production was the highest on soluble, low branched mannan substrates. The highest activity observed was on konjac glucomannan. Purification of the mannanase activity was then attempted using various methods. Ammonium sulfate precipitation, acetone precipitation, as well as centrifugal filtration device concentration was assessed for concentration of the mannanase activity.Concentration was not very successful due to low activity yields (≤ 20%). Anion exchange chromatography (AEC) and size exclusion chromatography (SEC) was used for purification. AEC gave good activity yield and fold purification, but SDS-PAGE analysis revealed the presence of many different proteins so further purification was necessary. SDS-PAGE analysis showed that there were only a few protein contaminants in the SEC fraction. However, the yield was too low to allow for biochemical characterization. The optimized purification procedure, which partially purified the mannanase activity, used 85% ammonium sulfate precipitation, followed by AEC. The fold purification was high (88.9) and the specific activity was 29.5 U.mg-1. A zymogram of the partially purified mannanase showed a mannanase active band with a MW of 40 - 41 kDa. A serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), was added during the purification steps. This indicated that the mannanase/s in the crude concentrate, without PMSF added, was hydrolyzed by serine protease activity. Native PAGE zymograms suggested that at least two different isoforms of mannanases were present. Additional purification would be required to determine the true characteristics of the mannanase/s. The biochemical characteristics of the crude and partially purified mannanases were similar. The pH optima of the partially purified mannanases were different; the pH optima were 6.0 and 9.0. The substrate specificities were similar, except that the partially purified mannanases displayed no cellulase and β-D-galactosidase activity, but showed a small amount of α-L-arabinase activity. The partially purified mannanase and a Cyamopsis tetragonolobus GH27 α-galactosidase synergistically hydrolyzed locust bean gum. The M50G50 combination displayed the highest extent of hydrolysis; after 24 h there was a 1.39 fold increase in reducing sugar release and the degree of synergy (DS) was 4.64. TLC analysis indicated that synergy increased the release of small MOS. These MOS could be useful as prebiotics. The synergy between the partially purified mannanase and the commercial cellulase mixture Cellic® CTec2 (Novozymes) on spent coffee grounds (SCG) was also determined. SCG is an abundant industrial waste product that has high mannan content. The SCG was pretreated using NaOH, and the monosaccharide, soluble phenolics and insoluble contents were determined. Glucose and mannose were the dominant monosaccharides in the SCG; the pretreated SCG contained 20.4% (w/w) glucose and 18.5% (w/w) mannose, respectively. The NaOH pretreatment improved mannanase hydrolysis of SCG. It resulted in the opening up and swelling of the SCG particles and removed some of the insoluble solids. The partially purified B. paralicheniformis SVD1 mannanase displayed no detectable activity on SCG, but showed synergy with CTec2, in terms of DS, on untreated and NaOH pretreated SCG. This is the first report of mannanasecellulase synergy on SCG; other studies found that increased hydrolysis was due to additive effects. The results obtained in this study are only an initial assessment of the biochemical properties of B. paralicheniformis SVD1 mannanase activity and its synergy with other enzymes. These results can be used to inform future studies.
- Full Text:
- Date Issued: 2019
- Authors: Clarke, Matthew David
- Date: 2019
- Subjects: Mycobacterium avium paratuberculosis , Enzymes -- Biotechnology , Lignocellulose -- Biotechnology
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/67570 , vital:29112
- Description: Products produced via the enzymatic hydrolysis of lignocellulosic biomass, the most abundant renewable terrestrial source of carbon, can potentially replace a lot of the fuels and chemicals currently produced using non-renewable hydrocarbons. Mannan is a polysaccharide component of lignocellulose that is abundant in softwoods and legume seeds. Enzymatic hydrolysis of mannan by β-mannanases has various industrial applications, including use in biofuel and prebiotic mannooligosaccharide (MOS) production for the improvement of human and animal health. The industrial use of β-mannanases depends on their biochemical characteristics, such as their activity, stability and substrate specificity. Knowledge of their synergistic interactions with other enzymes is also useful for effective hydrolysis. Bacillus paralicheniformis SVD1 was used as a source for β-mannanases. The two mannanases of B. paralicheniformis SVD1 have not been biochemically characterized apart from minor characterization of crude β-mannanase activity. The protein sequences of the two β-mannanases, of glycosyl hydrolase family 5 and 26, have a 95% - 96% identity to the β-mannanases of B. licheniformis DSM13T (=ATCC14580T). These small protein sequence differences could lead to quite different biochemical characteristics. These mannanases were characterized as these enzymes may have industrially useful characteristics. To induce mannanase production, B. paralicheniformis SVD1 was cultured in broth containing the mannan substrate locust bean gum. Various growth curve parameters were measured over 72 h. Mannanase activity was the highest after 48 h of growth - this was the time at which mannanase activity was concentrated, using 3 kDa centrifugal filtration devices, for biochemical characterization of the crude activity. Zymography revealed that the crude concentrated mannanase fraction consisted of at least two mannanases with relative molecular weights (MWs) of 29.6 kDa and 33 kDa. This was smaller than expected – based on their theoretical molecular masses. Protease activity, which was detected in the broth, was probably the reason. There were two pH optima, pH 5.0 and pH 7.0, which also indicated the presence of two mannanases. The concentrated mannanase displayed characteristics that were expected of a B. paralicheniformis β-mannanase. The temperature optimum was 50°C and the activity loss was less than 7% at 50°C after 24 h. Substrate specificity assays revealed that there was predominantly mannanase activity present. Thin layer chromatography (TLC) analysis of mannan and MOS hydrolysis showed that mainly M2 and M3 MOS were produced; only MOS with a degree of polymerization of 4 or higher were hydrolyzed. Hydrolysis was minimal on mannoligosaccharides with galactose substituents. Activity and MOS production was the highest on soluble, low branched mannan substrates. The highest activity observed was on konjac glucomannan. Purification of the mannanase activity was then attempted using various methods. Ammonium sulfate precipitation, acetone precipitation, as well as centrifugal filtration device concentration was assessed for concentration of the mannanase activity.Concentration was not very successful due to low activity yields (≤ 20%). Anion exchange chromatography (AEC) and size exclusion chromatography (SEC) was used for purification. AEC gave good activity yield and fold purification, but SDS-PAGE analysis revealed the presence of many different proteins so further purification was necessary. SDS-PAGE analysis showed that there were only a few protein contaminants in the SEC fraction. However, the yield was too low to allow for biochemical characterization. The optimized purification procedure, which partially purified the mannanase activity, used 85% ammonium sulfate precipitation, followed by AEC. The fold purification was high (88.9) and the specific activity was 29.5 U.mg-1. A zymogram of the partially purified mannanase showed a mannanase active band with a MW of 40 - 41 kDa. A serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), was added during the purification steps. This indicated that the mannanase/s in the crude concentrate, without PMSF added, was hydrolyzed by serine protease activity. Native PAGE zymograms suggested that at least two different isoforms of mannanases were present. Additional purification would be required to determine the true characteristics of the mannanase/s. The biochemical characteristics of the crude and partially purified mannanases were similar. The pH optima of the partially purified mannanases were different; the pH optima were 6.0 and 9.0. The substrate specificities were similar, except that the partially purified mannanases displayed no cellulase and β-D-galactosidase activity, but showed a small amount of α-L-arabinase activity. The partially purified mannanase and a Cyamopsis tetragonolobus GH27 α-galactosidase synergistically hydrolyzed locust bean gum. The M50G50 combination displayed the highest extent of hydrolysis; after 24 h there was a 1.39 fold increase in reducing sugar release and the degree of synergy (DS) was 4.64. TLC analysis indicated that synergy increased the release of small MOS. These MOS could be useful as prebiotics. The synergy between the partially purified mannanase and the commercial cellulase mixture Cellic® CTec2 (Novozymes) on spent coffee grounds (SCG) was also determined. SCG is an abundant industrial waste product that has high mannan content. The SCG was pretreated using NaOH, and the monosaccharide, soluble phenolics and insoluble contents were determined. Glucose and mannose were the dominant monosaccharides in the SCG; the pretreated SCG contained 20.4% (w/w) glucose and 18.5% (w/w) mannose, respectively. The NaOH pretreatment improved mannanase hydrolysis of SCG. It resulted in the opening up and swelling of the SCG particles and removed some of the insoluble solids. The partially purified B. paralicheniformis SVD1 mannanase displayed no detectable activity on SCG, but showed synergy with CTec2, in terms of DS, on untreated and NaOH pretreated SCG. This is the first report of mannanasecellulase synergy on SCG; other studies found that increased hydrolysis was due to additive effects. The results obtained in this study are only an initial assessment of the biochemical properties of B. paralicheniformis SVD1 mannanase activity and its synergy with other enzymes. These results can be used to inform future studies.
- Full Text:
- Date Issued: 2019
Characterization of termite Trinervitermes trinervoides metagenome-derived glycoside hydrolases, the formulation of synergistic core enzyme sets for effective sweet sorghum and corncob saccharification, and their potential industrial applications
- Authors: Mafa, Mpho Stephen
- Date: 2019
- Subjects: Termites , Metagenomics , Glucosides , Hydrolases , Enzymes , Feedstock
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/72414 , vital:30044 , DOI https://doi.org/10.21504/10962/72414
- Description: The current study investigated the biochemical properties of endo-glucanase (GH5E), exo-glucanase (GH5D), xylanase (GH5H) and endo-glucanase/xylanase (GH45), derived from the hindgut bacterial symbionts of a termite (Trinervitermes trinervoides) for their potential role in the biotechnology industry. All these enzymes, except GH5D, exhibited activities on cellulosic and xylan-rich polymeric substrates, which only displayed activity on p-nitrophenyl cellobioside. GH5D, GH5E, GH5H and GH45 enzymes retained more than 80% of their activities at pH 5.5 and also retained more than 80% of their activities at 40ºC. Furthermore, these enzymes were thermostable at 37ºC for 72 hours. GH5E, GH5H and GH45 were generally stable over a range of metal-ion. The kinetic parameters for GH5E were 5.68 mg/ml (KM) and 34.36 U/mg protein (Vmax). GH5D activity did not follow classical Michaelis-Menten kinetics, suggesting product inhibition. GH5H displayed KM values of 5.53, 95.03 and 2.10 mg/ml and Vmax values of 112.36, 144.45 and 180.32 U/mg protein on beechwood xylan, CMC, and xyloglucan, respectively. GH45 displayed a KM of 6.94 mg/ml and a Vmax of 12.30 U/mg protein on CMC. GH5D [cellobiohydrolase (CBH)] and a commercial CBHII (GH6) enzyme outperformed a commercial CBHI (GH7) enzyme when these enzymes hydrolysed β-glucan. GH5D and CBHII also displayed a higher degree of synergy on β-glucan but failed to show synergy on Avicel. We therefore concluded that GH5D and CBHII are β-glucan-specific cellobiohydrolases. The corncob (CC) and sweet sorghum bagasse (SSB) substrates were pretreated with lime, NaOH and NaClO2. Subsequent to pretreatment, these substrates were used to investigate if GH5D, GH5E, GH5H and GH45 could operate in synergy. Results revealed that out of 12 possible core enzyme sets constructed, only two (referred to as CES-E and CES-H) displayed higher activities on pretreated CC or SSB. Simultaneous synergy was generally the most effective mode of synergy during hydrolysis of alkaline pretreated SSB and CC samples by both CES-E and CES-H. Both core enzyme sets did not display synergy on oxidative pretreated substrates. These findings suggest that lime and NaOH are more effective pretreatments for CC and SSB substrates. We used PRotein Interactive MOdeling (PRIMO) software to demonstrate that GH5D protein structure is an (α/β)8 barrel with a tunnel-like active site. Enzymes with this type of protein structure are able to perform transglycosylation, a process in which GH5D produced methyl, ethyl and propyl cellobiosides. We concluded that the GH5D, GH5E, GH5H and GH45 enzymes possess novel biochemical properties and that they form synergy during the hydrolysis of complex substrates (SSB and CC). GH5D transglycosylation could be used to produce novel biodegradable chemicals with special properties (e.g. anti-microbial properties). In conclusion, our findings suggest that GH5D, GH5E, GH5H and GH45 can potentially be used to improve biorefinery processes. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2019
- Full Text:
- Date Issued: 2019
- Authors: Mafa, Mpho Stephen
- Date: 2019
- Subjects: Termites , Metagenomics , Glucosides , Hydrolases , Enzymes , Feedstock
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/72414 , vital:30044 , DOI https://doi.org/10.21504/10962/72414
- Description: The current study investigated the biochemical properties of endo-glucanase (GH5E), exo-glucanase (GH5D), xylanase (GH5H) and endo-glucanase/xylanase (GH45), derived from the hindgut bacterial symbionts of a termite (Trinervitermes trinervoides) for their potential role in the biotechnology industry. All these enzymes, except GH5D, exhibited activities on cellulosic and xylan-rich polymeric substrates, which only displayed activity on p-nitrophenyl cellobioside. GH5D, GH5E, GH5H and GH45 enzymes retained more than 80% of their activities at pH 5.5 and also retained more than 80% of their activities at 40ºC. Furthermore, these enzymes were thermostable at 37ºC for 72 hours. GH5E, GH5H and GH45 were generally stable over a range of metal-ion. The kinetic parameters for GH5E were 5.68 mg/ml (KM) and 34.36 U/mg protein (Vmax). GH5D activity did not follow classical Michaelis-Menten kinetics, suggesting product inhibition. GH5H displayed KM values of 5.53, 95.03 and 2.10 mg/ml and Vmax values of 112.36, 144.45 and 180.32 U/mg protein on beechwood xylan, CMC, and xyloglucan, respectively. GH45 displayed a KM of 6.94 mg/ml and a Vmax of 12.30 U/mg protein on CMC. GH5D [cellobiohydrolase (CBH)] and a commercial CBHII (GH6) enzyme outperformed a commercial CBHI (GH7) enzyme when these enzymes hydrolysed β-glucan. GH5D and CBHII also displayed a higher degree of synergy on β-glucan but failed to show synergy on Avicel. We therefore concluded that GH5D and CBHII are β-glucan-specific cellobiohydrolases. The corncob (CC) and sweet sorghum bagasse (SSB) substrates were pretreated with lime, NaOH and NaClO2. Subsequent to pretreatment, these substrates were used to investigate if GH5D, GH5E, GH5H and GH45 could operate in synergy. Results revealed that out of 12 possible core enzyme sets constructed, only two (referred to as CES-E and CES-H) displayed higher activities on pretreated CC or SSB. Simultaneous synergy was generally the most effective mode of synergy during hydrolysis of alkaline pretreated SSB and CC samples by both CES-E and CES-H. Both core enzyme sets did not display synergy on oxidative pretreated substrates. These findings suggest that lime and NaOH are more effective pretreatments for CC and SSB substrates. We used PRotein Interactive MOdeling (PRIMO) software to demonstrate that GH5D protein structure is an (α/β)8 barrel with a tunnel-like active site. Enzymes with this type of protein structure are able to perform transglycosylation, a process in which GH5D produced methyl, ethyl and propyl cellobiosides. We concluded that the GH5D, GH5E, GH5H and GH45 enzymes possess novel biochemical properties and that they form synergy during the hydrolysis of complex substrates (SSB and CC). GH5D transglycosylation could be used to produce novel biodegradable chemicals with special properties (e.g. anti-microbial properties). In conclusion, our findings suggest that GH5D, GH5E, GH5H and GH45 can potentially be used to improve biorefinery processes. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2019
- Full Text:
- Date Issued: 2019
Chitin hydrolysis with chitinolytic enzymes for the production of chitooligomers with antimicrobial properties
- Authors: Oree, Glynis
- Date: 2019
- Subjects: Chitin -- Biotechnology , Enzymes -- Biotechnology , Hydrolysis , Chitooligomers -- Biotechnology
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/67887 , vital:29165
- Description: There are many diseases and illnesses in the world that require new drug treatments and chitin has been shown to produce chitooligomeric derivatives which exhibit promising antimicrobial and immune-enhancing properties. However, the rate-limiting step is associated with the high recalcitrance of chitinous substrates, and low hydrolytic activities of chitinolytic enzymes, resulting in low product release. To improve and create a more sustainable and economical process, enhancing chitin hydrolysis through various treatment procedures is essential for obtaining high enzyme hydrolysis rates, resulting in a higher yield of chitooligomers (CHOS). In literature, pre-treatment of insoluble biomass is generally associated with an increase in accessibility of the carbohydrate to hydrolytic enzymes, thus generating more products. The first part of this study investigated the effect of alkali- (NaOH) and acid pre-treatments (HCl and phosphoric acid) on chitin biomass, and chemical and morphological modifications were assessed by the employment of scanning electron microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR), Energy-Dispersive X-ray spectrometery (EDX) and x-ray diffraction (XRD). Data obtained confirmed that pre-treated substrates were more chemically and morphologically modified. These results confirmed the fact that pre-treatment of chitin disrupts the structure of the biomass, rendering the polymer more accessible for enzymatic hydrolysis. The commercial chitinases from Bacillus cereus and Streptomyces griseus (CHB and CHS) are costly. Bio-prospecting for other chitin-degrading enzymes from alternate sources such as Oidiodendron maius, or the recombinant expression of CHOS, was a more economically feasible avenue. The chit1 gene from Thermomyces lanuginosus, expressed in Pichia pastoris, produced a large range CHOS with a degree of polymerisation (DP) ranging from 1 to above 6. TLC analysis showed that O. maius exhibited chitin-degrading properties by producing CHOS with a DP length of 1 to 3. These two sources were therefore successful in producing chitin-degrading enzymes. The physico-chemical properties of commercial (CHB and CHS) and expressed (Chit1) chitinolytic enzymes were investigated, to determine under which biochemical conditions and on which type of biomass they can function on optimally, for the production of value-added products such as CHOS. Substrate affinity assays were conducted on the un-treated and pre-treated biomass. TLC revealed that chitosan hydrolysis by the commercial chitinases produced the largest range of CHOS with a DP length ranging from 1 to 6. A range of temperatures (35-90oC) were investigated and CHB, CHS and Chit1 displayed optimum activities at 50, 40 and 45 oC, respectively. Thermostability studies that were conducted at 37 and 50oC revealed that CHB and CHS were most stable at 37oC. Chit1 showed great thermostablity at both temperatures, rendering this enzyme suitable for industrial processes at high temperatures. pH optima studies demonstrated that the pH optima for CHB, CHS and Chit1 was at a pH of 5.0, with specific activities of 33.459, 46.2 and 5.776 μmol/h/mg, respectively. The chain cleaving patterns of the commercial enzymes were determined and exo-chitinase activity was exhibited, due to the production of CHOS that were predominantly of a DP length of 2. Enzyme binary synergy studies were conducted with commercial chitinases (CHB and CHS) on colloidal chitin. Studies illustrated that the simultaneous combination of CHB 75%: CHS 25% produced the highest specific activity (3.526 μmol/h/mg), with no synergy. TLC analysis of this enzyme combination over time revealed that predominantly chitobiose was produced. This suggested that the substrate crystallinity and morphology played an important role in the way the enzymes cleaved the carbohydrate. Since CHOS have shown great promise for their antimicrobial properties, the CHOS generated from the chitinous substrates were tested for antimicrobial properties on Bacillus subtilis, Escherichia coli, Klebsiella and Staphlococcus aureus. This study revealed that certain CHOS produced have inhibitory effects on certain bacteria and could potentially be used in the pharamceutical or medical industries. In conclusion, this study revealed that chitinases can be produced and found in alternate sources and be used for the hydrolysis of chitinous biomass in a more sustainabe and economically viable manner. The chitinases investigated (CHB, CHS and Chit1) exhibited different cleaving patterns of the chitinous substrates due to the chemical and morphological properties of the biomass. CHOS produced from chitinous biomass exhibited some inhibitory effects on bacterial growth and show potential for use in the medical industry.
- Full Text:
- Date Issued: 2019
- Authors: Oree, Glynis
- Date: 2019
- Subjects: Chitin -- Biotechnology , Enzymes -- Biotechnology , Hydrolysis , Chitooligomers -- Biotechnology
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/67887 , vital:29165
- Description: There are many diseases and illnesses in the world that require new drug treatments and chitin has been shown to produce chitooligomeric derivatives which exhibit promising antimicrobial and immune-enhancing properties. However, the rate-limiting step is associated with the high recalcitrance of chitinous substrates, and low hydrolytic activities of chitinolytic enzymes, resulting in low product release. To improve and create a more sustainable and economical process, enhancing chitin hydrolysis through various treatment procedures is essential for obtaining high enzyme hydrolysis rates, resulting in a higher yield of chitooligomers (CHOS). In literature, pre-treatment of insoluble biomass is generally associated with an increase in accessibility of the carbohydrate to hydrolytic enzymes, thus generating more products. The first part of this study investigated the effect of alkali- (NaOH) and acid pre-treatments (HCl and phosphoric acid) on chitin biomass, and chemical and morphological modifications were assessed by the employment of scanning electron microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR), Energy-Dispersive X-ray spectrometery (EDX) and x-ray diffraction (XRD). Data obtained confirmed that pre-treated substrates were more chemically and morphologically modified. These results confirmed the fact that pre-treatment of chitin disrupts the structure of the biomass, rendering the polymer more accessible for enzymatic hydrolysis. The commercial chitinases from Bacillus cereus and Streptomyces griseus (CHB and CHS) are costly. Bio-prospecting for other chitin-degrading enzymes from alternate sources such as Oidiodendron maius, or the recombinant expression of CHOS, was a more economically feasible avenue. The chit1 gene from Thermomyces lanuginosus, expressed in Pichia pastoris, produced a large range CHOS with a degree of polymerisation (DP) ranging from 1 to above 6. TLC analysis showed that O. maius exhibited chitin-degrading properties by producing CHOS with a DP length of 1 to 3. These two sources were therefore successful in producing chitin-degrading enzymes. The physico-chemical properties of commercial (CHB and CHS) and expressed (Chit1) chitinolytic enzymes were investigated, to determine under which biochemical conditions and on which type of biomass they can function on optimally, for the production of value-added products such as CHOS. Substrate affinity assays were conducted on the un-treated and pre-treated biomass. TLC revealed that chitosan hydrolysis by the commercial chitinases produced the largest range of CHOS with a DP length ranging from 1 to 6. A range of temperatures (35-90oC) were investigated and CHB, CHS and Chit1 displayed optimum activities at 50, 40 and 45 oC, respectively. Thermostability studies that were conducted at 37 and 50oC revealed that CHB and CHS were most stable at 37oC. Chit1 showed great thermostablity at both temperatures, rendering this enzyme suitable for industrial processes at high temperatures. pH optima studies demonstrated that the pH optima for CHB, CHS and Chit1 was at a pH of 5.0, with specific activities of 33.459, 46.2 and 5.776 μmol/h/mg, respectively. The chain cleaving patterns of the commercial enzymes were determined and exo-chitinase activity was exhibited, due to the production of CHOS that were predominantly of a DP length of 2. Enzyme binary synergy studies were conducted with commercial chitinases (CHB and CHS) on colloidal chitin. Studies illustrated that the simultaneous combination of CHB 75%: CHS 25% produced the highest specific activity (3.526 μmol/h/mg), with no synergy. TLC analysis of this enzyme combination over time revealed that predominantly chitobiose was produced. This suggested that the substrate crystallinity and morphology played an important role in the way the enzymes cleaved the carbohydrate. Since CHOS have shown great promise for their antimicrobial properties, the CHOS generated from the chitinous substrates were tested for antimicrobial properties on Bacillus subtilis, Escherichia coli, Klebsiella and Staphlococcus aureus. This study revealed that certain CHOS produced have inhibitory effects on certain bacteria and could potentially be used in the pharamceutical or medical industries. In conclusion, this study revealed that chitinases can be produced and found in alternate sources and be used for the hydrolysis of chitinous biomass in a more sustainabe and economically viable manner. The chitinases investigated (CHB, CHS and Chit1) exhibited different cleaving patterns of the chitinous substrates due to the chemical and morphological properties of the biomass. CHOS produced from chitinous biomass exhibited some inhibitory effects on bacterial growth and show potential for use in the medical industry.
- Full Text:
- Date Issued: 2019
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:
- Date Issued: 2018
- 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:
- Date Issued: 2018
Formulation of an enzyme cocktail, HoloMix, using cellulolytic and xylanolytic enzyme core-sets for effective degradation of various pre-treated hardwoods
- Authors: Malgas, Samkelo
- Date: 2018
- Subjects: Biomass , Cellulase , Hardwoods , Xylanases
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/62827 , vital:28297 , DOI https://doi.org/10.21504/10962/62827
- Description: Currently, there is a growing interest in utilising hardwoods as feedstocks for bioethanol production due to the vast advantages they have over other feedstocks for fermentable sugar production. In this study, two selected hardwoods, Acacia and Populus spp., were subjected to two pre-treatment processes (Sodium chlorite delignification and Steam explosion) and compared with respect to how these pre-treatments affect their enzymatic saccharification. Hardwoods were selected for this study, because hardwoods are easier to delignify when compared to softwoods, and therefore their polysaccharides are more easily accessible by enzymes for the purpose of producing fermentable sugars. Currently available commercial enzyme mixtures have been developed for optimal hydrolysis of acid-pre-treated corn stover and are therefore not optimal for saccharification of pre-treated hardwoods. In this work, we attempted the empirical design of a hardwood specific enzyme cocktail, HoloMix. Firstly, a cellulolytic core-set, CelMix (in a ratio of Egl 68%: Cel7A 17%: Cel6A 6%: Bgl1 9%), for the optimal release of glucose, and a xylanolytic core-set, XynMix (in a ratio of Xyn2A 60%: XT6 20%: AguA 11%: SXA 9%), for the optimal release of xylose, were formulated using an empirical enzyme ratio approach after biochemically characterising these enzymes. As it is well ̶ known that biomass pre-treatment may result in the generation of compounds that hamper enzymatic hydrolysis and microbial fermentation, the effects of these compounds on CelMix and XynMix were evaluated. Using the optimised CelMix and XynMix cocktails, a HoloMix cocktail was established for optimal reducing sugar, glucose and xylose release from the various pre-treated hardwoods. For delignified biomass, the optimized HoloMix consisted of CelMix to XynMix at 75% to 25% protein loading, while for the untreated and steam exploded biomass the HoloMix consisted of CelMix to XynMix at 93.75% to 6.25% protein loading. Sugar release by the HoloMix at a loading of 27.5 mg protein/g of biomass (or 55 mg protein/g of glucan) after 24 h gave 70-100% sugar yield. Treatment of the hardwoods with a laccase from Agaricus bisporus, especially wood biomass with a higher proportion of lignin, significantly improved saccharification by the formulated HoloMix enzyme cocktails. This study provided insights into the enzymatic hydrolysis of various pre-treated hardwood substrates and assessed whether the same lignocellulolytic cocktail can be used to efficiently hydrolyse different hardwood species. The present study also demonstrated that the hydrolysis efficiency of the optimised HoloMix was comparable to (if not better) than commercial enzyme preparations during hardwood biomass saccharification. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
- Authors: Malgas, Samkelo
- Date: 2018
- Subjects: Biomass , Cellulase , Hardwoods , Xylanases
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/62827 , vital:28297 , DOI https://doi.org/10.21504/10962/62827
- Description: Currently, there is a growing interest in utilising hardwoods as feedstocks for bioethanol production due to the vast advantages they have over other feedstocks for fermentable sugar production. In this study, two selected hardwoods, Acacia and Populus spp., were subjected to two pre-treatment processes (Sodium chlorite delignification and Steam explosion) and compared with respect to how these pre-treatments affect their enzymatic saccharification. Hardwoods were selected for this study, because hardwoods are easier to delignify when compared to softwoods, and therefore their polysaccharides are more easily accessible by enzymes for the purpose of producing fermentable sugars. Currently available commercial enzyme mixtures have been developed for optimal hydrolysis of acid-pre-treated corn stover and are therefore not optimal for saccharification of pre-treated hardwoods. In this work, we attempted the empirical design of a hardwood specific enzyme cocktail, HoloMix. Firstly, a cellulolytic core-set, CelMix (in a ratio of Egl 68%: Cel7A 17%: Cel6A 6%: Bgl1 9%), for the optimal release of glucose, and a xylanolytic core-set, XynMix (in a ratio of Xyn2A 60%: XT6 20%: AguA 11%: SXA 9%), for the optimal release of xylose, were formulated using an empirical enzyme ratio approach after biochemically characterising these enzymes. As it is well ̶ known that biomass pre-treatment may result in the generation of compounds that hamper enzymatic hydrolysis and microbial fermentation, the effects of these compounds on CelMix and XynMix were evaluated. Using the optimised CelMix and XynMix cocktails, a HoloMix cocktail was established for optimal reducing sugar, glucose and xylose release from the various pre-treated hardwoods. For delignified biomass, the optimized HoloMix consisted of CelMix to XynMix at 75% to 25% protein loading, while for the untreated and steam exploded biomass the HoloMix consisted of CelMix to XynMix at 93.75% to 6.25% protein loading. Sugar release by the HoloMix at a loading of 27.5 mg protein/g of biomass (or 55 mg protein/g of glucan) after 24 h gave 70-100% sugar yield. Treatment of the hardwoods with a laccase from Agaricus bisporus, especially wood biomass with a higher proportion of lignin, significantly improved saccharification by the formulated HoloMix enzyme cocktails. This study provided insights into the enzymatic hydrolysis of various pre-treated hardwood substrates and assessed whether the same lignocellulolytic cocktail can be used to efficiently hydrolyse different hardwood species. The present study also demonstrated that the hydrolysis efficiency of the optimised HoloMix was comparable to (if not better) than commercial enzyme preparations during hardwood biomass saccharification. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
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:
- Date Issued: 2018
- 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:
- Date Issued: 2018
Bio-prospecting a Soil Metagenomic Library for Carbohydrate Active Esterases
- Authors: Shezi, Ntombifuthi
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4172 , http://hdl.handle.net/10962/d1021266
- Description: Lignocellulosic biomass is a promising renewable resource on earth. Plant biomass contains fermentable sugars and other moieties that can be converted to biofuels or other chemicals. Enzymatic hydrolysis of these biopolymers is significant in the liberation of sugars for fermentation into desired products. Owing to its complex structure, synergistic action of enzymes is required for its degradation. Enzymes that are involved in biomass degradation include cellulases, hemicellulases and the accessory enzymes acetyl xylan esterases and ferulic acid esterases. Ferulic acid esterases (FAEs, EC 3.1.1.73), represent a subclass of carboxylester hydrolases (EC 3.1.1.-) that catalyse the release of hydroxycinnamic acids (such as ferulic acid, p-coumaric, ferulic, sinapic and caffeic acid) that are generally found esterified to polysaccharides, such as arabinoxylans. Hydroxycinnamic acids have widespread potential applications due to their antimicrobial, photoprotectant and antioxidant properties, as well as their use as flavour precursors. Therefore, this interesting group of FAEs has a potentially wide variety of applications in agriculture, food and pharmaceutical industries. In the search for novel biocatalysts, metagenomics is considered as an alternative approach to conventional microbe screening, therefore, searching for novel biocatalysts from a soil metagenome that harbours a unique diversity of biocatalyst is significant. The aim of this study was to extract DNA from soil associated with cattle manure and construct a soil metagenomic library using a fosmid based plasmid vector and subsequently functionally screen for ferulic acid esterases using ethyl ferulate as a model substrate. A total of 59 recombinant fosmids conferring ferulic acid esterase phenotypes were identified (Hit rate 1:3122) and the two fosmids that consistently showed high FAE activities were selected for further study. Following nucleotide sequencing and translational analysis, two fae encoding open reading frames (FAE9 and FAE27) of approximately 274 and 322 aa, respectively, were identified. The amino acid sequence of the two ORFs contained a classical conserved esterase/lipase G-x-S-x-G sequence motif. The two genes (fae9 and fae27) were successfully expressed in Escherichia coli BL21 (DE3) and the purified enzymes exhibited respective temperature optima of 50 °C and 40 °C, and respective pH optima of 6.0 and 7.0. Further biochemical characterisation showed that FAE9 and FAE27 have high substrate specificity, following the fact that EFA is the preferred substrate for FAE9 (kcat/Km value of 128 s−1.mM-1) and also the preferred substrate for FAE27 (kcat/Km value of 137 s−1.mM-1). This work proves that soil is a valuable environmental source for novel esterase screening through functional based metagenomic approach. Therefore, this method may be used to screen for other valuable enzymes from environmental sources using inexpensive natural sources to encourage the screening of specific enzymes. Biochemistry of the two isolated enzymes makes these enzymes to be useful in industrial applications due to broad substrate activity that could replace the specialised enzymes to complete plant biomass degradation.
- Full Text:
- Date Issued: 2016
- Authors: Shezi, Ntombifuthi
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4172 , http://hdl.handle.net/10962/d1021266
- Description: Lignocellulosic biomass is a promising renewable resource on earth. Plant biomass contains fermentable sugars and other moieties that can be converted to biofuels or other chemicals. Enzymatic hydrolysis of these biopolymers is significant in the liberation of sugars for fermentation into desired products. Owing to its complex structure, synergistic action of enzymes is required for its degradation. Enzymes that are involved in biomass degradation include cellulases, hemicellulases and the accessory enzymes acetyl xylan esterases and ferulic acid esterases. Ferulic acid esterases (FAEs, EC 3.1.1.73), represent a subclass of carboxylester hydrolases (EC 3.1.1.-) that catalyse the release of hydroxycinnamic acids (such as ferulic acid, p-coumaric, ferulic, sinapic and caffeic acid) that are generally found esterified to polysaccharides, such as arabinoxylans. Hydroxycinnamic acids have widespread potential applications due to their antimicrobial, photoprotectant and antioxidant properties, as well as their use as flavour precursors. Therefore, this interesting group of FAEs has a potentially wide variety of applications in agriculture, food and pharmaceutical industries. In the search for novel biocatalysts, metagenomics is considered as an alternative approach to conventional microbe screening, therefore, searching for novel biocatalysts from a soil metagenome that harbours a unique diversity of biocatalyst is significant. The aim of this study was to extract DNA from soil associated with cattle manure and construct a soil metagenomic library using a fosmid based plasmid vector and subsequently functionally screen for ferulic acid esterases using ethyl ferulate as a model substrate. A total of 59 recombinant fosmids conferring ferulic acid esterase phenotypes were identified (Hit rate 1:3122) and the two fosmids that consistently showed high FAE activities were selected for further study. Following nucleotide sequencing and translational analysis, two fae encoding open reading frames (FAE9 and FAE27) of approximately 274 and 322 aa, respectively, were identified. The amino acid sequence of the two ORFs contained a classical conserved esterase/lipase G-x-S-x-G sequence motif. The two genes (fae9 and fae27) were successfully expressed in Escherichia coli BL21 (DE3) and the purified enzymes exhibited respective temperature optima of 50 °C and 40 °C, and respective pH optima of 6.0 and 7.0. Further biochemical characterisation showed that FAE9 and FAE27 have high substrate specificity, following the fact that EFA is the preferred substrate for FAE9 (kcat/Km value of 128 s−1.mM-1) and also the preferred substrate for FAE27 (kcat/Km value of 137 s−1.mM-1). This work proves that soil is a valuable environmental source for novel esterase screening through functional based metagenomic approach. Therefore, this method may be used to screen for other valuable enzymes from environmental sources using inexpensive natural sources to encourage the screening of specific enzymes. Biochemistry of the two isolated enzymes makes these enzymes to be useful in industrial applications due to broad substrate activity that could replace the specialised enzymes to complete plant biomass degradation.
- Full Text:
- Date Issued: 2016
The effect of kelp supplementation in formulated feed on the production performance and gut microbiota of South African abalone (Haliotis midae)
- Authors: Nel, Aldi
- Date: 2016
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/899 , vital:20001
- Description: Formulated feeds with a relatively low (< 5 % of dry mass) kelp (Ecklonia maxima) inclusion level are widely used on commercial abalone (Haliotis midae) farms in South Africa. Although the use of kelp, a major constituent of the natural diet of H. midae, as a dietary supplement is considered to enhance abalone growth and feed utilisation, there are no published studies which quantify the effects of kelp inclusion in formulated feeds. Furthermore, the physiological mechanisms by which kelp supplementation may positively influence abalone digestive physiology and growth are largely unknown. As the kelp supplement is comprised mostly of soluble fibres and abalone gut bacteria associated with macroalgae (and its fibrous polysaccharides) are known to play a key role in digestion, it was hypothesised that the kelp supplement influences the gut-bacterial community profiles of cultured abalone through prebiotic and other metabolic effects. The present thesis thus examined the effect of kelp supplementation on the performance of abalone (Haliotis midae) fed formulated feeds and explored the influence of a kelp supplement on the abalone gut microbiota and its function in the gastrointestinal tract. The key hypotheses of the study were that kelp supplementation in formulated feed: 1) enhances abalone growth; 2) causes a shift in abalone gut-bacterial community composition through a prebiotic-like effect; 3) may induce changes in crop morphology as a result of potential bacterial-associated increases in volatile short-chain fatty acids, and 4) alters digestive enzyme activities in the abalone gut through changes in bacterial-derived (exogenous) digestive enzymes. The growth-promoting efficacy of low-level kelp supplementation was tested by feeding isonitrogenous and isoenergetic experimental feeds containing 0.00 – 3.54 % kelp (dry mass) to sub-adult abalone (~43 mm shell length) for eight months under commercial farm conditions. The growth trial established that kelp supplementation (0.44 – 3.54 % of dry mass) promoted faster growth and improved feed conversion and protein efficiency ratios in cultured abalone compared to abalone fed the non-supplemented control diet, while there were no significant differences in growth for abalone fed the different kelp-supplemented diets (0.44, 0.88, 1.76 and 3.54 % of dry mass). Feed conversion and protein efficiency ratios displayed significant correlations with kelp level in the range of 0.00 – 3.54 % dry mass, and it is therefore recommended that kelp be included in the formulated feeds of cultured South African abalone at a rate of up to 3.54 % of dry mass. A kelp-supplemented (0.88 % dry weight inclusion) feed was fed to abalone under farm conditions to compare gut physiological parameters (crop morphology, digestive enzyme activities and the gut microbiota) in abalone against that of abalone fed an isonitrogenous and isoenergetic non-supplemented control feed. To establish if the observed higher abalone growth rates were related to improved gastrointestinal tract epithelium activity and integrity, as reflected by epithelial cell growth in response to potential changes in bacterial-derived short-chain fatty acid production, crop epithelial morphology was compared between abalone fed the kelp-supplemented and control feeds. Kelp supplementation did not induce any observable changes in crop epithelial cell height for farm-reared sub-adult abalone fed the experimental diets on-farm for seven weeks. This was attributed to the similar macronutrient compositions of kelp-supplemented and control diets and/ or the common diet history of experimental abalone from weaning to the initiation of the experiment. Digestive enzyme activity was compared between abalone fed a kelp-supplemented and a control feed during an on-farm feeding trial with sub-adult abalone. Gut samples were collected after seven weeks and colorimetric enzyme assays were performed for the polysaccharide-degrading enzymes amylase, alginate lyase, laminarinase and fucoidanase, and for acid protease, trypsin and chymotrypsin activity. Amylase and alginate lyase activities were relatively high, compared to the other enzymes. Polysaccharidase and acid protease activity levels did not differ significantly between abalone fed kelp-supplemented and control feeds, but a greater variability in enzyme activity levels was observed in abalone fed the control diet. It was hypothesised that this might be due to the kelp supplement promoting a more stable and less opportunistic gut-bacterial community than the control diet. Pooled gut samples of abalone fed the kelp-supplemented diet were used for proteomic analyses to identify the composition of enzyme proteins of both endogenous and exogenous origin in the abalone digestive system. The key polysaccharidases and proteases in the gut samples of kelp-supplemented formulated feed-fed abalone were all of abalone origin, whereas the bacterial enzymes were of the types that form part of intermediate reactions in metabolic pathways. The results suggested that bacterial enzymes play a different role to abalone endogenous enzymes in the digestion of formulated feed. While abalone enzymes appear to be the main degraders of carbohydrate and protein macromolecules, the profile of exogenous enzymes suggests that they perform bioconversions of smaller organic compounds. The profiles of gut-bacterial communities of farm-reared sub-adult abalone fed kelpsupplemented and control feeds on-farm for seven weeks were analysed with metagenomic pyrosequencing and DGGE analyses, using 16S rDNA-targeted amplified DNA. The results indicated a shift in gut-bacterial composition with a higher abundance of Mollicutes in abalone fed kelp-supplemented feed compared to those fed the control feed. DGGE band patterns displayed a greater within-group similarity in gut bacteria for abalone fed the kelpsupplemented diet and the presence of unique and variable bands for bacteria in the guts of abalone fed the control diet. It was concluded that when cultured abalone are fed kelpsupplemented formulated feeds, more stable gut bacterial communities are present compared to a more opportunistic gut-bacterial community in abalone fed non-supplemented feeds, and that the observed increase in Mollicutes could reflect the restoration of the abalone gut microbiota to a more natural state. The novel application of proteomics to abalone nutrition in the present study demonstrated that gut-bacterial enzymes may form part of many different metabolic pathways and suggests that the metabolism of the gut microbiota serves as an extension of the abalone’s digestive metabolism. Future studies should quantify the contribution of commensal gut-bacteria to cultured abalone nutrition by employing metabolomic studies to characterize the utilisation of bacterial-derived metabolites by the abalone host.
- Full Text:
- Date Issued: 2016
- Authors: Nel, Aldi
- Date: 2016
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/899 , vital:20001
- Description: Formulated feeds with a relatively low (< 5 % of dry mass) kelp (Ecklonia maxima) inclusion level are widely used on commercial abalone (Haliotis midae) farms in South Africa. Although the use of kelp, a major constituent of the natural diet of H. midae, as a dietary supplement is considered to enhance abalone growth and feed utilisation, there are no published studies which quantify the effects of kelp inclusion in formulated feeds. Furthermore, the physiological mechanisms by which kelp supplementation may positively influence abalone digestive physiology and growth are largely unknown. As the kelp supplement is comprised mostly of soluble fibres and abalone gut bacteria associated with macroalgae (and its fibrous polysaccharides) are known to play a key role in digestion, it was hypothesised that the kelp supplement influences the gut-bacterial community profiles of cultured abalone through prebiotic and other metabolic effects. The present thesis thus examined the effect of kelp supplementation on the performance of abalone (Haliotis midae) fed formulated feeds and explored the influence of a kelp supplement on the abalone gut microbiota and its function in the gastrointestinal tract. The key hypotheses of the study were that kelp supplementation in formulated feed: 1) enhances abalone growth; 2) causes a shift in abalone gut-bacterial community composition through a prebiotic-like effect; 3) may induce changes in crop morphology as a result of potential bacterial-associated increases in volatile short-chain fatty acids, and 4) alters digestive enzyme activities in the abalone gut through changes in bacterial-derived (exogenous) digestive enzymes. The growth-promoting efficacy of low-level kelp supplementation was tested by feeding isonitrogenous and isoenergetic experimental feeds containing 0.00 – 3.54 % kelp (dry mass) to sub-adult abalone (~43 mm shell length) for eight months under commercial farm conditions. The growth trial established that kelp supplementation (0.44 – 3.54 % of dry mass) promoted faster growth and improved feed conversion and protein efficiency ratios in cultured abalone compared to abalone fed the non-supplemented control diet, while there were no significant differences in growth for abalone fed the different kelp-supplemented diets (0.44, 0.88, 1.76 and 3.54 % of dry mass). Feed conversion and protein efficiency ratios displayed significant correlations with kelp level in the range of 0.00 – 3.54 % dry mass, and it is therefore recommended that kelp be included in the formulated feeds of cultured South African abalone at a rate of up to 3.54 % of dry mass. A kelp-supplemented (0.88 % dry weight inclusion) feed was fed to abalone under farm conditions to compare gut physiological parameters (crop morphology, digestive enzyme activities and the gut microbiota) in abalone against that of abalone fed an isonitrogenous and isoenergetic non-supplemented control feed. To establish if the observed higher abalone growth rates were related to improved gastrointestinal tract epithelium activity and integrity, as reflected by epithelial cell growth in response to potential changes in bacterial-derived short-chain fatty acid production, crop epithelial morphology was compared between abalone fed the kelp-supplemented and control feeds. Kelp supplementation did not induce any observable changes in crop epithelial cell height for farm-reared sub-adult abalone fed the experimental diets on-farm for seven weeks. This was attributed to the similar macronutrient compositions of kelp-supplemented and control diets and/ or the common diet history of experimental abalone from weaning to the initiation of the experiment. Digestive enzyme activity was compared between abalone fed a kelp-supplemented and a control feed during an on-farm feeding trial with sub-adult abalone. Gut samples were collected after seven weeks and colorimetric enzyme assays were performed for the polysaccharide-degrading enzymes amylase, alginate lyase, laminarinase and fucoidanase, and for acid protease, trypsin and chymotrypsin activity. Amylase and alginate lyase activities were relatively high, compared to the other enzymes. Polysaccharidase and acid protease activity levels did not differ significantly between abalone fed kelp-supplemented and control feeds, but a greater variability in enzyme activity levels was observed in abalone fed the control diet. It was hypothesised that this might be due to the kelp supplement promoting a more stable and less opportunistic gut-bacterial community than the control diet. Pooled gut samples of abalone fed the kelp-supplemented diet were used for proteomic analyses to identify the composition of enzyme proteins of both endogenous and exogenous origin in the abalone digestive system. The key polysaccharidases and proteases in the gut samples of kelp-supplemented formulated feed-fed abalone were all of abalone origin, whereas the bacterial enzymes were of the types that form part of intermediate reactions in metabolic pathways. The results suggested that bacterial enzymes play a different role to abalone endogenous enzymes in the digestion of formulated feed. While abalone enzymes appear to be the main degraders of carbohydrate and protein macromolecules, the profile of exogenous enzymes suggests that they perform bioconversions of smaller organic compounds. The profiles of gut-bacterial communities of farm-reared sub-adult abalone fed kelpsupplemented and control feeds on-farm for seven weeks were analysed with metagenomic pyrosequencing and DGGE analyses, using 16S rDNA-targeted amplified DNA. The results indicated a shift in gut-bacterial composition with a higher abundance of Mollicutes in abalone fed kelp-supplemented feed compared to those fed the control feed. DGGE band patterns displayed a greater within-group similarity in gut bacteria for abalone fed the kelpsupplemented diet and the presence of unique and variable bands for bacteria in the guts of abalone fed the control diet. It was concluded that when cultured abalone are fed kelpsupplemented formulated feeds, more stable gut bacterial communities are present compared to a more opportunistic gut-bacterial community in abalone fed non-supplemented feeds, and that the observed increase in Mollicutes could reflect the restoration of the abalone gut microbiota to a more natural state. The novel application of proteomics to abalone nutrition in the present study demonstrated that gut-bacterial enzymes may form part of many different metabolic pathways and suggests that the metabolism of the gut microbiota serves as an extension of the abalone’s digestive metabolism. Future studies should quantify the contribution of commensal gut-bacteria to cultured abalone nutrition by employing metabolomic studies to characterize the utilisation of bacterial-derived metabolites by the abalone host.
- Full Text:
- Date Issued: 2016
An investigation into the synergistic action of cellulose-degrading enzymes on complex substrates
- Authors: Thoresen, Mariska
- Date: 2015
- Subjects: Lignocellulose , Biomass energy , Cellulosic ethanol , Saccharomyces cerevisiae , Cellulase , Enzymes -- Biotechnology , Hydrolases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4154 , http://hdl.handle.net/10962/d1017915
- Full Text:
- Date Issued: 2015
- Authors: Thoresen, Mariska
- Date: 2015
- Subjects: Lignocellulose , Biomass energy , Cellulosic ethanol , Saccharomyces cerevisiae , Cellulase , Enzymes -- Biotechnology , Hydrolases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4154 , http://hdl.handle.net/10962/d1017915
- Full Text:
- Date Issued: 2015
Nanofiber immobilized cellulases and hemicellulases for fruit waste beneficiation
- Authors: Swart, Shanna
- Date: 2015
- Subjects: Agricultural wastes , Cellulase , Hemicellulose , Nanofibers , Electrospinning , Lignocellulose -- Biodegradation , Biomass conversion , Polysaccharides , Immobilized enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4153 , http://hdl.handle.net/10962/d1017914
- Full Text:
- Date Issued: 2015
- Authors: Swart, Shanna
- Date: 2015
- Subjects: Agricultural wastes , Cellulase , Hemicellulose , Nanofibers , Electrospinning , Lignocellulose -- Biodegradation , Biomass conversion , Polysaccharides , Immobilized enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4153 , http://hdl.handle.net/10962/d1017914
- Full Text:
- Date Issued: 2015
The detection of glyphosate and glyphosate-based herbicides in water, using nanotechnology
- De Almeida, Louise Kashiyavala Sophia
- Authors: De Almeida, Louise Kashiyavala Sophia
- Date: 2015
- Subjects: Water -- Glyphosate content , Aquatic herbicides -- South Africa , Aquatic herbicides -- Physiological effect , Nanotechnology , Invasive plants -- South Africa , Genetic toxicology , Thiazoles , Tetrazolium , Immunotoxicology , Colorimetry , Nanofibers
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4163 , http://hdl.handle.net/10962/d1019755
- Description: Glyphosate (N-phosphonomethylglycine) is an organophosphate compound which was developed by the Monsanto Company in 1971 and is the active ingredient found in several herbicide formulations. The use of glyphosate-based herbicides in South Africa for the control of alien invasive plants and weeds is well established, extensive and currently unregulated, which vastly increases the likelihood of glyphosate contamination in environmental water systems. Although the use of glyphosate-based herbicides is required for economic enhancement in industries such as agriculture, the presence of this compound in natural water systems presents a potential risk to human health. Glyphosate and glyphosate formulations were previously considered safe, however their toxicity has become a major focal point of research over recent years. The lack of monitoring protocols for pesticides in South Africa is primarily due to limited financial capacity and the lack of analytical techniques.
- Full Text:
- Date Issued: 2015
- Authors: De Almeida, Louise Kashiyavala Sophia
- Date: 2015
- Subjects: Water -- Glyphosate content , Aquatic herbicides -- South Africa , Aquatic herbicides -- Physiological effect , Nanotechnology , Invasive plants -- South Africa , Genetic toxicology , Thiazoles , Tetrazolium , Immunotoxicology , Colorimetry , Nanofibers
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4163 , http://hdl.handle.net/10962/d1019755
- Description: Glyphosate (N-phosphonomethylglycine) is an organophosphate compound which was developed by the Monsanto Company in 1971 and is the active ingredient found in several herbicide formulations. The use of glyphosate-based herbicides in South Africa for the control of alien invasive plants and weeds is well established, extensive and currently unregulated, which vastly increases the likelihood of glyphosate contamination in environmental water systems. Although the use of glyphosate-based herbicides is required for economic enhancement in industries such as agriculture, the presence of this compound in natural water systems presents a potential risk to human health. Glyphosate and glyphosate formulations were previously considered safe, however their toxicity has become a major focal point of research over recent years. The lack of monitoring protocols for pesticides in South Africa is primarily due to limited financial capacity and the lack of analytical techniques.
- Full Text:
- Date Issued: 2015
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
A lignocellulolytic enzyme system for fruit waste degradation : commercial enzyme mixture synergy and bioreactor design
- Authors: Gama, Repson
- Date: 2014
- Subjects: Enzymes -- Biotechnology , Enzymes -- Industrial applications , Lignocellulose -- Biodegradation , Biomass energy , Biomass conversion , Biochemical engineering , Agricultural wastes as fuel
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4114 , http://hdl.handle.net/10962/d1013073
- Description: Studies into sources of alternative liquid transport fuel energy have identified agro-industrial wastes, which are lignocellulosic in nature, as a potential feedstock for biofuel production against the background of depleting nonrenewable fossil fuels. In South Africa, large quantities of apple and other fruit wastes, called pomace, are generated from fruit and juice industries. Apple pomace is a rich source of cellulose, pectin and hemicellulose, making it a potential target for utilisation as a lignocellulosic feedstock for biofuel and biorefinery chemical production. Lignocellulosic biomass is recalcitrant in nature and therefore its degradation requires the synergistic action of a number of enzymes such as cellulases, hemicellulases, pectinases and ligninases. Commercial enzyme cocktails, containing some of these enzymes, are available and can be used for apple pomace degradation. In this study, the degradation of apple pomace using commercial enzyme cocktails was investigated. The main focus was the optimisation of the release of sugar monomers that could potentially be used for biofuel and biorefinery chemical production. There is no or little information reported in literature on the enzymatic degradation of fruit waste using commercial enzyme mixtures. This study first focused on the characterisation of the substrate (apple pomace) and the commercial enzyme cocktails. Apple pomace was found to contain mainly glucose, galacturonic acid, arabinose, galactose, lignin and low amounts of xylose and fructose. Three commercial enzyme cocktails were initially selected: Biocip Membrane, Viscozyme L (from Aspergillus aculeatus) and Celluclast 1.5L (a Trichoderma reesei ATCC 26921 cellulase preparation). The selection of the enzymes was based on activities declared by the manufacturers, cost and local availability. The enzymes were screened based on their synergistic cooperation in the degradation of apple pomace and the main enzymes present in each cocktail. Viscozyme L and Celluclast 1.5L, in a 50:50 ratio, resulted in the best degree of synergy (1.6) compared to any other combination. The enzyme ratios were determined on Viscozyme L and Celluclast 1.5L based on the protein ratio. Enzyme activity was determined as glucose equivalents using the dinitrosalicylic acid (DNS) method. Sugar monomers were determined using Megazyme assay kits. There is limited information available on the enzymes present in the commercial enzyme cocktails. Therefore, the main enzymes present in Viscozyme L and Celluclast 1.5L were identified using different substrates, each targeted for a specific enzyme and activity. Characterisation of the enzyme mixtures revealed a large number of enzymes required for apple pomace degradation and these included cellulases, pectinases, xylanases, arabinases and mannanases in different proportions. Viscozyme L contained mainly pectinases and hemicellulases, while Celluclast 1.5L displayed largely cellulase and xylanase activity, hence the high degree of synergy reported. The temperature optimum was 50ºC for both enzyme mixtures and pH optima were observed at pH 5.0 and pH 3.0 for Viscozyme L and Celluclast 1.5L, respectively. At 37ºC and pH 5.0, the enzymes retained more that 90% activity after 15 days of incubation, allowing the enzymes to be used together with less energy input. The enzymes were further characterised by determining the effect of various compounds, such as alcohols, sugars, phenolic compounds and metal ions at various concentrations on the activity of the enzymes during apple pomace hydrolysis. Apart from lignin, which had almost no effect on enzyme activity, all the compounds caused inhibition of the enzymes to varying degrees. The most inhibitory compounds were some organic acids and metal ions, as well as cellobiose and xylobiose. Using the best ratio for Viscozyme L and Celluclast 1.5L (50:50) for the hydrolysis of apple pomace, it was observed that synergy was highest at the initial stages of hydrolysis and decreased over time, though the sugar concentration increased. The type of synergy for optimal apple pomace hydrolysis was found to be simultaneous. There was no synergy observed between Viscozyme L and Celluclast 1.5L with ligninases - laccase, lignin peroxidase and manganese peroxidase. Hydrolysing apple pomace with ligninases prior to addition of Viscozyme L and Celluclast 1.5L did not improve degradation of the substrate. Immobilisation of the enzyme mixtures on different supports was performed with the aim of increasing stability and enabling reuse of the enzymes. Immobilisation methods were selected based on the chemical properties of the supports, availability, cost and applicability on heterogeneous and insoluble substrate like apple pomace. These methods included crosslinked enzyme aggregates (CLEAs), immobilisation on various supports such as nylon mesh, nylon beads, sodium alginate beads, chitin and silica gel beads. The immobilisation strategies were unsuccessful, mainly due to the low percentage of immobilisation of the enzyme on the matrix and loss of activity of the immobilised enzyme. Free enzymes were therefore used for the remainder of the study. Hydrolysis conditions for apple pomace degradation were optimised using different temperatures and buffer systems in 1 L volumes mixed with compressed air. Hydrolysis at room temperature, using an unbuffered system, gave a better performance as compared to a buffered system. Reactors operated in batch mode performed better (4.2 g/L (75% yield) glucose and 16.8 g/L (75%) reducing sugar) than fed-batch reactors (3.2 g/L (66%) glucose and 14.6 g/L (72.7% yield) reducing sugar) over 100 h using Viscozyme L and Celluclast 1.5L. Supplementation of β- glucosidase activity in Viscozyme L and Celluclast 1.5L with Novozyme 188 resulted in a doubling of the amount of glucose released. The main products released from apple pomace hydrolysis were galacturonic acid, glucose and arabinose and low amounts of galactose and xylose. These products are potential raw materials for biofuel and biorefinery chemical production. An artificial neural network (ANN) model was successfully developed and used for predicting the optimum conditions for apple pomace hydrolysis using Celluclast 1.5L, Viscozyme L and Novozyme 188. Four main conditions that affect apple pomace hydrolysis were selected, namely temperature, initial pH, enzyme loading and substrate loading, which were taken as inputs. The glucose and reducing sugars released as a result of each treatment and their combinations were taken as outputs for 1–100 h. An ANN with 20, 20 and 6 neurons in the first, second and third hidden layers, respectively, was constructed. The performance and predictive ability of the ANN was good, with a R² of 0.99 and a small mean square error (MSE). New data was successfully predicted and simulated. Optimal hydrolysis conditions predicted by ANN for apple pomace hydrolysis were at 30% substrate (wet w/v) and an enzyme loading of 0.5 mg/g and 0.2 mg/mL of substrate for glucose and reducing sugar, respectively, giving sugar concentrations of 6.5 mg/mL and 28.9 mg/mL for glucose and reducing sugar, respectively. ANN showed that enzyme and substrate loadings were the most important factors for the hydrolysis of apple pomace.
- Full Text:
- Date Issued: 2014
- Authors: Gama, Repson
- Date: 2014
- Subjects: Enzymes -- Biotechnology , Enzymes -- Industrial applications , Lignocellulose -- Biodegradation , Biomass energy , Biomass conversion , Biochemical engineering , Agricultural wastes as fuel
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4114 , http://hdl.handle.net/10962/d1013073
- Description: Studies into sources of alternative liquid transport fuel energy have identified agro-industrial wastes, which are lignocellulosic in nature, as a potential feedstock for biofuel production against the background of depleting nonrenewable fossil fuels. In South Africa, large quantities of apple and other fruit wastes, called pomace, are generated from fruit and juice industries. Apple pomace is a rich source of cellulose, pectin and hemicellulose, making it a potential target for utilisation as a lignocellulosic feedstock for biofuel and biorefinery chemical production. Lignocellulosic biomass is recalcitrant in nature and therefore its degradation requires the synergistic action of a number of enzymes such as cellulases, hemicellulases, pectinases and ligninases. Commercial enzyme cocktails, containing some of these enzymes, are available and can be used for apple pomace degradation. In this study, the degradation of apple pomace using commercial enzyme cocktails was investigated. The main focus was the optimisation of the release of sugar monomers that could potentially be used for biofuel and biorefinery chemical production. There is no or little information reported in literature on the enzymatic degradation of fruit waste using commercial enzyme mixtures. This study first focused on the characterisation of the substrate (apple pomace) and the commercial enzyme cocktails. Apple pomace was found to contain mainly glucose, galacturonic acid, arabinose, galactose, lignin and low amounts of xylose and fructose. Three commercial enzyme cocktails were initially selected: Biocip Membrane, Viscozyme L (from Aspergillus aculeatus) and Celluclast 1.5L (a Trichoderma reesei ATCC 26921 cellulase preparation). The selection of the enzymes was based on activities declared by the manufacturers, cost and local availability. The enzymes were screened based on their synergistic cooperation in the degradation of apple pomace and the main enzymes present in each cocktail. Viscozyme L and Celluclast 1.5L, in a 50:50 ratio, resulted in the best degree of synergy (1.6) compared to any other combination. The enzyme ratios were determined on Viscozyme L and Celluclast 1.5L based on the protein ratio. Enzyme activity was determined as glucose equivalents using the dinitrosalicylic acid (DNS) method. Sugar monomers were determined using Megazyme assay kits. There is limited information available on the enzymes present in the commercial enzyme cocktails. Therefore, the main enzymes present in Viscozyme L and Celluclast 1.5L were identified using different substrates, each targeted for a specific enzyme and activity. Characterisation of the enzyme mixtures revealed a large number of enzymes required for apple pomace degradation and these included cellulases, pectinases, xylanases, arabinases and mannanases in different proportions. Viscozyme L contained mainly pectinases and hemicellulases, while Celluclast 1.5L displayed largely cellulase and xylanase activity, hence the high degree of synergy reported. The temperature optimum was 50ºC for both enzyme mixtures and pH optima were observed at pH 5.0 and pH 3.0 for Viscozyme L and Celluclast 1.5L, respectively. At 37ºC and pH 5.0, the enzymes retained more that 90% activity after 15 days of incubation, allowing the enzymes to be used together with less energy input. The enzymes were further characterised by determining the effect of various compounds, such as alcohols, sugars, phenolic compounds and metal ions at various concentrations on the activity of the enzymes during apple pomace hydrolysis. Apart from lignin, which had almost no effect on enzyme activity, all the compounds caused inhibition of the enzymes to varying degrees. The most inhibitory compounds were some organic acids and metal ions, as well as cellobiose and xylobiose. Using the best ratio for Viscozyme L and Celluclast 1.5L (50:50) for the hydrolysis of apple pomace, it was observed that synergy was highest at the initial stages of hydrolysis and decreased over time, though the sugar concentration increased. The type of synergy for optimal apple pomace hydrolysis was found to be simultaneous. There was no synergy observed between Viscozyme L and Celluclast 1.5L with ligninases - laccase, lignin peroxidase and manganese peroxidase. Hydrolysing apple pomace with ligninases prior to addition of Viscozyme L and Celluclast 1.5L did not improve degradation of the substrate. Immobilisation of the enzyme mixtures on different supports was performed with the aim of increasing stability and enabling reuse of the enzymes. Immobilisation methods were selected based on the chemical properties of the supports, availability, cost and applicability on heterogeneous and insoluble substrate like apple pomace. These methods included crosslinked enzyme aggregates (CLEAs), immobilisation on various supports such as nylon mesh, nylon beads, sodium alginate beads, chitin and silica gel beads. The immobilisation strategies were unsuccessful, mainly due to the low percentage of immobilisation of the enzyme on the matrix and loss of activity of the immobilised enzyme. Free enzymes were therefore used for the remainder of the study. Hydrolysis conditions for apple pomace degradation were optimised using different temperatures and buffer systems in 1 L volumes mixed with compressed air. Hydrolysis at room temperature, using an unbuffered system, gave a better performance as compared to a buffered system. Reactors operated in batch mode performed better (4.2 g/L (75% yield) glucose and 16.8 g/L (75%) reducing sugar) than fed-batch reactors (3.2 g/L (66%) glucose and 14.6 g/L (72.7% yield) reducing sugar) over 100 h using Viscozyme L and Celluclast 1.5L. Supplementation of β- glucosidase activity in Viscozyme L and Celluclast 1.5L with Novozyme 188 resulted in a doubling of the amount of glucose released. The main products released from apple pomace hydrolysis were galacturonic acid, glucose and arabinose and low amounts of galactose and xylose. These products are potential raw materials for biofuel and biorefinery chemical production. An artificial neural network (ANN) model was successfully developed and used for predicting the optimum conditions for apple pomace hydrolysis using Celluclast 1.5L, Viscozyme L and Novozyme 188. Four main conditions that affect apple pomace hydrolysis were selected, namely temperature, initial pH, enzyme loading and substrate loading, which were taken as inputs. The glucose and reducing sugars released as a result of each treatment and their combinations were taken as outputs for 1–100 h. An ANN with 20, 20 and 6 neurons in the first, second and third hidden layers, respectively, was constructed. The performance and predictive ability of the ANN was good, with a R² of 0.99 and a small mean square error (MSE). New data was successfully predicted and simulated. Optimal hydrolysis conditions predicted by ANN for apple pomace hydrolysis were at 30% substrate (wet w/v) and an enzyme loading of 0.5 mg/g and 0.2 mg/mL of substrate for glucose and reducing sugar, respectively, giving sugar concentrations of 6.5 mg/mL and 28.9 mg/mL for glucose and reducing sugar, respectively. ANN showed that enzyme and substrate loadings were the most important factors for the hydrolysis of apple pomace.
- Full Text:
- Date Issued: 2014
The large scale bioinformatics analysis of auxiliary activity family 9 enzymes
- Authors: Moses, Vuyani
- Date: 2014
- Subjects: Bioinformatics -- Analysis , Cellulose -- Biodegradation , Biomass energy
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4145 , http://hdl.handle.net/10962/d1016356
- Description: Biofuels have been proposed to be a suitable replacement to the already depleting fossil fuels. The complex structures of plant biomasses present a challenge the production of biofuels due to recalcitrance. The complex cellulose structure and hydrogen bonding between repeat units of cellulose is believed to be a major contributor to the recalcitrance of cellulose. Fungal organisms come equipped with various oxidative enzymes involved in degradation of plant biomass. The exact mechanism of cellulose degradation remains elusive. The GH61 is a group of proteins which are PMOs. GH61 sequences where previously described as endoglucanases due to weak endoglucanase activity. These enzymes were later found not possess any enzyme activity of their own however they could enhance the activity of other cellulose degrading enzymes. As a result reclassification of these enzymes as AA9 has been implemented. AA9 proteins have been reported to share structural homology with the bacterial AA10 group of enzymes. Based on cleavage products that are produced when AA9 proteins interact with cellulose, AA9 proteins have been grouped into three types. To date the exact mechanism and the sequence and structural basis for differentiating between the various AA9 types remains unknown. Using various bionformatic techniques sequence and structural elements were identified for distinguishing between the AA9 types. A large dataset of sequences was obtained from the Pfam database from UNIPROT entries. Due to high divergence of AA9 sequences, a smaller dataset with the more divergent sequences removed was created. The inclusion of the reference sequences to the data set was done to observe which sequences belong to a certain type. Phylogenetic analysis was able to group AA9 proteins into three distinct groups. MSA and motif analysis revealed that the N-Terminus of these proteins is mostly responsible for type specificity. Structural analysis of AA9 PDB structures and homology models allowed the effect of physicochemical properties to be gauged structurally. The presence of 310 helices and aromatic residues the surface of AA9 sequences is an observation which still warrants further investigation.
- Full Text:
- Date Issued: 2014
- Authors: Moses, Vuyani
- Date: 2014
- Subjects: Bioinformatics -- Analysis , Cellulose -- Biodegradation , Biomass energy
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4145 , http://hdl.handle.net/10962/d1016356
- Description: Biofuels have been proposed to be a suitable replacement to the already depleting fossil fuels. The complex structures of plant biomasses present a challenge the production of biofuels due to recalcitrance. The complex cellulose structure and hydrogen bonding between repeat units of cellulose is believed to be a major contributor to the recalcitrance of cellulose. Fungal organisms come equipped with various oxidative enzymes involved in degradation of plant biomass. The exact mechanism of cellulose degradation remains elusive. The GH61 is a group of proteins which are PMOs. GH61 sequences where previously described as endoglucanases due to weak endoglucanase activity. These enzymes were later found not possess any enzyme activity of their own however they could enhance the activity of other cellulose degrading enzymes. As a result reclassification of these enzymes as AA9 has been implemented. AA9 proteins have been reported to share structural homology with the bacterial AA10 group of enzymes. Based on cleavage products that are produced when AA9 proteins interact with cellulose, AA9 proteins have been grouped into three types. To date the exact mechanism and the sequence and structural basis for differentiating between the various AA9 types remains unknown. Using various bionformatic techniques sequence and structural elements were identified for distinguishing between the AA9 types. A large dataset of sequences was obtained from the Pfam database from UNIPROT entries. Due to high divergence of AA9 sequences, a smaller dataset with the more divergent sequences removed was created. The inclusion of the reference sequences to the data set was done to observe which sequences belong to a certain type. Phylogenetic analysis was able to group AA9 proteins into three distinct groups. MSA and motif analysis revealed that the N-Terminus of these proteins is mostly responsible for type specificity. Structural analysis of AA9 PDB structures and homology models allowed the effect of physicochemical properties to be gauged structurally. The presence of 310 helices and aromatic residues the surface of AA9 sequences is an observation which still warrants further investigation.
- Full Text:
- Date Issued: 2014
The effect of sewage effluent from De Beers marine diamond mining operations on the expression of cytochrome P450 (CYP1A) and vitellogenin (vtg)
- Authors: De Almeida, Louise
- Date: 2013-09-20
- Subjects: De Beers Consolidated Mines , Sewage disposal in rivers, lakes, etc. -- Namibia , Endocrine disrupting chemicals in water -- Namibia , Merluccius capensis -- Namibia , Merluccius -- Namibia , Water -- Pollution -- Namibia
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4096 , http://hdl.handle.net/10962/d1009440 , De Beers Consolidated Mines , Sewage disposal in rivers, lakes, etc. -- Namibia , Endocrine disrupting chemicals in water -- Namibia , Merluccius capensis -- Namibia , Merluccius -- Namibia , Water -- Pollution -- Namibia
- Description: Sewage effluents disposed into the marine environment from De Beers Marine Namibia diamond mining vessels have the potential to cause endocrine disruptive effects in marine organisms. Endocrine disruption refers to the alteration of the normal functioning of the endocrine system and various chemicals have the ability to mimic hormones, effecting endogenous hormone synthesis, transport, receptor interaction and intracellular signaling. The potential endocrine disruptive effects, caused by the release of different types of sewage effluents into the ocean, on fish species is a concern due to the commercial importance of fish species found in the mining area e.g. hake, sole, horse mackerel. Increased awareness of marine environmental degradation due to the presence of chemical contaminants has resulted in research being done on early warning systems, in the form of biomarkers. Cytochrome P450 monooxygenase 1A (CYP1A) and vitellogenin (vtg) are important proteins found in fish liver and blood, that have been used as biomarkers for the detection of pollutants in fish. CYP1A is a subfamily of the P450 superfamily of enzymes and catalyzes the oxidation, hydrolysis and reduction of exogenous and endogenous compounds (phase I reactions) and thus has the capacity to regulate the metabolism of several organic contaminants. CYP1A expression is altered by exposure to planar xenobiotic compounds e.g. polyaromatic hydrocarbons. Vtg is an important precursor for egg yolk proteins and plays a role in the growth and development of an oocyte. Expression of this protein is altered upon exposure to estrogenic compounds. The aim of this project was to isolate CYP1A from fish liver by differential centrifugation and optimize conditions for the CYP1A-mediated ethoxyresorufin-Odeethylase (EROD) assay and western blot analysis (to assess CYP1A expression). Another aim of this study was to evaluate the potential effects of biologically disruptive chemicals from sewage effluents, discharged into the marine environment, on the expression of CYP1A in two species of hake, Merluccius capensis and M. paradoxus (Cape hake). CYP1A in Cape hake is approximately a 60 kDa protein and the highest EROD activity was detected in the microsomal fraction after differential centrifugation. Optimal EROD assay conditions were observed at pH 7.5, a temperature of 25 °C, 10 μl of sample and a reaction time of 30 seconds. Enzyme stability assays indicated a drastic decrease in enzyme activity after 30 seconds. The EROD assay was not NADPH dependent but was limited by NADPH supply, with an increase of 300% in EROD activity being observed with the addition of 0.1 M exogenous NADPH. The addition of dicumarol (40 μM), a phase II enzyme inhibitor, showed a 232% increase in EROD activity. This is because dicumarol inhibited enzymes with the capacity to metabolize the product (resorufin) of the EROD reaction. With regard to western blot analysis, the optimal primary (rabbit antifish CYP1A peptide) and secondary (anti-mouse/rabbit antibody-horseradish peroxidase conjugate (POD)) antibody dilutions were determined to be 1:1000 and 1:5000, respectively. The comparison of CYP1A expression in Cape hake samples from De Beers Marine mining area and reference sites showed higher EROD activity (16.29 ± 0.91 pmol/min) in fish samples from the mining area in comparison to the reference site (10.42 ± 2.65 pmol/min). Western blot analysis was in agreement with the EROD assay results and a higher CYP1A expression was observed in fish from the mining sites. The increased CYP1A expression observed in fish from the mining area is not definitively an indication of a pollutant effect in the environment, as several environmental and biological factors (e.g. photoperiod and age) must also be considered before reaching this conclusion. Another aim of this study was to purify vtg from Cape hake blood samples. Cape hake vtg was purified from fish plasma by selective precipitation with MgCl2 and EDTA. Precipitated sample was subjected to anion exchange chromatography using fast protein liquid chromatography (FPLC). Vtg eluted as two broad peaks and had a molecular weight above 200 kDa. SDS-PAGE analysis also resolved smaller molecular weight proteins below 70 kDa, which were thought to be vitellogenin cleavage proteins, lipovitellin and phosphovitins. Western blot analysis was performed; however, it did not produce any conclusive results. The purification of vtg enables further studies in characterizing this protein and developing assay aimed at detecting estrogenic pollutants in the marine environment
- Full Text:
- Authors: De Almeida, Louise
- Date: 2013-09-20
- Subjects: De Beers Consolidated Mines , Sewage disposal in rivers, lakes, etc. -- Namibia , Endocrine disrupting chemicals in water -- Namibia , Merluccius capensis -- Namibia , Merluccius -- Namibia , Water -- Pollution -- Namibia
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4096 , http://hdl.handle.net/10962/d1009440 , De Beers Consolidated Mines , Sewage disposal in rivers, lakes, etc. -- Namibia , Endocrine disrupting chemicals in water -- Namibia , Merluccius capensis -- Namibia , Merluccius -- Namibia , Water -- Pollution -- Namibia
- Description: Sewage effluents disposed into the marine environment from De Beers Marine Namibia diamond mining vessels have the potential to cause endocrine disruptive effects in marine organisms. Endocrine disruption refers to the alteration of the normal functioning of the endocrine system and various chemicals have the ability to mimic hormones, effecting endogenous hormone synthesis, transport, receptor interaction and intracellular signaling. The potential endocrine disruptive effects, caused by the release of different types of sewage effluents into the ocean, on fish species is a concern due to the commercial importance of fish species found in the mining area e.g. hake, sole, horse mackerel. Increased awareness of marine environmental degradation due to the presence of chemical contaminants has resulted in research being done on early warning systems, in the form of biomarkers. Cytochrome P450 monooxygenase 1A (CYP1A) and vitellogenin (vtg) are important proteins found in fish liver and blood, that have been used as biomarkers for the detection of pollutants in fish. CYP1A is a subfamily of the P450 superfamily of enzymes and catalyzes the oxidation, hydrolysis and reduction of exogenous and endogenous compounds (phase I reactions) and thus has the capacity to regulate the metabolism of several organic contaminants. CYP1A expression is altered by exposure to planar xenobiotic compounds e.g. polyaromatic hydrocarbons. Vtg is an important precursor for egg yolk proteins and plays a role in the growth and development of an oocyte. Expression of this protein is altered upon exposure to estrogenic compounds. The aim of this project was to isolate CYP1A from fish liver by differential centrifugation and optimize conditions for the CYP1A-mediated ethoxyresorufin-Odeethylase (EROD) assay and western blot analysis (to assess CYP1A expression). Another aim of this study was to evaluate the potential effects of biologically disruptive chemicals from sewage effluents, discharged into the marine environment, on the expression of CYP1A in two species of hake, Merluccius capensis and M. paradoxus (Cape hake). CYP1A in Cape hake is approximately a 60 kDa protein and the highest EROD activity was detected in the microsomal fraction after differential centrifugation. Optimal EROD assay conditions were observed at pH 7.5, a temperature of 25 °C, 10 μl of sample and a reaction time of 30 seconds. Enzyme stability assays indicated a drastic decrease in enzyme activity after 30 seconds. The EROD assay was not NADPH dependent but was limited by NADPH supply, with an increase of 300% in EROD activity being observed with the addition of 0.1 M exogenous NADPH. The addition of dicumarol (40 μM), a phase II enzyme inhibitor, showed a 232% increase in EROD activity. This is because dicumarol inhibited enzymes with the capacity to metabolize the product (resorufin) of the EROD reaction. With regard to western blot analysis, the optimal primary (rabbit antifish CYP1A peptide) and secondary (anti-mouse/rabbit antibody-horseradish peroxidase conjugate (POD)) antibody dilutions were determined to be 1:1000 and 1:5000, respectively. The comparison of CYP1A expression in Cape hake samples from De Beers Marine mining area and reference sites showed higher EROD activity (16.29 ± 0.91 pmol/min) in fish samples from the mining area in comparison to the reference site (10.42 ± 2.65 pmol/min). Western blot analysis was in agreement with the EROD assay results and a higher CYP1A expression was observed in fish from the mining sites. The increased CYP1A expression observed in fish from the mining area is not definitively an indication of a pollutant effect in the environment, as several environmental and biological factors (e.g. photoperiod and age) must also be considered before reaching this conclusion. Another aim of this study was to purify vtg from Cape hake blood samples. Cape hake vtg was purified from fish plasma by selective precipitation with MgCl2 and EDTA. Precipitated sample was subjected to anion exchange chromatography using fast protein liquid chromatography (FPLC). Vtg eluted as two broad peaks and had a molecular weight above 200 kDa. SDS-PAGE analysis also resolved smaller molecular weight proteins below 70 kDa, which were thought to be vitellogenin cleavage proteins, lipovitellin and phosphovitins. Western blot analysis was performed; however, it did not produce any conclusive results. The purification of vtg enables further studies in characterizing this protein and developing assay aimed at detecting estrogenic pollutants in the marine environment
- Full Text: