A lignocellulolytic enzyme system for fruit waste degradation : commercial enzyme mixture synergy and bioreactor design
- Authors: Gama, Repson
- Date: 2014
- Subjects: Enzymes -- Biotechnology , Enzymes -- Industrial applications , Lignocellulose -- Biodegradation , Biomass energy , Biomass conversion , Biochemical engineering , Agricultural wastes as fuel
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4114 , http://hdl.handle.net/10962/d1013073
- Description: Studies into sources of alternative liquid transport fuel energy have identified agro-industrial wastes, which are lignocellulosic in nature, as a potential feedstock for biofuel production against the background of depleting nonrenewable fossil fuels. In South Africa, large quantities of apple and other fruit wastes, called pomace, are generated from fruit and juice industries. Apple pomace is a rich source of cellulose, pectin and hemicellulose, making it a potential target for utilisation as a lignocellulosic feedstock for biofuel and biorefinery chemical production. Lignocellulosic biomass is recalcitrant in nature and therefore its degradation requires the synergistic action of a number of enzymes such as cellulases, hemicellulases, pectinases and ligninases. Commercial enzyme cocktails, containing some of these enzymes, are available and can be used for apple pomace degradation. In this study, the degradation of apple pomace using commercial enzyme cocktails was investigated. The main focus was the optimisation of the release of sugar monomers that could potentially be used for biofuel and biorefinery chemical production. There is no or little information reported in literature on the enzymatic degradation of fruit waste using commercial enzyme mixtures. This study first focused on the characterisation of the substrate (apple pomace) and the commercial enzyme cocktails. Apple pomace was found to contain mainly glucose, galacturonic acid, arabinose, galactose, lignin and low amounts of xylose and fructose. Three commercial enzyme cocktails were initially selected: Biocip Membrane, Viscozyme L (from Aspergillus aculeatus) and Celluclast 1.5L (a Trichoderma reesei ATCC 26921 cellulase preparation). The selection of the enzymes was based on activities declared by the manufacturers, cost and local availability. The enzymes were screened based on their synergistic cooperation in the degradation of apple pomace and the main enzymes present in each cocktail. Viscozyme L and Celluclast 1.5L, in a 50:50 ratio, resulted in the best degree of synergy (1.6) compared to any other combination. The enzyme ratios were determined on Viscozyme L and Celluclast 1.5L based on the protein ratio. Enzyme activity was determined as glucose equivalents using the dinitrosalicylic acid (DNS) method. Sugar monomers were determined using Megazyme assay kits. There is limited information available on the enzymes present in the commercial enzyme cocktails. Therefore, the main enzymes present in Viscozyme L and Celluclast 1.5L were identified using different substrates, each targeted for a specific enzyme and activity. Characterisation of the enzyme mixtures revealed a large number of enzymes required for apple pomace degradation and these included cellulases, pectinases, xylanases, arabinases and mannanases in different proportions. Viscozyme L contained mainly pectinases and hemicellulases, while Celluclast 1.5L displayed largely cellulase and xylanase activity, hence the high degree of synergy reported. The temperature optimum was 50ºC for both enzyme mixtures and pH optima were observed at pH 5.0 and pH 3.0 for Viscozyme L and Celluclast 1.5L, respectively. At 37ºC and pH 5.0, the enzymes retained more that 90% activity after 15 days of incubation, allowing the enzymes to be used together with less energy input. The enzymes were further characterised by determining the effect of various compounds, such as alcohols, sugars, phenolic compounds and metal ions at various concentrations on the activity of the enzymes during apple pomace hydrolysis. Apart from lignin, which had almost no effect on enzyme activity, all the compounds caused inhibition of the enzymes to varying degrees. The most inhibitory compounds were some organic acids and metal ions, as well as cellobiose and xylobiose. Using the best ratio for Viscozyme L and Celluclast 1.5L (50:50) for the hydrolysis of apple pomace, it was observed that synergy was highest at the initial stages of hydrolysis and decreased over time, though the sugar concentration increased. The type of synergy for optimal apple pomace hydrolysis was found to be simultaneous. There was no synergy observed between Viscozyme L and Celluclast 1.5L with ligninases - laccase, lignin peroxidase and manganese peroxidase. Hydrolysing apple pomace with ligninases prior to addition of Viscozyme L and Celluclast 1.5L did not improve degradation of the substrate. Immobilisation of the enzyme mixtures on different supports was performed with the aim of increasing stability and enabling reuse of the enzymes. Immobilisation methods were selected based on the chemical properties of the supports, availability, cost and applicability on heterogeneous and insoluble substrate like apple pomace. These methods included crosslinked enzyme aggregates (CLEAs), immobilisation on various supports such as nylon mesh, nylon beads, sodium alginate beads, chitin and silica gel beads. The immobilisation strategies were unsuccessful, mainly due to the low percentage of immobilisation of the enzyme on the matrix and loss of activity of the immobilised enzyme. Free enzymes were therefore used for the remainder of the study. Hydrolysis conditions for apple pomace degradation were optimised using different temperatures and buffer systems in 1 L volumes mixed with compressed air. Hydrolysis at room temperature, using an unbuffered system, gave a better performance as compared to a buffered system. Reactors operated in batch mode performed better (4.2 g/L (75% yield) glucose and 16.8 g/L (75%) reducing sugar) than fed-batch reactors (3.2 g/L (66%) glucose and 14.6 g/L (72.7% yield) reducing sugar) over 100 h using Viscozyme L and Celluclast 1.5L. Supplementation of β- glucosidase activity in Viscozyme L and Celluclast 1.5L with Novozyme 188 resulted in a doubling of the amount of glucose released. The main products released from apple pomace hydrolysis were galacturonic acid, glucose and arabinose and low amounts of galactose and xylose. These products are potential raw materials for biofuel and biorefinery chemical production. An artificial neural network (ANN) model was successfully developed and used for predicting the optimum conditions for apple pomace hydrolysis using Celluclast 1.5L, Viscozyme L and Novozyme 188. Four main conditions that affect apple pomace hydrolysis were selected, namely temperature, initial pH, enzyme loading and substrate loading, which were taken as inputs. The glucose and reducing sugars released as a result of each treatment and their combinations were taken as outputs for 1–100 h. An ANN with 20, 20 and 6 neurons in the first, second and third hidden layers, respectively, was constructed. The performance and predictive ability of the ANN was good, with a R² of 0.99 and a small mean square error (MSE). New data was successfully predicted and simulated. Optimal hydrolysis conditions predicted by ANN for apple pomace hydrolysis were at 30% substrate (wet w/v) and an enzyme loading of 0.5 mg/g and 0.2 mg/mL of substrate for glucose and reducing sugar, respectively, giving sugar concentrations of 6.5 mg/mL and 28.9 mg/mL for glucose and reducing sugar, respectively. ANN showed that enzyme and substrate loadings were the most important factors for the hydrolysis of apple pomace.
- Full Text:
- Date Issued: 2014
- Authors: Gama, Repson
- Date: 2014
- Subjects: Enzymes -- Biotechnology , Enzymes -- Industrial applications , Lignocellulose -- Biodegradation , Biomass energy , Biomass conversion , Biochemical engineering , Agricultural wastes as fuel
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4114 , http://hdl.handle.net/10962/d1013073
- Description: Studies into sources of alternative liquid transport fuel energy have identified agro-industrial wastes, which are lignocellulosic in nature, as a potential feedstock for biofuel production against the background of depleting nonrenewable fossil fuels. In South Africa, large quantities of apple and other fruit wastes, called pomace, are generated from fruit and juice industries. Apple pomace is a rich source of cellulose, pectin and hemicellulose, making it a potential target for utilisation as a lignocellulosic feedstock for biofuel and biorefinery chemical production. Lignocellulosic biomass is recalcitrant in nature and therefore its degradation requires the synergistic action of a number of enzymes such as cellulases, hemicellulases, pectinases and ligninases. Commercial enzyme cocktails, containing some of these enzymes, are available and can be used for apple pomace degradation. In this study, the degradation of apple pomace using commercial enzyme cocktails was investigated. The main focus was the optimisation of the release of sugar monomers that could potentially be used for biofuel and biorefinery chemical production. There is no or little information reported in literature on the enzymatic degradation of fruit waste using commercial enzyme mixtures. This study first focused on the characterisation of the substrate (apple pomace) and the commercial enzyme cocktails. Apple pomace was found to contain mainly glucose, galacturonic acid, arabinose, galactose, lignin and low amounts of xylose and fructose. Three commercial enzyme cocktails were initially selected: Biocip Membrane, Viscozyme L (from Aspergillus aculeatus) and Celluclast 1.5L (a Trichoderma reesei ATCC 26921 cellulase preparation). The selection of the enzymes was based on activities declared by the manufacturers, cost and local availability. The enzymes were screened based on their synergistic cooperation in the degradation of apple pomace and the main enzymes present in each cocktail. Viscozyme L and Celluclast 1.5L, in a 50:50 ratio, resulted in the best degree of synergy (1.6) compared to any other combination. The enzyme ratios were determined on Viscozyme L and Celluclast 1.5L based on the protein ratio. Enzyme activity was determined as glucose equivalents using the dinitrosalicylic acid (DNS) method. Sugar monomers were determined using Megazyme assay kits. There is limited information available on the enzymes present in the commercial enzyme cocktails. Therefore, the main enzymes present in Viscozyme L and Celluclast 1.5L were identified using different substrates, each targeted for a specific enzyme and activity. Characterisation of the enzyme mixtures revealed a large number of enzymes required for apple pomace degradation and these included cellulases, pectinases, xylanases, arabinases and mannanases in different proportions. Viscozyme L contained mainly pectinases and hemicellulases, while Celluclast 1.5L displayed largely cellulase and xylanase activity, hence the high degree of synergy reported. The temperature optimum was 50ºC for both enzyme mixtures and pH optima were observed at pH 5.0 and pH 3.0 for Viscozyme L and Celluclast 1.5L, respectively. At 37ºC and pH 5.0, the enzymes retained more that 90% activity after 15 days of incubation, allowing the enzymes to be used together with less energy input. The enzymes were further characterised by determining the effect of various compounds, such as alcohols, sugars, phenolic compounds and metal ions at various concentrations on the activity of the enzymes during apple pomace hydrolysis. Apart from lignin, which had almost no effect on enzyme activity, all the compounds caused inhibition of the enzymes to varying degrees. The most inhibitory compounds were some organic acids and metal ions, as well as cellobiose and xylobiose. Using the best ratio for Viscozyme L and Celluclast 1.5L (50:50) for the hydrolysis of apple pomace, it was observed that synergy was highest at the initial stages of hydrolysis and decreased over time, though the sugar concentration increased. The type of synergy for optimal apple pomace hydrolysis was found to be simultaneous. There was no synergy observed between Viscozyme L and Celluclast 1.5L with ligninases - laccase, lignin peroxidase and manganese peroxidase. Hydrolysing apple pomace with ligninases prior to addition of Viscozyme L and Celluclast 1.5L did not improve degradation of the substrate. Immobilisation of the enzyme mixtures on different supports was performed with the aim of increasing stability and enabling reuse of the enzymes. Immobilisation methods were selected based on the chemical properties of the supports, availability, cost and applicability on heterogeneous and insoluble substrate like apple pomace. These methods included crosslinked enzyme aggregates (CLEAs), immobilisation on various supports such as nylon mesh, nylon beads, sodium alginate beads, chitin and silica gel beads. The immobilisation strategies were unsuccessful, mainly due to the low percentage of immobilisation of the enzyme on the matrix and loss of activity of the immobilised enzyme. Free enzymes were therefore used for the remainder of the study. Hydrolysis conditions for apple pomace degradation were optimised using different temperatures and buffer systems in 1 L volumes mixed with compressed air. Hydrolysis at room temperature, using an unbuffered system, gave a better performance as compared to a buffered system. Reactors operated in batch mode performed better (4.2 g/L (75% yield) glucose and 16.8 g/L (75%) reducing sugar) than fed-batch reactors (3.2 g/L (66%) glucose and 14.6 g/L (72.7% yield) reducing sugar) over 100 h using Viscozyme L and Celluclast 1.5L. Supplementation of β- glucosidase activity in Viscozyme L and Celluclast 1.5L with Novozyme 188 resulted in a doubling of the amount of glucose released. The main products released from apple pomace hydrolysis were galacturonic acid, glucose and arabinose and low amounts of galactose and xylose. These products are potential raw materials for biofuel and biorefinery chemical production. An artificial neural network (ANN) model was successfully developed and used for predicting the optimum conditions for apple pomace hydrolysis using Celluclast 1.5L, Viscozyme L and Novozyme 188. Four main conditions that affect apple pomace hydrolysis were selected, namely temperature, initial pH, enzyme loading and substrate loading, which were taken as inputs. The glucose and reducing sugars released as a result of each treatment and their combinations were taken as outputs for 1–100 h. An ANN with 20, 20 and 6 neurons in the first, second and third hidden layers, respectively, was constructed. The performance and predictive ability of the ANN was good, with a R² of 0.99 and a small mean square error (MSE). New data was successfully predicted and simulated. Optimal hydrolysis conditions predicted by ANN for apple pomace hydrolysis were at 30% substrate (wet w/v) and an enzyme loading of 0.5 mg/g and 0.2 mg/mL of substrate for glucose and reducing sugar, respectively, giving sugar concentrations of 6.5 mg/mL and 28.9 mg/mL for glucose and reducing sugar, respectively. ANN showed that enzyme and substrate loadings were the most important factors for the hydrolysis of apple pomace.
- Full Text:
- Date Issued: 2014
Development of a bench scale single batch biomass to liquid fuel facility
- Authors: Zhang, Yusheng
- Date: 2014
- Subjects: Biomass energy , Renewable energy sources , Energy conversion , Electric power production
- Language: English
- Type: Thesis , Masters , MSc (Chemistry)
- Identifier: http://hdl.handle.net/10353/811 , vital:26499 , Biomass energy , Renewable energy sources , Energy conversion , Electric power production
- Description: The research described in this dissertation was motivated by the global demand for energy that is not dependent on coal, oil, natural gas and other non-renewable fossil fuels. The technology used in this project is related to the use of biomass to produce a viable alternative to conventional sources of fuel. A bench scale biomass to liquid (BTL) facility was built and tested. This produced results confirming the feasibility of the BTL process. The findings of the pilot study outlined in this dissertation justified the conclusion that the next step will be to expand the capacity and productivity of the BTL pilot plant to an industrial scale. Biomass comes from a variety of renewable sources that are readily available. In this case, the material used in the fixed bed biomass gasification facility to generate wood gas was agricultural and forestry waste, such as straw and wood chips. The gasifier had the capacity to produce up to 10 cubic metres/hr of gas with a carbon monoxide and hydrogen content of between 20–40% by volume, when it was operated at ambient pressure and with air as the oxidizer. The gas, produced at a temperature above 700º C, was cooled in a quench/water scrubber in order to remove most of the mechanical impurities (tars and water-soluble inorganic particles), condensed and dried with corn cobs before being compressed in cylinders at over 100 bar (g) for use in the Fischer-Tropsch Synthesis (FTS). The syngas was subjected further to a series of refining processes which included removal of sulphur and oxygen. The sulphur removal technology chosen entailed applying modified activated carbon to adsorb H2S with the help of hydrolysis in order to convert organic sulphur impurities into H2S which reduced the sulphur content of the gas to less than 5 ppbv. Supported cobalt catalyst (100 grams), were loaded into a single-tube fixed bed FT reactor with an inner diameter of 50 mm. The reactor was fitted with a heating jacket through which, heated oil ran to cool the reactor during a normal reaction occurring at < 250 ºC, while nitrogen was used in the heating jacket during reduction, which occurred at temperatures up ~ 350 ºC. The FTS reaction was carried out at different pressures and temperatures. Liquid and wax products were produced from the facility. The properties of the liquid and solid hydrocarbons produced were found to be the same as FT products from other feed stocks, such as natural gas and coal.
- Full Text:
- Date Issued: 2014
- Authors: Zhang, Yusheng
- Date: 2014
- Subjects: Biomass energy , Renewable energy sources , Energy conversion , Electric power production
- Language: English
- Type: Thesis , Masters , MSc (Chemistry)
- Identifier: http://hdl.handle.net/10353/811 , vital:26499 , Biomass energy , Renewable energy sources , Energy conversion , Electric power production
- Description: The research described in this dissertation was motivated by the global demand for energy that is not dependent on coal, oil, natural gas and other non-renewable fossil fuels. The technology used in this project is related to the use of biomass to produce a viable alternative to conventional sources of fuel. A bench scale biomass to liquid (BTL) facility was built and tested. This produced results confirming the feasibility of the BTL process. The findings of the pilot study outlined in this dissertation justified the conclusion that the next step will be to expand the capacity and productivity of the BTL pilot plant to an industrial scale. Biomass comes from a variety of renewable sources that are readily available. In this case, the material used in the fixed bed biomass gasification facility to generate wood gas was agricultural and forestry waste, such as straw and wood chips. The gasifier had the capacity to produce up to 10 cubic metres/hr of gas with a carbon monoxide and hydrogen content of between 20–40% by volume, when it was operated at ambient pressure and with air as the oxidizer. The gas, produced at a temperature above 700º C, was cooled in a quench/water scrubber in order to remove most of the mechanical impurities (tars and water-soluble inorganic particles), condensed and dried with corn cobs before being compressed in cylinders at over 100 bar (g) for use in the Fischer-Tropsch Synthesis (FTS). The syngas was subjected further to a series of refining processes which included removal of sulphur and oxygen. The sulphur removal technology chosen entailed applying modified activated carbon to adsorb H2S with the help of hydrolysis in order to convert organic sulphur impurities into H2S which reduced the sulphur content of the gas to less than 5 ppbv. Supported cobalt catalyst (100 grams), were loaded into a single-tube fixed bed FT reactor with an inner diameter of 50 mm. The reactor was fitted with a heating jacket through which, heated oil ran to cool the reactor during a normal reaction occurring at < 250 ºC, while nitrogen was used in the heating jacket during reduction, which occurred at temperatures up ~ 350 ºC. The FTS reaction was carried out at different pressures and temperatures. Liquid and wax products were produced from the facility. The properties of the liquid and solid hydrocarbons produced were found to be the same as FT products from other feed stocks, such as natural gas and coal.
- Full Text:
- Date Issued: 2014
Ethanol production from lignocellulosic sugarcane leaves and tops
- Authors: Dodo, Charlie Marembu
- Date: 2014
- Subjects: Biomass energy , Ethanol as fuel , Lignocellulose
- Language: English
- Type: Thesis , Masters , MSc (Chemistry)
- Identifier: vital:11347 , http://hdl.handle.net/10353/d1019839 , Biomass energy , Ethanol as fuel , Lignocellulose
- Description: Various methods for the production of bioethanol using different feedstocks have been researched on. In most work on bioethanol synthesis from sugar cane, tops and leaves have been regarded as waste and generally removed and thrown away. In this work, lignocellulosic sugarcane leaves and tops were not discarded but instead used as biomass to evaluate their hydrolyzate content. The leaves and tops were hydrolysed using different methods, namely concentrated acid, dilute acid pre-treatment with subsequent enzyme hydrolysis and compared with a combination of oxidative alkali pretreatment and enzyme hydrolysis. Subsequent fermentation of the hydrolyzates into bioethanol was done using the yeast saccharomyces cerevisae. Acid hydrolysis has the problem of producing inhibitors, which have to be removed and this was done using overliming with calcium hydroxide and compared to sodium hydroxide neutralization. Oxidative alkali pre-treatment with enzyme hydrolysis gave the highest yields of fermentable sugars of 38% (g/g) using 7% (v/v) peroxide pre-treated biomass than 36% (g/g) for 5% (v/v) with the least inhibitors. Concentrated and dilute acid hydrolysis each gave yields of25% (g/g) and 22% (g/g) yields respectively although for acid a neutralization step was necessary and resulted in dilution. Alkaline neutralization of acid hydrolyzates using sodium hydroxide resulted in less dilution and loss of fermentable sugars as compared to overliming. Higher yields of bioethanol, 13.7 (g/l) were obtained from enzyme hydrolyzates than 6.9 (g/l) bioethanol from dilute acid hydrolyzates. There was more bioethanol yield 13.7 (g/l) after 72h of fermentation with the yeast than 7.0 (g/l) bioethanol after 24h. However, the longer fermentation period diminishes the value of the increase in yield by lowering the efficiency of the process.
- Full Text:
- Date Issued: 2014
- Authors: Dodo, Charlie Marembu
- Date: 2014
- Subjects: Biomass energy , Ethanol as fuel , Lignocellulose
- Language: English
- Type: Thesis , Masters , MSc (Chemistry)
- Identifier: vital:11347 , http://hdl.handle.net/10353/d1019839 , Biomass energy , Ethanol as fuel , Lignocellulose
- Description: Various methods for the production of bioethanol using different feedstocks have been researched on. In most work on bioethanol synthesis from sugar cane, tops and leaves have been regarded as waste and generally removed and thrown away. In this work, lignocellulosic sugarcane leaves and tops were not discarded but instead used as biomass to evaluate their hydrolyzate content. The leaves and tops were hydrolysed using different methods, namely concentrated acid, dilute acid pre-treatment with subsequent enzyme hydrolysis and compared with a combination of oxidative alkali pretreatment and enzyme hydrolysis. Subsequent fermentation of the hydrolyzates into bioethanol was done using the yeast saccharomyces cerevisae. Acid hydrolysis has the problem of producing inhibitors, which have to be removed and this was done using overliming with calcium hydroxide and compared to sodium hydroxide neutralization. Oxidative alkali pre-treatment with enzyme hydrolysis gave the highest yields of fermentable sugars of 38% (g/g) using 7% (v/v) peroxide pre-treated biomass than 36% (g/g) for 5% (v/v) with the least inhibitors. Concentrated and dilute acid hydrolysis each gave yields of25% (g/g) and 22% (g/g) yields respectively although for acid a neutralization step was necessary and resulted in dilution. Alkaline neutralization of acid hydrolyzates using sodium hydroxide resulted in less dilution and loss of fermentable sugars as compared to overliming. Higher yields of bioethanol, 13.7 (g/l) were obtained from enzyme hydrolyzates than 6.9 (g/l) bioethanol from dilute acid hydrolyzates. There was more bioethanol yield 13.7 (g/l) after 72h of fermentation with the yeast than 7.0 (g/l) bioethanol after 24h. However, the longer fermentation period diminishes the value of the increase in yield by lowering the efficiency of the process.
- Full Text:
- Date Issued: 2014
The large scale bioinformatics analysis of auxiliary activity family 9 enzymes
- Authors: Moses, Vuyani
- Date: 2014
- Subjects: Bioinformatics -- Analysis , Cellulose -- Biodegradation , Biomass energy
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4145 , http://hdl.handle.net/10962/d1016356
- Description: Biofuels have been proposed to be a suitable replacement to the already depleting fossil fuels. The complex structures of plant biomasses present a challenge the production of biofuels due to recalcitrance. The complex cellulose structure and hydrogen bonding between repeat units of cellulose is believed to be a major contributor to the recalcitrance of cellulose. Fungal organisms come equipped with various oxidative enzymes involved in degradation of plant biomass. The exact mechanism of cellulose degradation remains elusive. The GH61 is a group of proteins which are PMOs. GH61 sequences where previously described as endoglucanases due to weak endoglucanase activity. These enzymes were later found not possess any enzyme activity of their own however they could enhance the activity of other cellulose degrading enzymes. As a result reclassification of these enzymes as AA9 has been implemented. AA9 proteins have been reported to share structural homology with the bacterial AA10 group of enzymes. Based on cleavage products that are produced when AA9 proteins interact with cellulose, AA9 proteins have been grouped into three types. To date the exact mechanism and the sequence and structural basis for differentiating between the various AA9 types remains unknown. Using various bionformatic techniques sequence and structural elements were identified for distinguishing between the AA9 types. A large dataset of sequences was obtained from the Pfam database from UNIPROT entries. Due to high divergence of AA9 sequences, a smaller dataset with the more divergent sequences removed was created. The inclusion of the reference sequences to the data set was done to observe which sequences belong to a certain type. Phylogenetic analysis was able to group AA9 proteins into three distinct groups. MSA and motif analysis revealed that the N-Terminus of these proteins is mostly responsible for type specificity. Structural analysis of AA9 PDB structures and homology models allowed the effect of physicochemical properties to be gauged structurally. The presence of 310 helices and aromatic residues the surface of AA9 sequences is an observation which still warrants further investigation.
- Full Text:
- Date Issued: 2014
- Authors: Moses, Vuyani
- Date: 2014
- Subjects: Bioinformatics -- Analysis , Cellulose -- Biodegradation , Biomass energy
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4145 , http://hdl.handle.net/10962/d1016356
- Description: Biofuels have been proposed to be a suitable replacement to the already depleting fossil fuels. The complex structures of plant biomasses present a challenge the production of biofuels due to recalcitrance. The complex cellulose structure and hydrogen bonding between repeat units of cellulose is believed to be a major contributor to the recalcitrance of cellulose. Fungal organisms come equipped with various oxidative enzymes involved in degradation of plant biomass. The exact mechanism of cellulose degradation remains elusive. The GH61 is a group of proteins which are PMOs. GH61 sequences where previously described as endoglucanases due to weak endoglucanase activity. These enzymes were later found not possess any enzyme activity of their own however they could enhance the activity of other cellulose degrading enzymes. As a result reclassification of these enzymes as AA9 has been implemented. AA9 proteins have been reported to share structural homology with the bacterial AA10 group of enzymes. Based on cleavage products that are produced when AA9 proteins interact with cellulose, AA9 proteins have been grouped into three types. To date the exact mechanism and the sequence and structural basis for differentiating between the various AA9 types remains unknown. Using various bionformatic techniques sequence and structural elements were identified for distinguishing between the AA9 types. A large dataset of sequences was obtained from the Pfam database from UNIPROT entries. Due to high divergence of AA9 sequences, a smaller dataset with the more divergent sequences removed was created. The inclusion of the reference sequences to the data set was done to observe which sequences belong to a certain type. Phylogenetic analysis was able to group AA9 proteins into three distinct groups. MSA and motif analysis revealed that the N-Terminus of these proteins is mostly responsible for type specificity. Structural analysis of AA9 PDB structures and homology models allowed the effect of physicochemical properties to be gauged structurally. The presence of 310 helices and aromatic residues the surface of AA9 sequences is an observation which still warrants further investigation.
- Full Text:
- Date Issued: 2014
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