The effect of various substrate pretreatment methods on the enzymatic degradability of a Eucalyptus sp. – a potential feedstock for producing fermentable sugars
- Authors: Thoresen, Mariska
- Date: 2021-04
- Subjects: Cellulose , Cellulase , Enzymes , Hydrolysis , Eucalyptus , Biomass energy
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/178580 , vital:42952 , DOI 10.21504/10962/178580
- Description: Over the past few years, there has been a global urgency to make the transition from conventional fossil fuels to renewable energy in order to meet the world’s increasing energy demands. Lignocellulosic biomass is currently at the forefront of intensive biofuel research due to its renewable nature. Lignocellulose valorisation into value added products such as bio-ethanol is a multistep process. The first step requires the biomass to go through a recalcitrance-reducing step (pretreatment), after which, enzymatic hydrolysis is required to break down the polysaccharides into simple sugars for fermentation. However, the recalcitrant structure of biomass and the low hydrolytic activities of the enzymes (glycoside hydrolases) on the substrate pose major technical and economic obstacles to the biomass conversion process. Since this process remains more expensive compared to petroleum-based fuels, lignocellulose has been intensively investigated in terms of its cost efficiency and effective decomposition. Although improvements to this process are ongoing, with some of the first commercial facilities producing cellulosic ethanol in 2013 and 2014, there is still a deep sense of urgency to render the facilities more economically feasible. Some important factors that determine the yield and rate of enzymatic hydrolysis include the type of enzymes used, enzyme recognition with the substrate, substrate composition and crystallinity. In this context, the major focus of this study was to develop a deeper understanding of how enzymes co-operate (synergise) at a molecular level using model substrates. This knowledge was then used as a basis for understanding how these enzymes synergise on more natural, complex substrates. This study specifically focused on how different pretreatments affect the chemical and structural properties of Eucalyptus. Lastly, we wanted to develop an effective method of enzyme recycling as a means to reduce the high process costs in biomass saccharification. Enhancing cellulose hydrolysis through enzyme synergy is essential for achieving higher hydrolysis rates, and numerous research efforts have focused on trying to elucidate the enzyme mechanisms required to design optimal enzyme cocktails. Despite the extensive amount of research carried out over the past few years, little is known about the enzymatic machinery underpinning the synergistic interactions between bacterial and fungal cellulases - neither is it understood why only a limited number of Cellobiohydrolases (CBHs) and Endoglucanases (EGs) exhibit synergism. Therefore, the first part of the study evaluated and compared the synergistic relationships between cellulases from different GH families and microbial sources (cross-synergism), i.e. cellobiohydrolase I (CBHI) from Hypocrea jecorina (Cel7A), CBHI from Trichoderma longibrachiatum (Cel7A), CBHI from Clostridium stercorarium (Cel48A), CBHII from a microbial source, CBHII from Clostridium thermocellum (Cel5A), endoglucanases (EG) from Bacillus amyloliquefaciens (Cel5A), EG from Thermotaga maritima (Cel5A), EG from Trichoderma reesei and a β-glucosidase from Aspergillus niger (Novozyme 188). An aim of this study was to provide insights into how the molecular mechanisms of different GH families govern synergism. The results showed that cellulases from different GH families and microbial sources exhibit different substrate specificities, which influence their synergistic interactions with other enzymes. Based on these observations, this study agreed with evidence that not all endo- and exo-cellulase interactions are synergistic, and that the extent of synergism is dependent on the composition of the cellulase systems from various sources and their compatibility in the cellulase cocktail. From the enzymes assessed in this study, an optimal enzyme cocktail (CelMix) was formulated which was composed of Egl 68%, Cel7A 17%, Cel6A 6%, βgl 9%. This method of screening for maximal compatibility between exo- and endo-cellulases from different GH families constituted a critical step towards a better understanding of the specific interactions between the enzymes of interest and how they synergise at the molecular level. Consequently, this information may assist in the design of improved synergistic cellulose-degrading cocktails for industrial-scale biomass degradation. The enzyme synergy studies provided a basis for the second part of this study, where it was assessed how these optimised enzyme cocktails would perform on complex substrates. It is well-known that lignocellulosic substrates are highly recalcitrant to microbial degradation, and although extensive research has been performed to understand biomass recalcitrance, the key features of biomass which hinder enzymatic hydrolysis are yet to be elucidated. In this study, we explored the effect of eight (8) different pretreatment methods on the enzymatic hydrolysis of a Eucalyptus sp. – a potential feedstock for biofuel production. This study was performed to increase our understanding of the relationship between biomass architecture and hydrolysis yield potential. Our results demonstrated that pretreatments induce changes at a micro- and macro-level in the cell walls of Eucalyptus, and that cellulose accessibility, cellulose crystallinity and the changes in the lignin S/G ratio played an important role in the enzymatic activity on the biomass. Thus, this study provided insight into important cellulose structural features related to biomass recalcitrance arising from various pretreatment methods, which may ultimately be used for the development of more efficient conversion technologies for better, more competitive bio-refineries. Lastly, a simple and yet effective method for desorbing the adsorbed cellulases on lignocellulosic substrates was established for better understanding cellulase adsorption and desorption in order to develop an effective enzyme recycling strategy. Various reagents were assessed to determine how effective they were in promoting enzyme desorption. Tris-HCl buffer (pH 9.0; 0.05 M) was the most effective method for promoting enzyme desorption and retained a substantial amount of hydrolytic activity after elution. However, minor activity loss was observed due to irreversible binding, which was further confirmed by SDS-PAGE analysis. With this information available, the feasibility of recovering the enzymes from the solid fraction after enzymatic hydrolysis of steam pretreated Eucalyptus was evaluated by two different approaches, i.e.: i) re-adsorption of the entire hydrolysed insoluble biomass fraction (no desorption) to fresh biomass (recycling approach 1 - RA1) and ii) re-adsorption of alkaline elution desorbed enzymes from hydrolysed biomass to fresh biomass (recycling approach 2 - RA2). The recycling performance of RA1 and RA2 achieved > 95% of the initial sugar liberation for three continuous rounds, whilst successfully reducing enzyme loadings by 50% and 40% for RA1 and RA2, respectively. This study presented a simple and effective pathway for improving the economic feasibility of fermentable sugar production for biofuels. In conclusion, this study has contributed to expanding our knowledge and providing new insights into factors relating to the biomass conversion process, including enzyme synergism, pretreatment methods and enzyme recycling strategies. Ultimately, the knowledge and information gained from this study can be used as a platform for the development of more efficient conversion technologies for better, more competitive bio-refineries. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-04
- Authors: Thoresen, Mariska
- Date: 2021-04
- Subjects: Cellulose , Cellulase , Enzymes , Hydrolysis , Eucalyptus , Biomass energy
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/178580 , vital:42952 , DOI 10.21504/10962/178580
- Description: Over the past few years, there has been a global urgency to make the transition from conventional fossil fuels to renewable energy in order to meet the world’s increasing energy demands. Lignocellulosic biomass is currently at the forefront of intensive biofuel research due to its renewable nature. Lignocellulose valorisation into value added products such as bio-ethanol is a multistep process. The first step requires the biomass to go through a recalcitrance-reducing step (pretreatment), after which, enzymatic hydrolysis is required to break down the polysaccharides into simple sugars for fermentation. However, the recalcitrant structure of biomass and the low hydrolytic activities of the enzymes (glycoside hydrolases) on the substrate pose major technical and economic obstacles to the biomass conversion process. Since this process remains more expensive compared to petroleum-based fuels, lignocellulose has been intensively investigated in terms of its cost efficiency and effective decomposition. Although improvements to this process are ongoing, with some of the first commercial facilities producing cellulosic ethanol in 2013 and 2014, there is still a deep sense of urgency to render the facilities more economically feasible. Some important factors that determine the yield and rate of enzymatic hydrolysis include the type of enzymes used, enzyme recognition with the substrate, substrate composition and crystallinity. In this context, the major focus of this study was to develop a deeper understanding of how enzymes co-operate (synergise) at a molecular level using model substrates. This knowledge was then used as a basis for understanding how these enzymes synergise on more natural, complex substrates. This study specifically focused on how different pretreatments affect the chemical and structural properties of Eucalyptus. Lastly, we wanted to develop an effective method of enzyme recycling as a means to reduce the high process costs in biomass saccharification. Enhancing cellulose hydrolysis through enzyme synergy is essential for achieving higher hydrolysis rates, and numerous research efforts have focused on trying to elucidate the enzyme mechanisms required to design optimal enzyme cocktails. Despite the extensive amount of research carried out over the past few years, little is known about the enzymatic machinery underpinning the synergistic interactions between bacterial and fungal cellulases - neither is it understood why only a limited number of Cellobiohydrolases (CBHs) and Endoglucanases (EGs) exhibit synergism. Therefore, the first part of the study evaluated and compared the synergistic relationships between cellulases from different GH families and microbial sources (cross-synergism), i.e. cellobiohydrolase I (CBHI) from Hypocrea jecorina (Cel7A), CBHI from Trichoderma longibrachiatum (Cel7A), CBHI from Clostridium stercorarium (Cel48A), CBHII from a microbial source, CBHII from Clostridium thermocellum (Cel5A), endoglucanases (EG) from Bacillus amyloliquefaciens (Cel5A), EG from Thermotaga maritima (Cel5A), EG from Trichoderma reesei and a β-glucosidase from Aspergillus niger (Novozyme 188). An aim of this study was to provide insights into how the molecular mechanisms of different GH families govern synergism. The results showed that cellulases from different GH families and microbial sources exhibit different substrate specificities, which influence their synergistic interactions with other enzymes. Based on these observations, this study agreed with evidence that not all endo- and exo-cellulase interactions are synergistic, and that the extent of synergism is dependent on the composition of the cellulase systems from various sources and their compatibility in the cellulase cocktail. From the enzymes assessed in this study, an optimal enzyme cocktail (CelMix) was formulated which was composed of Egl 68%, Cel7A 17%, Cel6A 6%, βgl 9%. This method of screening for maximal compatibility between exo- and endo-cellulases from different GH families constituted a critical step towards a better understanding of the specific interactions between the enzymes of interest and how they synergise at the molecular level. Consequently, this information may assist in the design of improved synergistic cellulose-degrading cocktails for industrial-scale biomass degradation. The enzyme synergy studies provided a basis for the second part of this study, where it was assessed how these optimised enzyme cocktails would perform on complex substrates. It is well-known that lignocellulosic substrates are highly recalcitrant to microbial degradation, and although extensive research has been performed to understand biomass recalcitrance, the key features of biomass which hinder enzymatic hydrolysis are yet to be elucidated. In this study, we explored the effect of eight (8) different pretreatment methods on the enzymatic hydrolysis of a Eucalyptus sp. – a potential feedstock for biofuel production. This study was performed to increase our understanding of the relationship between biomass architecture and hydrolysis yield potential. Our results demonstrated that pretreatments induce changes at a micro- and macro-level in the cell walls of Eucalyptus, and that cellulose accessibility, cellulose crystallinity and the changes in the lignin S/G ratio played an important role in the enzymatic activity on the biomass. Thus, this study provided insight into important cellulose structural features related to biomass recalcitrance arising from various pretreatment methods, which may ultimately be used for the development of more efficient conversion technologies for better, more competitive bio-refineries. Lastly, a simple and yet effective method for desorbing the adsorbed cellulases on lignocellulosic substrates was established for better understanding cellulase adsorption and desorption in order to develop an effective enzyme recycling strategy. Various reagents were assessed to determine how effective they were in promoting enzyme desorption. Tris-HCl buffer (pH 9.0; 0.05 M) was the most effective method for promoting enzyme desorption and retained a substantial amount of hydrolytic activity after elution. However, minor activity loss was observed due to irreversible binding, which was further confirmed by SDS-PAGE analysis. With this information available, the feasibility of recovering the enzymes from the solid fraction after enzymatic hydrolysis of steam pretreated Eucalyptus was evaluated by two different approaches, i.e.: i) re-adsorption of the entire hydrolysed insoluble biomass fraction (no desorption) to fresh biomass (recycling approach 1 - RA1) and ii) re-adsorption of alkaline elution desorbed enzymes from hydrolysed biomass to fresh biomass (recycling approach 2 - RA2). The recycling performance of RA1 and RA2 achieved > 95% of the initial sugar liberation for three continuous rounds, whilst successfully reducing enzyme loadings by 50% and 40% for RA1 and RA2, respectively. This study presented a simple and effective pathway for improving the economic feasibility of fermentable sugar production for biofuels. In conclusion, this study has contributed to expanding our knowledge and providing new insights into factors relating to the biomass conversion process, including enzyme synergism, pretreatment methods and enzyme recycling strategies. Ultimately, the knowledge and information gained from this study can be used as a platform for the development of more efficient conversion technologies for better, more competitive bio-refineries. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-04
The investigation of type-specific features of the copper coordinating AA9 proteins and their effect on the interaction with crystalline cellulose using molecular dynamics studies
- Authors: Moses, Vuyani
- Date: 2018
- Subjects: Copper proteins , Cellulose , Molecular dynamics , Cellulose -- Biodegradation , Bioinformatics
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/58327 , vital:27230
- Description: AA9 proteins are metallo-enzymes which are crucial for the early stages of cellulose degradation. AA9 proteins have been suggested to cleave glycosidic bonds linking cellulose through the use of their Cu2+ coordinating active site. AA9 proteins possess different regioselectivities depending on the resulting cleavage they form and as result, are grouped accordingly. Type 1 AA9 proteins cleave the C1 carbon of cellulose while Type 2 AA9 proteins cleave the C4 carbon and Type 3 AA9 proteins cleave either C1 or C4 carbons. The steric congestion of the AA9 active site has been proposed to be a contributor to the observed regioselectivity. As such, a bioinformatics characterisation of type-specific sequence and structural features was performed. Initially AA9 protein sequences were obtained from the Pfam database and multiple sequence alignment was performed. The sequences were phylogenetically characterised and sequences were grouped into their respective types and sub-groups were identified. A selection analysis was performed on AA9 LPMO types to determine the selective pressure acting on AA9 protein residues. Motif discovery was then performed to identify conserved sequence motifs in AA9 proteins. Once type-specific sequence features were identified structural mapping was performed to assess possible effects on substrate interaction. Physicochemical property analysis was also performed to assess biochemical differences between AA9 LPMO types. Molecular dynamics (MD) simulations were then employed to dynamically assess the consequences of the discovered type-specific features on AA9-cellulose interaction. Due to the absence of AA9 specific force field parameters MD simulations were not readily applicable. As a result, Potential Energy Surface (PES) scans were performed to evaluate the force field parameters for the AA9 active site using the PM6 semi empirical approach and least squares fitting. A Type 1 AA9 active site was constructed from the crystal structure 4B5Q, encompassing only the Cu2+ coordinating residues, the Cu2+ ion and two water residues. Due to the similarity in AA9 active sites, the Type force field parameters were validated on all three AA9 LPMO types. Two MD simulations for each AA9 LPMO types were conducted using two separate Lennard-Jones parameter sets. Once completed, the MD trajectories were analysed for various features including the RMSD, RMSF, radius of gyration, coordination during simulation, hydrogen bonding, secondary structure conservation and overall protein movement. Force field parameters were successfully evaluated and validated for AA9 proteins. MD simulations of AA9 proteins were able to reveal the presence of unique type-specific binding modes of AA9 active sites to cellulose. These binding modes were characterised by the presence of unique type-specific loops which were present in Type 2 and 3 AA9 proteins but not in Type 1 AA9 proteins. The loops were found to result in steric congestion that affects how the Cu2+ ion interacts with cellulose. As a result, Cu2+ binding to cellulose was observed for Type 1 and not Type 2 and 3 AA9 proteins. In this study force field parameters have been evaluated for the Type 1 active site of AA9 proteins and this parameters were evaluated on all three types and binding. Future work will focus on identifying the nature of the reactive oxygen species and performing QM/MM calculations to elucidate the reactive mechanism of all three AA9 LPMO types.
- Full Text:
- Date Issued: 2018
- Authors: Moses, Vuyani
- Date: 2018
- Subjects: Copper proteins , Cellulose , Molecular dynamics , Cellulose -- Biodegradation , Bioinformatics
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/58327 , vital:27230
- Description: AA9 proteins are metallo-enzymes which are crucial for the early stages of cellulose degradation. AA9 proteins have been suggested to cleave glycosidic bonds linking cellulose through the use of their Cu2+ coordinating active site. AA9 proteins possess different regioselectivities depending on the resulting cleavage they form and as result, are grouped accordingly. Type 1 AA9 proteins cleave the C1 carbon of cellulose while Type 2 AA9 proteins cleave the C4 carbon and Type 3 AA9 proteins cleave either C1 or C4 carbons. The steric congestion of the AA9 active site has been proposed to be a contributor to the observed regioselectivity. As such, a bioinformatics characterisation of type-specific sequence and structural features was performed. Initially AA9 protein sequences were obtained from the Pfam database and multiple sequence alignment was performed. The sequences were phylogenetically characterised and sequences were grouped into their respective types and sub-groups were identified. A selection analysis was performed on AA9 LPMO types to determine the selective pressure acting on AA9 protein residues. Motif discovery was then performed to identify conserved sequence motifs in AA9 proteins. Once type-specific sequence features were identified structural mapping was performed to assess possible effects on substrate interaction. Physicochemical property analysis was also performed to assess biochemical differences between AA9 LPMO types. Molecular dynamics (MD) simulations were then employed to dynamically assess the consequences of the discovered type-specific features on AA9-cellulose interaction. Due to the absence of AA9 specific force field parameters MD simulations were not readily applicable. As a result, Potential Energy Surface (PES) scans were performed to evaluate the force field parameters for the AA9 active site using the PM6 semi empirical approach and least squares fitting. A Type 1 AA9 active site was constructed from the crystal structure 4B5Q, encompassing only the Cu2+ coordinating residues, the Cu2+ ion and two water residues. Due to the similarity in AA9 active sites, the Type force field parameters were validated on all three AA9 LPMO types. Two MD simulations for each AA9 LPMO types were conducted using two separate Lennard-Jones parameter sets. Once completed, the MD trajectories were analysed for various features including the RMSD, RMSF, radius of gyration, coordination during simulation, hydrogen bonding, secondary structure conservation and overall protein movement. Force field parameters were successfully evaluated and validated for AA9 proteins. MD simulations of AA9 proteins were able to reveal the presence of unique type-specific binding modes of AA9 active sites to cellulose. These binding modes were characterised by the presence of unique type-specific loops which were present in Type 2 and 3 AA9 proteins but not in Type 1 AA9 proteins. The loops were found to result in steric congestion that affects how the Cu2+ ion interacts with cellulose. As a result, Cu2+ binding to cellulose was observed for Type 1 and not Type 2 and 3 AA9 proteins. In this study force field parameters have been evaluated for the Type 1 active site of AA9 proteins and this parameters were evaluated on all three types and binding. Future work will focus on identifying the nature of the reactive oxygen species and performing QM/MM calculations to elucidate the reactive mechanism of all three AA9 LPMO types.
- Full Text:
- Date Issued: 2018
Isolation of xylanolytic multi-enzyme complexes from Bacillus subtilis SJ01
- Authors: Jones, Sarah Melissa Jane
- Date: 2010
- Subjects: Bacillus subtilis , Xylans , Multienzyme complexes , Botanical chemistry , Cellulose , Hemicellulose , Polysaccharides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3974 , http://hdl.handle.net/10962/d1004033 , Bacillus subtilis , Xylans , Multienzyme complexes , Botanical chemistry , Cellulose , Hemicellulose , Polysaccharides
- Description: Cellulose and hemicellulose account for a large portion of the world‘s plant biomass. In nature, these polysaccharides are intertwined forming complex materials that require multiple enzymes to degrade them. Multi-enzyme complexes (MECs) consist of a number of enzymes working in close proximity and synergistically to degrade complex substrates with higher efficiency than individual enzymes. The cellulosome is a cellulolytic MEC produced by anaerobic bacteria that has been studied extensively since its discovery in 1983. The aim of this study was to purify a cellulolytic and/or hemicellulolytic MEC from an aerobic bacterium of the Bacillus genus. Several bacterial isolates were identified using morphological characteristics and 16S rDNA sequencing, and screened for their ability to degrade cellulose and xylan using a MEC. The isolate that produced a high molecular weight protein fraction with the greatest ability to degrade Avicel®, carboxymethyl cellulose (CMC) and birchwood xylan was identified as Bacillus subtilis SJ01. An optimised growth medium, consisting of vitamins, trace elements, birchwood xylan (as the carbon source), and yeast and ammonium sulphate (as the nitrogen sources), increased the production of CMCase and xylanase enzymes from this bacterium. The removal of a competing bacterial strain from the culture and the inhibition of proteases also increased enzyme activities. A growth curve of B. subtilis SJ01 indicated that xylanase production was highest in early stationary growth phase and thus 84 hours was chosen as the best cell harvesting time. To purify the MECs produced by B. subtilis SJ01 size-exclusion chromatography on a Sephacryl S-400 column was used. It was concluded that (for the purposes of this study) the best method of concentrating the culture supernatant prior to loading onto Sephacryl S-400 was the use of ultrafiltration with a 50 kDa cut-off membrane. Two MECs, named C1 and C2 of 371 and 267 kDa, respectively, were purified from the culture supernatant of B. subtilis SJ01. Electrophoretic analysis revealed that these MECs consisted of 16 and 18 subunits, respectively, 4 of which degraded birchwood xylan and 5 of which degraded oat spelt xylan. The MECs degraded xylan substrates (C1: 0.24 U/mg, C2: 0.14 U/mg birchwood xylan) with higher efficiency than cellulose substrates (C1: 0.002 U/mg, C2: 0.01 U/mg CMC), and could therefore be considered xylanosomes. Interestingly, the MECs did not bind to insoluble birchwood xylan or Avicel® and did not contain glycosylated proteins, which are common features of cellulosomes. This study is, therefore, important in revealing the presence of MECs that differ from the cellulosome and that may have particular application in industries requiring high xylanase activity, such as the paper and pulp industry. The abundant genetic information available on B. subtilis means that this organism could also be used for genetic engineering of cellulolytic/hemicellulolytic MECs.
- Full Text:
- Date Issued: 2010
- Authors: Jones, Sarah Melissa Jane
- Date: 2010
- Subjects: Bacillus subtilis , Xylans , Multienzyme complexes , Botanical chemistry , Cellulose , Hemicellulose , Polysaccharides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3974 , http://hdl.handle.net/10962/d1004033 , Bacillus subtilis , Xylans , Multienzyme complexes , Botanical chemistry , Cellulose , Hemicellulose , Polysaccharides
- Description: Cellulose and hemicellulose account for a large portion of the world‘s plant biomass. In nature, these polysaccharides are intertwined forming complex materials that require multiple enzymes to degrade them. Multi-enzyme complexes (MECs) consist of a number of enzymes working in close proximity and synergistically to degrade complex substrates with higher efficiency than individual enzymes. The cellulosome is a cellulolytic MEC produced by anaerobic bacteria that has been studied extensively since its discovery in 1983. The aim of this study was to purify a cellulolytic and/or hemicellulolytic MEC from an aerobic bacterium of the Bacillus genus. Several bacterial isolates were identified using morphological characteristics and 16S rDNA sequencing, and screened for their ability to degrade cellulose and xylan using a MEC. The isolate that produced a high molecular weight protein fraction with the greatest ability to degrade Avicel®, carboxymethyl cellulose (CMC) and birchwood xylan was identified as Bacillus subtilis SJ01. An optimised growth medium, consisting of vitamins, trace elements, birchwood xylan (as the carbon source), and yeast and ammonium sulphate (as the nitrogen sources), increased the production of CMCase and xylanase enzymes from this bacterium. The removal of a competing bacterial strain from the culture and the inhibition of proteases also increased enzyme activities. A growth curve of B. subtilis SJ01 indicated that xylanase production was highest in early stationary growth phase and thus 84 hours was chosen as the best cell harvesting time. To purify the MECs produced by B. subtilis SJ01 size-exclusion chromatography on a Sephacryl S-400 column was used. It was concluded that (for the purposes of this study) the best method of concentrating the culture supernatant prior to loading onto Sephacryl S-400 was the use of ultrafiltration with a 50 kDa cut-off membrane. Two MECs, named C1 and C2 of 371 and 267 kDa, respectively, were purified from the culture supernatant of B. subtilis SJ01. Electrophoretic analysis revealed that these MECs consisted of 16 and 18 subunits, respectively, 4 of which degraded birchwood xylan and 5 of which degraded oat spelt xylan. The MECs degraded xylan substrates (C1: 0.24 U/mg, C2: 0.14 U/mg birchwood xylan) with higher efficiency than cellulose substrates (C1: 0.002 U/mg, C2: 0.01 U/mg CMC), and could therefore be considered xylanosomes. Interestingly, the MECs did not bind to insoluble birchwood xylan or Avicel® and did not contain glycosylated proteins, which are common features of cellulosomes. This study is, therefore, important in revealing the presence of MECs that differ from the cellulosome and that may have particular application in industries requiring high xylanase activity, such as the paper and pulp industry. The abundant genetic information available on B. subtilis means that this organism could also be used for genetic engineering of cellulolytic/hemicellulolytic MECs.
- Full Text:
- Date Issued: 2010
An investigation into the synergistic association between the major Clostridium cellulovorans cellulosomal endoglucanase and two hemicellulases on plant cell wall degradation
- Authors: Beukes, Natasha
- Date: 2008
- Subjects: Clostridium , Cellulose , Hemicellulose , Cellulase , Biomass conversion , Biomass energy -- South Africa , Energy crops -- South Africa , Bagasse -- Biodegradation , Pineapple -- Biodegradation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3968 , http://hdl.handle.net/10962/d1004027 , Clostridium , Cellulose , Hemicellulose , Cellulase , Biomass conversion , Biomass energy -- South Africa , Energy crops -- South Africa , Bagasse -- Biodegradation , Pineapple -- Biodegradation
- Description: The cellulosome is a multimeric enzyme complex that has the ability to metabolise a wide variety of carbonaceous compounds. Cellulosomal composition may vary according to the microbe’s nutritional requirement and allows for the anaerobic degradation of complex substrates. The complex substrates of interest in this research study were sugarcane bagasse and pineapple fibre waste, as they represent two important lignocellulosic, South African agricultural crops. The effective degradation of complex plant biomass wastes may present a valuable source of renewable compounds for the production of a variety of biofuels, for example bioethanol, and a variety of biocomposites of industrial importance. The identification of renewable energy sources for the production of biofuels is becoming increasingly important, as a result of the rapid depletion of the fossil fuels that are traditionally used as energy sources. An effective means of completely degrading lignocellulose biomass still remains elusive due to the complex heterogeneity of the substrate structure, and the fact that the effective degradation of the substrate requires a consortium of enzymes. The cellulosome not only provides a variety of enzymes with varying specificities, but also promote a close proximity between the catalytic components (enzymes). The close proximity between the enzymes promotes the synergistic degradation of complex plant biomass for the production of valuable energy products. Previous synergy studies have focused predominantly on the synergistic associations between cellulases; however, the synergy between hemicellulases has occasionally been documented. This research project established the synergistic associations between two Clostridium cellulovorans hemicellulases that may be incorporated into the cellulosome and a cellulosomal endoglucanase that is conserved in all cellulosomes. This research study indicated that there was indeed a synergistic degradation of the complex plant biomass (sugarcane bagasse and pineapple fibre). The degrees of synergy and the ratio of the enzymes varied between the two complex substrates. The initial degradation of the bagasse required the presence of all the enzymes and proceeded at an enhanced rate under sulphidogenic conditions; however, there was a low production of fermentable sugars. The low quantity of fermentable sugars produced by the degradation of the bagasse may be related to the chemical composition of the substrate. The sugarcane contains a high percentage of lignin forming a protective layer around the holocellulose, thus the glycosidic bonds are shielded extensively from enzymatic attack. In comparison, the initial degradation of the pineapple fibre required the action of hemicellulases, and proceeded at an enhanced rate under sulphidogenic conditions. The initial degradation of the pineapple fibre produced a substantially larger quantity of fermentable sugars in comparison to the bagasse. The higher production of fermentable sugars from the degradation of the pineapple fibre may be explained by the fact that this substrate may have a lower percentage of lignin than the bagasse, thus allowing a larger percentage of the glycosidic bonds to be exposed to enzymatic attack. The data obtained also indicated that the glycosidic bonds from the hemicellulosic components of the pineapple fibre shielded the glycosidic bonds of the cellulose component. The identification of the chemical components of the different substrates may allow for the initial development of an ideal enzyme complex (designer cellulosome) with enzymes in an ideal ratio with optimal synergy that will effectively degrade the complex plant biomass substrate.
- Full Text:
- Date Issued: 2008
- Authors: Beukes, Natasha
- Date: 2008
- Subjects: Clostridium , Cellulose , Hemicellulose , Cellulase , Biomass conversion , Biomass energy -- South Africa , Energy crops -- South Africa , Bagasse -- Biodegradation , Pineapple -- Biodegradation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3968 , http://hdl.handle.net/10962/d1004027 , Clostridium , Cellulose , Hemicellulose , Cellulase , Biomass conversion , Biomass energy -- South Africa , Energy crops -- South Africa , Bagasse -- Biodegradation , Pineapple -- Biodegradation
- Description: The cellulosome is a multimeric enzyme complex that has the ability to metabolise a wide variety of carbonaceous compounds. Cellulosomal composition may vary according to the microbe’s nutritional requirement and allows for the anaerobic degradation of complex substrates. The complex substrates of interest in this research study were sugarcane bagasse and pineapple fibre waste, as they represent two important lignocellulosic, South African agricultural crops. The effective degradation of complex plant biomass wastes may present a valuable source of renewable compounds for the production of a variety of biofuels, for example bioethanol, and a variety of biocomposites of industrial importance. The identification of renewable energy sources for the production of biofuels is becoming increasingly important, as a result of the rapid depletion of the fossil fuels that are traditionally used as energy sources. An effective means of completely degrading lignocellulose biomass still remains elusive due to the complex heterogeneity of the substrate structure, and the fact that the effective degradation of the substrate requires a consortium of enzymes. The cellulosome not only provides a variety of enzymes with varying specificities, but also promote a close proximity between the catalytic components (enzymes). The close proximity between the enzymes promotes the synergistic degradation of complex plant biomass for the production of valuable energy products. Previous synergy studies have focused predominantly on the synergistic associations between cellulases; however, the synergy between hemicellulases has occasionally been documented. This research project established the synergistic associations between two Clostridium cellulovorans hemicellulases that may be incorporated into the cellulosome and a cellulosomal endoglucanase that is conserved in all cellulosomes. This research study indicated that there was indeed a synergistic degradation of the complex plant biomass (sugarcane bagasse and pineapple fibre). The degrees of synergy and the ratio of the enzymes varied between the two complex substrates. The initial degradation of the bagasse required the presence of all the enzymes and proceeded at an enhanced rate under sulphidogenic conditions; however, there was a low production of fermentable sugars. The low quantity of fermentable sugars produced by the degradation of the bagasse may be related to the chemical composition of the substrate. The sugarcane contains a high percentage of lignin forming a protective layer around the holocellulose, thus the glycosidic bonds are shielded extensively from enzymatic attack. In comparison, the initial degradation of the pineapple fibre required the action of hemicellulases, and proceeded at an enhanced rate under sulphidogenic conditions. The initial degradation of the pineapple fibre produced a substantially larger quantity of fermentable sugars in comparison to the bagasse. The higher production of fermentable sugars from the degradation of the pineapple fibre may be explained by the fact that this substrate may have a lower percentage of lignin than the bagasse, thus allowing a larger percentage of the glycosidic bonds to be exposed to enzymatic attack. The data obtained also indicated that the glycosidic bonds from the hemicellulosic components of the pineapple fibre shielded the glycosidic bonds of the cellulose component. The identification of the chemical components of the different substrates may allow for the initial development of an ideal enzyme complex (designer cellulosome) with enzymes in an ideal ratio with optimal synergy that will effectively degrade the complex plant biomass substrate.
- Full Text:
- Date Issued: 2008
Isolation of a Clostridium Beijerinckii sLM01 cellulosome and the effect of sulphide on anaerobic digestion
- Authors: Mayende, Lungisa
- Date: 2007
- Subjects: Cellulose , Clostridium , Cellulase , Sulfides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3973 , http://hdl.handle.net/10962/d1004032 , Cellulose , Clostridium , Cellulase , Sulfides
- Description: Cellulose is the most abundant and the most resistant and stable natural organic compound on earth. Enzyme hydrolysis is difficult because of its insolubility and heterogeneity. Some (anaerobic) microorganisms have overcome this by having a multienzyme system called the cellulosome. The aims of the study were to isolate a mesophilic Clostridium sp. from a biosulphidogenic bioreactor, to purify the cellulosome from this culture, to determine the cellulase and endoglucanase activities using Avicel and carboxymethylcellulose (CMC) as substrates and the dinitrosalicyclic (DNS) method. The organism was identified using 16S rDNA sequence analysis. The sequence obtained indicated that a strain of Clostridium beijerinckii was isolated. The cellulosome was purified from the putative C. beijerinckii sLM01 host culture using affinity chromatography purification and affinity digestion purification procedures. The cellulosomal and non-cellulosomal fractions of C. beijerinckii sLM01 were separated successfully, but the majority of the endoglucanase activity was lost during the Sepharose 4B chromatography step. These cellulosomal and non-cellulosomal fractions were characterised with regards to their pH and temperature optima and effector sensitivity. Increased additions of sulphide activated the cellulase activity of the cellulosomal and non-cellulosomal fractions up to 700 %, while increased additions of sulphate either increased the activity slightly or inhibited it dramatically, depending on the cellulosomal and non-cellulosomal fractions. Increased additions of cellobiose, glucose and acetate inhibited the cellulase and endoglucanase activities. pH optima of 5.0 and 7.5 were observed for cellulases and 5.0 for endoglucanases of the cellulosomal fraction. The noncellulosomal fraction exhibited a pH optimum of 7.5 for both cellulase and endoglucanase activities. Both fractions and enzymes exhibited a temperature optimum of 30 °C. The fundamental knowledge gained from the characterisation was applied to anaerobic digestion, where the effect of sulphide on the rate-limiting step was determined. Sulphide activated cellulase and endoglucanase activities and increased the % chemical oxygen demand (COD) removal rate. Levels of volatile fatty acids (VFAs) were higher in the bioreactor containing sulphide, substrate and C. beijerinckii. Sulphide therefore accelerated the rate-limiting step of anaerobic digestion.
- Full Text:
- Date Issued: 2007
- Authors: Mayende, Lungisa
- Date: 2007
- Subjects: Cellulose , Clostridium , Cellulase , Sulfides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3973 , http://hdl.handle.net/10962/d1004032 , Cellulose , Clostridium , Cellulase , Sulfides
- Description: Cellulose is the most abundant and the most resistant and stable natural organic compound on earth. Enzyme hydrolysis is difficult because of its insolubility and heterogeneity. Some (anaerobic) microorganisms have overcome this by having a multienzyme system called the cellulosome. The aims of the study were to isolate a mesophilic Clostridium sp. from a biosulphidogenic bioreactor, to purify the cellulosome from this culture, to determine the cellulase and endoglucanase activities using Avicel and carboxymethylcellulose (CMC) as substrates and the dinitrosalicyclic (DNS) method. The organism was identified using 16S rDNA sequence analysis. The sequence obtained indicated that a strain of Clostridium beijerinckii was isolated. The cellulosome was purified from the putative C. beijerinckii sLM01 host culture using affinity chromatography purification and affinity digestion purification procedures. The cellulosomal and non-cellulosomal fractions of C. beijerinckii sLM01 were separated successfully, but the majority of the endoglucanase activity was lost during the Sepharose 4B chromatography step. These cellulosomal and non-cellulosomal fractions were characterised with regards to their pH and temperature optima and effector sensitivity. Increased additions of sulphide activated the cellulase activity of the cellulosomal and non-cellulosomal fractions up to 700 %, while increased additions of sulphate either increased the activity slightly or inhibited it dramatically, depending on the cellulosomal and non-cellulosomal fractions. Increased additions of cellobiose, glucose and acetate inhibited the cellulase and endoglucanase activities. pH optima of 5.0 and 7.5 were observed for cellulases and 5.0 for endoglucanases of the cellulosomal fraction. The noncellulosomal fraction exhibited a pH optimum of 7.5 for both cellulase and endoglucanase activities. Both fractions and enzymes exhibited a temperature optimum of 30 °C. The fundamental knowledge gained from the characterisation was applied to anaerobic digestion, where the effect of sulphide on the rate-limiting step was determined. Sulphide activated cellulase and endoglucanase activities and increased the % chemical oxygen demand (COD) removal rate. Levels of volatile fatty acids (VFAs) were higher in the bioreactor containing sulphide, substrate and C. beijerinckii. Sulphide therefore accelerated the rate-limiting step of anaerobic digestion.
- Full Text:
- Date Issued: 2007
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