Sequence, structure, dynamics, and substrate specificity analyses of bacterial Glycoside Hydrolase 1 enzymes from several activities
- Authors: Veldman, Wayde Michael
- Date: 2022-04-08
- Subjects: Glycosidases , Bioinformatics , Molecular dynamics , Ligands (Biochemistry) , Enzymes , Ligand binding (Biochemistry) , Sequence alignment (Bioinformatics) , Structural bioinformatics
- Language: English
- Type: Doctoral thesis , text
- Identifier: http://hdl.handle.net/10962/233805 , vital:50129 , DOI 10.21504/10962/233810
- Description: Glycoside hydrolase 1 (GH1) enzymes are a ubiquitous family of enzymes that hydrolyse the glycosidic bond between two or more carbohydrates, or between a carbohydrate and a non-carbohydrate moiety. Despite their conserved catalytic domain, these enzymes have many different enzyme activities and/or substrate specificities as a change of only a few residues in the active site can alter their function. Most GH1 active site residues are situated in loop regions, and it is known that enzymes are more likely to develop new functions (broad specificity) if they possess an active site with a high proportion of loops. Furthermore, the GH1 active site consists of several subsites and cooperative binding makes the binding affinity of sites difficult to measure because the properties of one subsite are influenced by the binding of the other subsites. Extensive knowledge of protein-ligand interactions is critical to the comprehension of biology at the molecular level. However, the structural determinants and molecular details of GH1 ligand specificity and affinity are very broad, highly complex, not well understood, and therefore still need to be clarified. The aim of this study was to computationally characterise the activity of three newly solved GH1 crystallographic structures sent to us by our collaborators, and to provide evidence for their ligand-binding specificities. In addition, the differences in structural and biochemical contributions to enzyme specificity and/or function between different GH1 activities/enzymes was assessed, and the sequence/structure/function relationship of several activities of GH1 enzymes was analysed and compared. To accomplish the research aims, sequence analyses involving sequence identity, phylogenetics, and motif discovery were performed. As protein structure is more conserved than sequence, the discovered motifs were mapped to 3D structures for structural analysis and comparisons. To obtain information on enzyme mechanism or mode of action, as well as structure-function relationship, computational methods such as docking, molecular dynamics, binding free energy calculations, and essential dynamics were implemented. These computational approaches can provide information on the active site, binding residues, protein-ligand interactions, binding affinity, conformational change, and most structural or dynamic elements that play a role in enzyme function. The three new structures received from our collaborators are the first GH1 crystallographic structures from Bacillus licheniformis ever determined. As phospho-glycoside compounds were unavailable for purchase for use in activity assays, and as the active sites of the structures were absent of ligand, in silico docking and MD simulations were performed to provide evidence for their GH1 activities and substrate specificities. First though, the amino acid sequences of all known characterised bacterial GH1 enzymes were retrieved from the CAZy database and compared to the sequences of the three new B. licheniformis crystallographic structures which provided evidence of the putative 6Pβ-glucosidase activity of enzyme BlBglH, and dual 6Pβ-glucosidase/6Pβ-galactosidase (dual-phospho) activity of enzymes BlBglB and BlBglC. As all three enzymes were determined to be putative 6Pβ-glycosidase activity enzymes, much of the thesis focused on the overall analysis and comparison of the 6Pβ-glucosidase, 6Pβ-galactosidase, and dual-phospho activities that make up the 6Pβ-glycosidases. The 6Pβ-glycosidase active site residues were identified through consensus of binding interactions using all known 6Pβ-glycosidase PDB structures complexed complete ligand substrates. With regards to the 6Pβ-glucosidase activity, it was found that the L8b loop is longer and forms extra interactions with the L8a loop likely leading to increased L8 loop rigidity which would prevent the displacement of residue Ala423 ensuring a steric clash with galactoconfigured ligands and may engender substrate specificity for gluco-configured ligands only. Also, during molecular dynamics simulations using enzyme BlBglH (6Pβ-glucosidase activity), it was revealed that the favourable binding of substrate stabilises the loops that surround and make up the enzyme active site. Using the BlBglC (dual-phospho activity) enzyme structure with either galacto- (PNP6Pgal) or gluco-configured (PNP6Pglc) ligands, MD simulations in triplicate revealed important details of the broad specificity of dual-phospho activity enzymes. The ligand O4 hydroxyl position is the only difference between PNP6Pgal and PNP6Pgal, and it was found that residues Gln23 and Trp433 bind strongly to the ligand O3 hydroxyl group in the PNP6Pgal-enzyme complex, but to the ligand O4 hydroxyl group in the PNP6Pglc-enzyme complex. Also, His124 formed many hydrogen bonds with the PNP6Pgal O3 hydroxyl group but had none with PNP6Pglc. Alternatively, residues Tyr173, Tyr301, Gln302 and Thr321 formed hydrogen bonds with PNP6Pglc but not PNP6Pgal. Lastly, using multiple 3D structures from various GH1 activities, a large network of conserved interactions between active site residues (and other important residues) was uncovered, which most likely stabilise the loop regions that contain these residues, helping to retain their positions needed for binding molecules. Alternatively, there exists several differing residue-residue interactions when comparing each of the activities which could contribute towards individual activity substrate specificity by causing slightly different overall structure and malleability of the active site. Altogether, the findings in this thesis shed light on the function, mechanisms, dynamics, and ligand-binding of GH1 enzymes – particularly of the 6Pβ-glycosidase activities. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-04-08
- Authors: Veldman, Wayde Michael
- Date: 2022-04-08
- Subjects: Glycosidases , Bioinformatics , Molecular dynamics , Ligands (Biochemistry) , Enzymes , Ligand binding (Biochemistry) , Sequence alignment (Bioinformatics) , Structural bioinformatics
- Language: English
- Type: Doctoral thesis , text
- Identifier: http://hdl.handle.net/10962/233805 , vital:50129 , DOI 10.21504/10962/233810
- Description: Glycoside hydrolase 1 (GH1) enzymes are a ubiquitous family of enzymes that hydrolyse the glycosidic bond between two or more carbohydrates, or between a carbohydrate and a non-carbohydrate moiety. Despite their conserved catalytic domain, these enzymes have many different enzyme activities and/or substrate specificities as a change of only a few residues in the active site can alter their function. Most GH1 active site residues are situated in loop regions, and it is known that enzymes are more likely to develop new functions (broad specificity) if they possess an active site with a high proportion of loops. Furthermore, the GH1 active site consists of several subsites and cooperative binding makes the binding affinity of sites difficult to measure because the properties of one subsite are influenced by the binding of the other subsites. Extensive knowledge of protein-ligand interactions is critical to the comprehension of biology at the molecular level. However, the structural determinants and molecular details of GH1 ligand specificity and affinity are very broad, highly complex, not well understood, and therefore still need to be clarified. The aim of this study was to computationally characterise the activity of three newly solved GH1 crystallographic structures sent to us by our collaborators, and to provide evidence for their ligand-binding specificities. In addition, the differences in structural and biochemical contributions to enzyme specificity and/or function between different GH1 activities/enzymes was assessed, and the sequence/structure/function relationship of several activities of GH1 enzymes was analysed and compared. To accomplish the research aims, sequence analyses involving sequence identity, phylogenetics, and motif discovery were performed. As protein structure is more conserved than sequence, the discovered motifs were mapped to 3D structures for structural analysis and comparisons. To obtain information on enzyme mechanism or mode of action, as well as structure-function relationship, computational methods such as docking, molecular dynamics, binding free energy calculations, and essential dynamics were implemented. These computational approaches can provide information on the active site, binding residues, protein-ligand interactions, binding affinity, conformational change, and most structural or dynamic elements that play a role in enzyme function. The three new structures received from our collaborators are the first GH1 crystallographic structures from Bacillus licheniformis ever determined. As phospho-glycoside compounds were unavailable for purchase for use in activity assays, and as the active sites of the structures were absent of ligand, in silico docking and MD simulations were performed to provide evidence for their GH1 activities and substrate specificities. First though, the amino acid sequences of all known characterised bacterial GH1 enzymes were retrieved from the CAZy database and compared to the sequences of the three new B. licheniformis crystallographic structures which provided evidence of the putative 6Pβ-glucosidase activity of enzyme BlBglH, and dual 6Pβ-glucosidase/6Pβ-galactosidase (dual-phospho) activity of enzymes BlBglB and BlBglC. As all three enzymes were determined to be putative 6Pβ-glycosidase activity enzymes, much of the thesis focused on the overall analysis and comparison of the 6Pβ-glucosidase, 6Pβ-galactosidase, and dual-phospho activities that make up the 6Pβ-glycosidases. The 6Pβ-glycosidase active site residues were identified through consensus of binding interactions using all known 6Pβ-glycosidase PDB structures complexed complete ligand substrates. With regards to the 6Pβ-glucosidase activity, it was found that the L8b loop is longer and forms extra interactions with the L8a loop likely leading to increased L8 loop rigidity which would prevent the displacement of residue Ala423 ensuring a steric clash with galactoconfigured ligands and may engender substrate specificity for gluco-configured ligands only. Also, during molecular dynamics simulations using enzyme BlBglH (6Pβ-glucosidase activity), it was revealed that the favourable binding of substrate stabilises the loops that surround and make up the enzyme active site. Using the BlBglC (dual-phospho activity) enzyme structure with either galacto- (PNP6Pgal) or gluco-configured (PNP6Pglc) ligands, MD simulations in triplicate revealed important details of the broad specificity of dual-phospho activity enzymes. The ligand O4 hydroxyl position is the only difference between PNP6Pgal and PNP6Pgal, and it was found that residues Gln23 and Trp433 bind strongly to the ligand O3 hydroxyl group in the PNP6Pgal-enzyme complex, but to the ligand O4 hydroxyl group in the PNP6Pglc-enzyme complex. Also, His124 formed many hydrogen bonds with the PNP6Pgal O3 hydroxyl group but had none with PNP6Pglc. Alternatively, residues Tyr173, Tyr301, Gln302 and Thr321 formed hydrogen bonds with PNP6Pglc but not PNP6Pgal. Lastly, using multiple 3D structures from various GH1 activities, a large network of conserved interactions between active site residues (and other important residues) was uncovered, which most likely stabilise the loop regions that contain these residues, helping to retain their positions needed for binding molecules. Alternatively, there exists several differing residue-residue interactions when comparing each of the activities which could contribute towards individual activity substrate specificity by causing slightly different overall structure and malleability of the active site. Altogether, the findings in this thesis shed light on the function, mechanisms, dynamics, and ligand-binding of GH1 enzymes – particularly of the 6Pβ-glycosidase activities. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-04-08
Exploring the effect of the indigenous technology of oil extraction on Grade 10 Biology learners’ perspectives and sense making of enzymes
- Authors: Nyamakuti, Martha Ndeyatila
- Date: 2021-10-29
- Subjects: Biology Study and teaching (Secondary) , Enzymes , Traditional ecological knowledge Namibia , Culturally relevant pedagogy Namibia , Reasoning , Contiguity Argumentation Theory (CAT) , Namibian National Curriculum for Basic Education(NCBE) , Socio-cultural theory
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192019 , vital:45188
- Description: The current status quo is that African learners’ local indigenous knowledge (IK) and experiences from home (cultural heritage) are not considered in science classrooms. In the context of Namibia, it seems that the Namibian curriculum policies do little or nothing to decolonise and address the issues of equity and social injustice in Namibian schools. For instance, although the Namibian National Curriculum for Basic Education (NCBE) claims that it “embraces traditional knowledge”, it does not specify how this should be done in schools. As a result, little or no integration of IK is enacted in many of our schools in Namibia. Resultantly, learners seem to find science inaccessible and irrelevant to their everyday lives. It is against this background that in this study I sought to explore how the mobilisation of the indigenous technology of oil extraction (okuyenga) from marula nuts and melon seeds influences (or not) Grade 10 Biology learners’ conceptions, dispositions and sense making of the topic of enzymes. The study was located within the interpretive and indigenous research paradigms. Central to the interpretive paradigm is the development of a greater understanding of how people make sense of the contexts in which they live and work. On the other hand, central to indigenous research paradigms are belief systems based on the lived experiences, values, and histories of the participants. The Ubuntu perspective or approach in which respect and humble togetherness is emphasised is critical in indigenous research paradigms especially when researchers are working with and in communities as I did in this study. The study was conducted in an under resourced school in Walvis Bay, Namibia. A qualitative case study approach was used and data were derived from a focus group interview, participatory classroom observations, learners’ reflections, and a stimulated recall interview. Vygotsky’s socio-cultural theory was employed as a theoretical framework. Within the socio-cultural theory, mediation of learning, social interactions, and the zone of proximal development (ZPD) were used as analytical lenses. The conceptual framework comprised of perspectives (conceptions, dispositions) and sense making of enzymes by the learners. Criteria were also adapted from Atallah, Bryant, and Dada to analyse learners’ perspectives. A thematic approach to analysis was employed to come up with categories and sub-themes and thereafter, common sub-themes were combined to form themes. The findings of the study revealed that the presentations on the indigenous technology of oil extraction by the expert community members influenced learners’ conceptions, dispositions, and sense making of enzymes and other associated science concepts. This was noticed when learners extracted emerging science concepts from the indigenous technology of oil extraction. The implication of this study is that there is a need for science teachers to consider learners’ cultural heritage and integrate local IK in their Biology classrooms in order to make science accessible and relevant to learners. Moreover, the integration of local IK is critical for learners to embrace and respect their cultural heritage. This study thus recommends that teachers should make efforts to collaborate with expert community members who are the custodians of local IK and tap into their cultural heritage and wisdom to enrich teaching in their science classrooms. , Thesis (MEd) -- Faculty of Education, Education, 2021
- Full Text:
- Date Issued: 2021-10-29
- Authors: Nyamakuti, Martha Ndeyatila
- Date: 2021-10-29
- Subjects: Biology Study and teaching (Secondary) , Enzymes , Traditional ecological knowledge Namibia , Culturally relevant pedagogy Namibia , Reasoning , Contiguity Argumentation Theory (CAT) , Namibian National Curriculum for Basic Education(NCBE) , Socio-cultural theory
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192019 , vital:45188
- Description: The current status quo is that African learners’ local indigenous knowledge (IK) and experiences from home (cultural heritage) are not considered in science classrooms. In the context of Namibia, it seems that the Namibian curriculum policies do little or nothing to decolonise and address the issues of equity and social injustice in Namibian schools. For instance, although the Namibian National Curriculum for Basic Education (NCBE) claims that it “embraces traditional knowledge”, it does not specify how this should be done in schools. As a result, little or no integration of IK is enacted in many of our schools in Namibia. Resultantly, learners seem to find science inaccessible and irrelevant to their everyday lives. It is against this background that in this study I sought to explore how the mobilisation of the indigenous technology of oil extraction (okuyenga) from marula nuts and melon seeds influences (or not) Grade 10 Biology learners’ conceptions, dispositions and sense making of the topic of enzymes. The study was located within the interpretive and indigenous research paradigms. Central to the interpretive paradigm is the development of a greater understanding of how people make sense of the contexts in which they live and work. On the other hand, central to indigenous research paradigms are belief systems based on the lived experiences, values, and histories of the participants. The Ubuntu perspective or approach in which respect and humble togetherness is emphasised is critical in indigenous research paradigms especially when researchers are working with and in communities as I did in this study. The study was conducted in an under resourced school in Walvis Bay, Namibia. A qualitative case study approach was used and data were derived from a focus group interview, participatory classroom observations, learners’ reflections, and a stimulated recall interview. Vygotsky’s socio-cultural theory was employed as a theoretical framework. Within the socio-cultural theory, mediation of learning, social interactions, and the zone of proximal development (ZPD) were used as analytical lenses. The conceptual framework comprised of perspectives (conceptions, dispositions) and sense making of enzymes by the learners. Criteria were also adapted from Atallah, Bryant, and Dada to analyse learners’ perspectives. A thematic approach to analysis was employed to come up with categories and sub-themes and thereafter, common sub-themes were combined to form themes. The findings of the study revealed that the presentations on the indigenous technology of oil extraction by the expert community members influenced learners’ conceptions, dispositions, and sense making of enzymes and other associated science concepts. This was noticed when learners extracted emerging science concepts from the indigenous technology of oil extraction. The implication of this study is that there is a need for science teachers to consider learners’ cultural heritage and integrate local IK in their Biology classrooms in order to make science accessible and relevant to learners. Moreover, the integration of local IK is critical for learners to embrace and respect their cultural heritage. This study thus recommends that teachers should make efforts to collaborate with expert community members who are the custodians of local IK and tap into their cultural heritage and wisdom to enrich teaching in their science classrooms. , Thesis (MEd) -- Faculty of Education, Education, 2021
- Full Text:
- Date Issued: 2021-10-29
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:
- Date Issued: 2021-04
- 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:
- Date Issued: 2021-04
Characterization of termite Trinervitermes trinervoides metagenome-derived glycoside hydrolases, the formulation of synergistic core enzyme sets for effective sweet sorghum and corncob saccharification, and their potential industrial applications
- Authors: Mafa, Mpho Stephen
- Date: 2019
- Subjects: Termites , Metagenomics , Glucosides , Hydrolases , Enzymes , Feedstock
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/72414 , vital:30044 , DOI https://doi.org/10.21504/10962/72414
- Description: The current study investigated the biochemical properties of endo-glucanase (GH5E), exo-glucanase (GH5D), xylanase (GH5H) and endo-glucanase/xylanase (GH45), derived from the hindgut bacterial symbionts of a termite (Trinervitermes trinervoides) for their potential role in the biotechnology industry. All these enzymes, except GH5D, exhibited activities on cellulosic and xylan-rich polymeric substrates, which only displayed activity on p-nitrophenyl cellobioside. GH5D, GH5E, GH5H and GH45 enzymes retained more than 80% of their activities at pH 5.5 and also retained more than 80% of their activities at 40ºC. Furthermore, these enzymes were thermostable at 37ºC for 72 hours. GH5E, GH5H and GH45 were generally stable over a range of metal-ion. The kinetic parameters for GH5E were 5.68 mg/ml (KM) and 34.36 U/mg protein (Vmax). GH5D activity did not follow classical Michaelis-Menten kinetics, suggesting product inhibition. GH5H displayed KM values of 5.53, 95.03 and 2.10 mg/ml and Vmax values of 112.36, 144.45 and 180.32 U/mg protein on beechwood xylan, CMC, and xyloglucan, respectively. GH45 displayed a KM of 6.94 mg/ml and a Vmax of 12.30 U/mg protein on CMC. GH5D [cellobiohydrolase (CBH)] and a commercial CBHII (GH6) enzyme outperformed a commercial CBHI (GH7) enzyme when these enzymes hydrolysed β-glucan. GH5D and CBHII also displayed a higher degree of synergy on β-glucan but failed to show synergy on Avicel. We therefore concluded that GH5D and CBHII are β-glucan-specific cellobiohydrolases. The corncob (CC) and sweet sorghum bagasse (SSB) substrates were pretreated with lime, NaOH and NaClO2. Subsequent to pretreatment, these substrates were used to investigate if GH5D, GH5E, GH5H and GH45 could operate in synergy. Results revealed that out of 12 possible core enzyme sets constructed, only two (referred to as CES-E and CES-H) displayed higher activities on pretreated CC or SSB. Simultaneous synergy was generally the most effective mode of synergy during hydrolysis of alkaline pretreated SSB and CC samples by both CES-E and CES-H. Both core enzyme sets did not display synergy on oxidative pretreated substrates. These findings suggest that lime and NaOH are more effective pretreatments for CC and SSB substrates. We used PRotein Interactive MOdeling (PRIMO) software to demonstrate that GH5D protein structure is an (α/β)8 barrel with a tunnel-like active site. Enzymes with this type of protein structure are able to perform transglycosylation, a process in which GH5D produced methyl, ethyl and propyl cellobiosides. We concluded that the GH5D, GH5E, GH5H and GH45 enzymes possess novel biochemical properties and that they form synergy during the hydrolysis of complex substrates (SSB and CC). GH5D transglycosylation could be used to produce novel biodegradable chemicals with special properties (e.g. anti-microbial properties). In conclusion, our findings suggest that GH5D, GH5E, GH5H and GH45 can potentially be used to improve biorefinery processes. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2019
- Full Text:
- Date Issued: 2019
- Authors: Mafa, Mpho Stephen
- Date: 2019
- Subjects: Termites , Metagenomics , Glucosides , Hydrolases , Enzymes , Feedstock
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/72414 , vital:30044 , DOI https://doi.org/10.21504/10962/72414
- Description: The current study investigated the biochemical properties of endo-glucanase (GH5E), exo-glucanase (GH5D), xylanase (GH5H) and endo-glucanase/xylanase (GH45), derived from the hindgut bacterial symbionts of a termite (Trinervitermes trinervoides) for their potential role in the biotechnology industry. All these enzymes, except GH5D, exhibited activities on cellulosic and xylan-rich polymeric substrates, which only displayed activity on p-nitrophenyl cellobioside. GH5D, GH5E, GH5H and GH45 enzymes retained more than 80% of their activities at pH 5.5 and also retained more than 80% of their activities at 40ºC. Furthermore, these enzymes were thermostable at 37ºC for 72 hours. GH5E, GH5H and GH45 were generally stable over a range of metal-ion. The kinetic parameters for GH5E were 5.68 mg/ml (KM) and 34.36 U/mg protein (Vmax). GH5D activity did not follow classical Michaelis-Menten kinetics, suggesting product inhibition. GH5H displayed KM values of 5.53, 95.03 and 2.10 mg/ml and Vmax values of 112.36, 144.45 and 180.32 U/mg protein on beechwood xylan, CMC, and xyloglucan, respectively. GH45 displayed a KM of 6.94 mg/ml and a Vmax of 12.30 U/mg protein on CMC. GH5D [cellobiohydrolase (CBH)] and a commercial CBHII (GH6) enzyme outperformed a commercial CBHI (GH7) enzyme when these enzymes hydrolysed β-glucan. GH5D and CBHII also displayed a higher degree of synergy on β-glucan but failed to show synergy on Avicel. We therefore concluded that GH5D and CBHII are β-glucan-specific cellobiohydrolases. The corncob (CC) and sweet sorghum bagasse (SSB) substrates were pretreated with lime, NaOH and NaClO2. Subsequent to pretreatment, these substrates were used to investigate if GH5D, GH5E, GH5H and GH45 could operate in synergy. Results revealed that out of 12 possible core enzyme sets constructed, only two (referred to as CES-E and CES-H) displayed higher activities on pretreated CC or SSB. Simultaneous synergy was generally the most effective mode of synergy during hydrolysis of alkaline pretreated SSB and CC samples by both CES-E and CES-H. Both core enzyme sets did not display synergy on oxidative pretreated substrates. These findings suggest that lime and NaOH are more effective pretreatments for CC and SSB substrates. We used PRotein Interactive MOdeling (PRIMO) software to demonstrate that GH5D protein structure is an (α/β)8 barrel with a tunnel-like active site. Enzymes with this type of protein structure are able to perform transglycosylation, a process in which GH5D produced methyl, ethyl and propyl cellobiosides. We concluded that the GH5D, GH5E, GH5H and GH45 enzymes possess novel biochemical properties and that they form synergy during the hydrolysis of complex substrates (SSB and CC). GH5D transglycosylation could be used to produce novel biodegradable chemicals with special properties (e.g. anti-microbial properties). In conclusion, our findings suggest that GH5D, GH5E, GH5H and GH45 can potentially be used to improve biorefinery processes. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2019
- Full Text:
- Date Issued: 2019
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