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
Development & evaluation of modified lignocellulose-clinoptilolite composites for water treatment
- Authors: Vala, Mavula Kikwe Remy
- Date: 2012-12
- Subjects: Lignocellulose , Lignocellulose -- Biotechnology
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/24521 , vital:63051
- Description: Municipalities, mining, textile and many other industries release wastewater into water bodies. Thus, the entire ecosystem (biota and abiota) including drinking water is affected by polluted effluents. The growing environmental concern over water pollution (due to inorganic and persistent organic compounds) attracts a significant amount of research in the removal of pollutants from water. In this study, lignocellulose and clinoptilolite were modified for the preparation of composites, with high adsorption properties, suitable for the removal of pollutants. Grass (Kikuyu grass) material was first treated with boiling water in order to remove soluble compounds and then with sulfuric acid in order to free functional groups within lignocellulose. The lignocellulose obtained was then chemically modified with three different siloxanes (3-aminopropyl-terminated poly (di)methylsiloxanes) of different molecular weights. For clinoptilolite, impurities were removed by reflux in hydrochloric acid before chemical modification with siloxanes. Grafting of siloxanes onto lignocellulose and clinoptilolite as well as the preparation of composites were successfully achieved by means of dibutyltin dilaurate (catalyst) after reflux under nitrogen. The modified materials were characterized by FT-IR, XRD, SEM and TGA and results confirmed successful modification of the materials. Solid state 29Si and 13C NMR were used to investigate the nature of the composite prepared with siloxane NH40D (CNH40D). The investigation revealed a possible bond between the modified lignocellulose and the modified clinoptilolite in the composite. The sorptive and/or ion exchange properties of the materials prepared for the removal of pollutants from water were then investigated. Phenol red, used motor (engine) oil and cyanide were used (with regard to textile, oil spill and gold mining effluents respectively) to simulate water pollution in the laboratory. It was found that adsorption properties of lignocellulose were significantly increased after sulfuric acid treatment, suggesting the availability of lignocellulose functional groups as adsorption sites. When further modified with siloxanes, lignocellulose showed less efficiency in adsorbing phenol red. The general mechanism of phenol red uptake onto lignocellulose and clinoptilolite modified with siloxane or composites was: rapid initial adsorption, slow uptake, small rate increase and then equilibrium. The mechanism of phenol red uptake could be well represented by the pseudo second-order kinetic model with equilibrium being reached after a period of time, ranging between 1-5 hours. The linear Langmuir model was the best model for describing adsorption of phenol red onto lignocellulose modified with siloxanes and composites while the Freundlich model appeared to be best for clinoptilolite modified with siloxanes. The general mechanism of used motor oil uptake onto lignocellulose and clinoptilolite modified with siloxane or composites was: rapid uptake, equilibrium and the process occurs over a short period (10 min). The pseudo second-order kinetic model appeared to be the best representation of this adsorption. The linear Langmuir isotherms are the best fitted model for used motor oil uptake onto the adsorbents prepared. Adsorption of cyanide occurred very quickly (10 to 30 min). For lignocellulose and clinoptilolite modified with siloxanes, desorption occurred soon after adsorption and thus no kinetic model nor isotherms of adsorption were deduced. However, adsorption of cyanide onto composites could be represented by the pseudo second-order kinetic model. Nanofibres were fabricated by electrospinning of the modified lignocellulose and composites by blending them with PAN in a solvent mixture of DMF-DMSO. Nanofiltration was achieved by packing the nanofibres prepared into a pipette and filtering polluted water. Nanofiltration was assessed by measurement of the turbidity of water which dropped from 63 NTU for polluted water to 3.06 NTU for filtered water. , Thesis (PhD) -- Faculty of Science and Agriculture, 2012
- Full Text:
- Date Issued: 2012-12
- Authors: Vala, Mavula Kikwe Remy
- Date: 2012-12
- Subjects: Lignocellulose , Lignocellulose -- Biotechnology
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/24521 , vital:63051
- Description: Municipalities, mining, textile and many other industries release wastewater into water bodies. Thus, the entire ecosystem (biota and abiota) including drinking water is affected by polluted effluents. The growing environmental concern over water pollution (due to inorganic and persistent organic compounds) attracts a significant amount of research in the removal of pollutants from water. In this study, lignocellulose and clinoptilolite were modified for the preparation of composites, with high adsorption properties, suitable for the removal of pollutants. Grass (Kikuyu grass) material was first treated with boiling water in order to remove soluble compounds and then with sulfuric acid in order to free functional groups within lignocellulose. The lignocellulose obtained was then chemically modified with three different siloxanes (3-aminopropyl-terminated poly (di)methylsiloxanes) of different molecular weights. For clinoptilolite, impurities were removed by reflux in hydrochloric acid before chemical modification with siloxanes. Grafting of siloxanes onto lignocellulose and clinoptilolite as well as the preparation of composites were successfully achieved by means of dibutyltin dilaurate (catalyst) after reflux under nitrogen. The modified materials were characterized by FT-IR, XRD, SEM and TGA and results confirmed successful modification of the materials. Solid state 29Si and 13C NMR were used to investigate the nature of the composite prepared with siloxane NH40D (CNH40D). The investigation revealed a possible bond between the modified lignocellulose and the modified clinoptilolite in the composite. The sorptive and/or ion exchange properties of the materials prepared for the removal of pollutants from water were then investigated. Phenol red, used motor (engine) oil and cyanide were used (with regard to textile, oil spill and gold mining effluents respectively) to simulate water pollution in the laboratory. It was found that adsorption properties of lignocellulose were significantly increased after sulfuric acid treatment, suggesting the availability of lignocellulose functional groups as adsorption sites. When further modified with siloxanes, lignocellulose showed less efficiency in adsorbing phenol red. The general mechanism of phenol red uptake onto lignocellulose and clinoptilolite modified with siloxane or composites was: rapid initial adsorption, slow uptake, small rate increase and then equilibrium. The mechanism of phenol red uptake could be well represented by the pseudo second-order kinetic model with equilibrium being reached after a period of time, ranging between 1-5 hours. The linear Langmuir model was the best model for describing adsorption of phenol red onto lignocellulose modified with siloxanes and composites while the Freundlich model appeared to be best for clinoptilolite modified with siloxanes. The general mechanism of used motor oil uptake onto lignocellulose and clinoptilolite modified with siloxane or composites was: rapid uptake, equilibrium and the process occurs over a short period (10 min). The pseudo second-order kinetic model appeared to be the best representation of this adsorption. The linear Langmuir isotherms are the best fitted model for used motor oil uptake onto the adsorbents prepared. Adsorption of cyanide occurred very quickly (10 to 30 min). For lignocellulose and clinoptilolite modified with siloxanes, desorption occurred soon after adsorption and thus no kinetic model nor isotherms of adsorption were deduced. However, adsorption of cyanide onto composites could be represented by the pseudo second-order kinetic model. Nanofibres were fabricated by electrospinning of the modified lignocellulose and composites by blending them with PAN in a solvent mixture of DMF-DMSO. Nanofiltration was achieved by packing the nanofibres prepared into a pipette and filtering polluted water. Nanofiltration was assessed by measurement of the turbidity of water which dropped from 63 NTU for polluted water to 3.06 NTU for filtered water. , Thesis (PhD) -- Faculty of Science and Agriculture, 2012
- Full Text:
- Date Issued: 2012-12
Synthesis of bioethanol from lignocellulosic materials: A focus on grass and waste paper as raw materials
- Authors: Vala, Mavula Kikwe
- Date: 2009-12
- Subjects: Ethanol as fuel , Biomass energy , Lignocellulose -- Biotechnology
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/24499 , vital:63049
- Description: Biofuels are currently recognized as not only a necessity, but an inevitable pathway to secure the planet future energy needs. Food crops have been used (so far) as the biomass for bioethanol and biodiesel production. This has increased concerns over food security and led to the search for diversification and alternative feedstocks for biofuel production. The use of lignocellulosic materials, the most abundant, low cost and easy feedstock to harvest for bioethanol purpose, involves challenging production processes. Several approaches have been used to facilitate the breakdown of the biopolymer structure to produce fermentable sugars that can be converted to ethanol. Most of the approaches have used high temperatures and pressures and have often led to the production of inhibitors of fermentation. In this study, lignocellulosic materials from grass and newsprint were investigated as sources of biomass for bioethanol production using a chemical route (sulfuric acid hydrolysis) which made use of temperatures below 100°C at normal atmospheric pressure. Fermentation of toxic lignocellulosic hydrolyzates was possible after the development of a method for inhibitors removal. The method used treated wood chips as a stationary phase in a chromatographic column to remove inhibitors. This method is expected to be extended to applications such as in municipal wastewater treatment. Sugar yields of 22.26 and 8.9 g/L of hydrolyzate; and an ethanol yield of 184.5 and 130.4 mg/mL of must were achieved for 5g grass and newsprint respectively using optimum conditions of 2percent H2SO4 at 97.5°C for grass and 0.5percent H2SO4 at 97.5°C for newsprint during the hydrolysis process. Pure cellulose was used as a control for the biomass where 254.1 g/L of fermentable sugars were recovered from soluble cellulose and the yield of ethanol was 201.8 mg/mL. , Thesis (MSc) -- Faculty of Science and Agriculture, 2009
- Full Text:
- Date Issued: 2009-12
- Authors: Vala, Mavula Kikwe
- Date: 2009-12
- Subjects: Ethanol as fuel , Biomass energy , Lignocellulose -- Biotechnology
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/24499 , vital:63049
- Description: Biofuels are currently recognized as not only a necessity, but an inevitable pathway to secure the planet future energy needs. Food crops have been used (so far) as the biomass for bioethanol and biodiesel production. This has increased concerns over food security and led to the search for diversification and alternative feedstocks for biofuel production. The use of lignocellulosic materials, the most abundant, low cost and easy feedstock to harvest for bioethanol purpose, involves challenging production processes. Several approaches have been used to facilitate the breakdown of the biopolymer structure to produce fermentable sugars that can be converted to ethanol. Most of the approaches have used high temperatures and pressures and have often led to the production of inhibitors of fermentation. In this study, lignocellulosic materials from grass and newsprint were investigated as sources of biomass for bioethanol production using a chemical route (sulfuric acid hydrolysis) which made use of temperatures below 100°C at normal atmospheric pressure. Fermentation of toxic lignocellulosic hydrolyzates was possible after the development of a method for inhibitors removal. The method used treated wood chips as a stationary phase in a chromatographic column to remove inhibitors. This method is expected to be extended to applications such as in municipal wastewater treatment. Sugar yields of 22.26 and 8.9 g/L of hydrolyzate; and an ethanol yield of 184.5 and 130.4 mg/mL of must were achieved for 5g grass and newsprint respectively using optimum conditions of 2percent H2SO4 at 97.5°C for grass and 0.5percent H2SO4 at 97.5°C for newsprint during the hydrolysis process. Pure cellulose was used as a control for the biomass where 254.1 g/L of fermentable sugars were recovered from soluble cellulose and the yield of ethanol was 201.8 mg/mL. , Thesis (MSc) -- Faculty of Science and Agriculture, 2009
- Full Text:
- Date Issued: 2009-12
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