An evaluation of synergistic interactions between feruloyl esterases and xylanases during the hydrolysis of various pre-treated agricultural residues
- Authors: Mkabayi, Lithalethu
- Date: 2021-04
- Subjects: Esterases , Xylanases , Hydrolysis , Agricultural wastes -- Recycling , Enzymes , Lignocellulose -- Biodegradation , Escherichia coli , Oligosaccharides , Hydroxycinnamic acids
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/178224 , vital:42922 , 10.21504/10962/178224
- Description: Agricultural residues are readily available and inexpensive renewable resources that can be used as raw materials for the production of value-added chemicals. The application of enzymes to facilitate the degradation of agricultural residues has long been considered the most environmentally friendly strategy for converting this material into good quality value-added chemicals. However, agricultural residues are typically lignocellulosic in composition and recalcitrant to enzymatic hydrolysis. Due to this recalcitrant nature, the complete degradation of biomass residues requires the synergistic action of a broad range of enzymes. The development and optimisation of synergistic enzyme cocktails is an effective approach for achieving high hydrolysis efficiency of lignocellulosic biomass. The aim of the current study was to evaluate the synergistic interactions between two termite metagenome-derived feruloyl esterases (FAE6 and FAE5) and endo-xylanases for the production of hydroxycinnamic acids and xylo-oligosaccharides (XOS) from model substrates, and untreated and pre-treated agricultural residues. Firstly, the two fae genes were heterologously expressed in Escherichia coli, and the recombinant enzymes were purified to homogeneity. The biochemical properties of the purified recombinant FAEs and xylanases (XT6 and Xyn11) were then assessed to determine the factors which influenced their activities and to select suitable operating conditions for synergy studies. An optimal protein loading ratio of xylanases to FAEs required to maximise the release of both reducing sugar and ferulic acid (FA) was established using 0.5% (w/v) insoluble wheat arabinoxylan (a model substrate). The enzyme combination of 66% xylanase and 33% FAE (on a protein loading basis) produced the highest amounts of reducing sugars and FA. The enzyme combination of XT6 (GH10 xylanase) and FAE5 or FAE6 liberated the highest amount of FA while a combination of Xyn11 (GH11 xylanase) and FAE5 or FAE6 produced the highest reducing sugar content. The synergistic interactions which were established between the xylanases and FAEs were further investigated using agricultural residues (corn cobs, rice straw and sugarcane bagasse). The three substrates were subjected to hydrothermal and dilute acid pre-treatment prior to synergy studies. It is generally known that, during pre-treatment, many compounds can be produced which may influence enzymatic hydrolysis. The effects of these by-products were assessed and it was found that lignin and its degradation products were the most inhibitory to the FAEs. The optimised enzyme cocktail was then applied to 1% (w/v) of untreated and pre-treated substrates for the efficient production of XOS and hydroxycinnamic acids. A significant improvement in xylanase substrate degradation was observed, especially with the combination of 66% Xyn11 and 33% FAE6 which displayed an improvement in reducing sugars of approximately 1.9-fold and 3.4-fold for hydrothermal and acid pre-treated corn cobs (compared to when Xyn11 was used alone), respectively. The study demonstrated that pre-treatment substantially enhanced the enzymatic hydrolysis of corn cobs and rice straw. Analysis of the hydrolysate product profiles revealed that the optimised enzyme cocktail displayed great potential for releasing XOS with a low degree of polymerisation. In conclusion, this study provided significant insights into the mechanism of synergistic interactions between xylanases and metagenome-derived FAEs during the hydrolysis of various substrates. The study also demonstrated that optimised enzyme cocktails combined with low severity pre-treatment can facilitate the potential use of xylan-rich lignocellulosic biomass for the production of valuable products in the future. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Authors: Mkabayi, Lithalethu
- Date: 2021-04
- Subjects: Esterases , Xylanases , Hydrolysis , Agricultural wastes -- Recycling , Enzymes , Lignocellulose -- Biodegradation , Escherichia coli , Oligosaccharides , Hydroxycinnamic acids
- Language: English
- Type: thesis , text , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/178224 , vital:42922 , 10.21504/10962/178224
- Description: Agricultural residues are readily available and inexpensive renewable resources that can be used as raw materials for the production of value-added chemicals. The application of enzymes to facilitate the degradation of agricultural residues has long been considered the most environmentally friendly strategy for converting this material into good quality value-added chemicals. However, agricultural residues are typically lignocellulosic in composition and recalcitrant to enzymatic hydrolysis. Due to this recalcitrant nature, the complete degradation of biomass residues requires the synergistic action of a broad range of enzymes. The development and optimisation of synergistic enzyme cocktails is an effective approach for achieving high hydrolysis efficiency of lignocellulosic biomass. The aim of the current study was to evaluate the synergistic interactions between two termite metagenome-derived feruloyl esterases (FAE6 and FAE5) and endo-xylanases for the production of hydroxycinnamic acids and xylo-oligosaccharides (XOS) from model substrates, and untreated and pre-treated agricultural residues. Firstly, the two fae genes were heterologously expressed in Escherichia coli, and the recombinant enzymes were purified to homogeneity. The biochemical properties of the purified recombinant FAEs and xylanases (XT6 and Xyn11) were then assessed to determine the factors which influenced their activities and to select suitable operating conditions for synergy studies. An optimal protein loading ratio of xylanases to FAEs required to maximise the release of both reducing sugar and ferulic acid (FA) was established using 0.5% (w/v) insoluble wheat arabinoxylan (a model substrate). The enzyme combination of 66% xylanase and 33% FAE (on a protein loading basis) produced the highest amounts of reducing sugars and FA. The enzyme combination of XT6 (GH10 xylanase) and FAE5 or FAE6 liberated the highest amount of FA while a combination of Xyn11 (GH11 xylanase) and FAE5 or FAE6 produced the highest reducing sugar content. The synergistic interactions which were established between the xylanases and FAEs were further investigated using agricultural residues (corn cobs, rice straw and sugarcane bagasse). The three substrates were subjected to hydrothermal and dilute acid pre-treatment prior to synergy studies. It is generally known that, during pre-treatment, many compounds can be produced which may influence enzymatic hydrolysis. The effects of these by-products were assessed and it was found that lignin and its degradation products were the most inhibitory to the FAEs. The optimised enzyme cocktail was then applied to 1% (w/v) of untreated and pre-treated substrates for the efficient production of XOS and hydroxycinnamic acids. A significant improvement in xylanase substrate degradation was observed, especially with the combination of 66% Xyn11 and 33% FAE6 which displayed an improvement in reducing sugars of approximately 1.9-fold and 3.4-fold for hydrothermal and acid pre-treated corn cobs (compared to when Xyn11 was used alone), respectively. The study demonstrated that pre-treatment substantially enhanced the enzymatic hydrolysis of corn cobs and rice straw. Analysis of the hydrolysate product profiles revealed that the optimised enzyme cocktail displayed great potential for releasing XOS with a low degree of polymerisation. In conclusion, this study provided significant insights into the mechanism of synergistic interactions between xylanases and metagenome-derived FAEs during the hydrolysis of various substrates. The study also demonstrated that optimised enzyme cocktails combined with low severity pre-treatment can facilitate the potential use of xylan-rich lignocellulosic biomass for the production of valuable products in the future. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
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:
- 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:
- «
- ‹
- 1
- ›
- »