Lignocellulosic waste degradation using enzyme synergy with commercially available enzymes and Clostridium cellulovorans XylanaseA and MannanaseA
- Authors: Morrison, David Graham
- Date: 2014
- Subjects: Lignocellulose -- Biodegradation , Enzymes -- Biotechnology , Agricultural wastes as fuel , Polysaccharides -- Biotechnology , Sugar -- Inversion , Clostridium , Xylanases , Monomers
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4119 , http://hdl.handle.net/10962/d1013292
- Description: The launch of national and international initiatives to reduce pollution, reliance on fossil fuels and increase the beneficiation of agricultural wastes has prompted research into sugar monomer production from lignocellulosic wastes. These sugars can subsequently be used in the production of biofuels and environmentally degradable plastics. This study investigated the use of synergistic combinations of commercial and pure enzymes to lower enzyme costs and loadings, while increasing enzyme activity in the hydrolysis of agricultural waste. Pineapple pomace was selected due to its current underutilisation and the substantial quantities of it produced annually, as a by-product of pineapple canning. One of the primary costs in beneficiating agricultural wastes, such as pineapple pomace, is the high cost of enzyme solutions used to generate reducing sugars. This can be lowered through the use of synergistic combinations of enzymes. Studies related to the inclusion of hemicellulose degrading enzymes with commercial enzyme solutions have been limited and investigation of these solutions in select combinations, together with pineapple pomace substrate, allows for novel research. The use of synergistic combinations of purified cellulosomal enzymes has previously been shown to be effective at releasing reducing sugars from agricultural wastes. For the present study, MannanaseA and XylanaseA from Clostridium cellulovorans were heterologously expressed in Escherichia coli BL21 (DE3) cells and purified with immobilised metal affinity chromatography. These enzymes, in addition to two commercially available enzyme solutions (Celluclast 1.5L® and Pectinex® 3XL), were assayed on defined polysaccharides that are present in pineapple pomace to determine their substrate specificities. The degree(s) of synergy and specific activities of selected combinations of these enzymes were tested under both simultaneous and sequential conditions. It was observed that several synergistic combinations of enzyme solutions in select ratios, such as C20P60X20 (20% cellulose, 60% pectinase and 20% xylanse), C20P40X40 (20% cellulose, 40% pectinase and 40% xylanase) and C20P80 (20% cellulose, 80% pectinase) with pineapple pomace could both decrease the protein loading, while raising the level of activity compared to individual enzyme solutions. The highest quantity of reducing sugars to protein weight used on pineapple pomace was recorded at 3, 9 and 18 hours with combinations of Pectinex® 3XL and Celluclast 1.5L®, but for 27 h it was combinations of both these commercial solutions with XynA. The contribution of XynA was significant as C20P60X20 displayed the second highest reducing sugar production of 1.521 mg/mL, at 36 h from 12.875 μg/mL of protein, which was the second lowest protein loading. It was also shown that certain enzyme combinations, such as Pectinex® 3XL, Celluclast 1.5L® and XynA, did not generate synergy when combined in solution at the initial stages of hydrolysis, and instead generated a form of competition called anti-synergy. This was due to Pectinex® 3XL which had anti-synergy relationships in select combinations with the other enzyme solutions assayed. It was also observed that the degree of synergy and specific activity for a combination changed over time. Some solutions displayed the highest levels of synergy at the commencement of hydrolysis, namely Celluclast 1.5L®, ManA and XynA. Other combinations exhibited the highest levels of synergy at the end of the assay period, such as Pectinex® 3XL and Celluclast 1.5L®. Whether greater synergy was generated at the start or end of hydrolysis was a function of the stability of the enzymes in solution and whether enzyme activity increased substrate accessibility or generated competition between enzymes in solution. Sequential synergy studies demonstrated an anti-synergy relationship between Pectinex® 3XL and XynA or ManA, as well as Pectinex® 3 XL and Celluclast 1.5L®. It was found that under sequential synergy conditions with Pectinex® 3 XL, XynA and ManA, that anti-synergy could be negated and high degrees of synergy attained when the enzymes were added in specific loading orders and not inhibited by the presence of other active enzymes. The importance of loading order was demonstrated under sequential synergy conditions when XynA was added before ManA followed by Pectinex® 3 XL, which increased the activity and synergy of the solution by 50%. This equates to a 60% increase in reducing sugar release from the same concentrations of enzymes and emphasises the importance of removing anti-synergy relationships from combinations of enzymes. It can be concluded that a C20P60X20 combination (based on activity) can both synergistically increase the reducing sugar production and lower the protein loading required for pineapple pomace hydrolysis. This study also highlights the importance of reducing anti-synergy in customised enzyme cocktails and how sequential synergy can demonstrate the order in which a lignocellulosic waste is degraded.
- Full Text:
- Date Issued: 2014
- Authors: Morrison, David Graham
- Date: 2014
- Subjects: Lignocellulose -- Biodegradation , Enzymes -- Biotechnology , Agricultural wastes as fuel , Polysaccharides -- Biotechnology , Sugar -- Inversion , Clostridium , Xylanases , Monomers
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4119 , http://hdl.handle.net/10962/d1013292
- Description: The launch of national and international initiatives to reduce pollution, reliance on fossil fuels and increase the beneficiation of agricultural wastes has prompted research into sugar monomer production from lignocellulosic wastes. These sugars can subsequently be used in the production of biofuels and environmentally degradable plastics. This study investigated the use of synergistic combinations of commercial and pure enzymes to lower enzyme costs and loadings, while increasing enzyme activity in the hydrolysis of agricultural waste. Pineapple pomace was selected due to its current underutilisation and the substantial quantities of it produced annually, as a by-product of pineapple canning. One of the primary costs in beneficiating agricultural wastes, such as pineapple pomace, is the high cost of enzyme solutions used to generate reducing sugars. This can be lowered through the use of synergistic combinations of enzymes. Studies related to the inclusion of hemicellulose degrading enzymes with commercial enzyme solutions have been limited and investigation of these solutions in select combinations, together with pineapple pomace substrate, allows for novel research. The use of synergistic combinations of purified cellulosomal enzymes has previously been shown to be effective at releasing reducing sugars from agricultural wastes. For the present study, MannanaseA and XylanaseA from Clostridium cellulovorans were heterologously expressed in Escherichia coli BL21 (DE3) cells and purified with immobilised metal affinity chromatography. These enzymes, in addition to two commercially available enzyme solutions (Celluclast 1.5L® and Pectinex® 3XL), were assayed on defined polysaccharides that are present in pineapple pomace to determine their substrate specificities. The degree(s) of synergy and specific activities of selected combinations of these enzymes were tested under both simultaneous and sequential conditions. It was observed that several synergistic combinations of enzyme solutions in select ratios, such as C20P60X20 (20% cellulose, 60% pectinase and 20% xylanse), C20P40X40 (20% cellulose, 40% pectinase and 40% xylanase) and C20P80 (20% cellulose, 80% pectinase) with pineapple pomace could both decrease the protein loading, while raising the level of activity compared to individual enzyme solutions. The highest quantity of reducing sugars to protein weight used on pineapple pomace was recorded at 3, 9 and 18 hours with combinations of Pectinex® 3XL and Celluclast 1.5L®, but for 27 h it was combinations of both these commercial solutions with XynA. The contribution of XynA was significant as C20P60X20 displayed the second highest reducing sugar production of 1.521 mg/mL, at 36 h from 12.875 μg/mL of protein, which was the second lowest protein loading. It was also shown that certain enzyme combinations, such as Pectinex® 3XL, Celluclast 1.5L® and XynA, did not generate synergy when combined in solution at the initial stages of hydrolysis, and instead generated a form of competition called anti-synergy. This was due to Pectinex® 3XL which had anti-synergy relationships in select combinations with the other enzyme solutions assayed. It was also observed that the degree of synergy and specific activity for a combination changed over time. Some solutions displayed the highest levels of synergy at the commencement of hydrolysis, namely Celluclast 1.5L®, ManA and XynA. Other combinations exhibited the highest levels of synergy at the end of the assay period, such as Pectinex® 3XL and Celluclast 1.5L®. Whether greater synergy was generated at the start or end of hydrolysis was a function of the stability of the enzymes in solution and whether enzyme activity increased substrate accessibility or generated competition between enzymes in solution. Sequential synergy studies demonstrated an anti-synergy relationship between Pectinex® 3XL and XynA or ManA, as well as Pectinex® 3 XL and Celluclast 1.5L®. It was found that under sequential synergy conditions with Pectinex® 3 XL, XynA and ManA, that anti-synergy could be negated and high degrees of synergy attained when the enzymes were added in specific loading orders and not inhibited by the presence of other active enzymes. The importance of loading order was demonstrated under sequential synergy conditions when XynA was added before ManA followed by Pectinex® 3 XL, which increased the activity and synergy of the solution by 50%. This equates to a 60% increase in reducing sugar release from the same concentrations of enzymes and emphasises the importance of removing anti-synergy relationships from combinations of enzymes. It can be concluded that a C20P60X20 combination (based on activity) can both synergistically increase the reducing sugar production and lower the protein loading required for pineapple pomace hydrolysis. This study also highlights the importance of reducing anti-synergy in customised enzyme cocktails and how sequential synergy can demonstrate the order in which a lignocellulosic waste is degraded.
- Full Text:
- Date Issued: 2014
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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