Characterisation of the cellulolytic and hemicellulolytic system of Bacillus Licheniformis SVD1 and the isolation and characterisation of a multi-enzyme complex
- Authors: Van Dyk, Jacoba Susanna
- Date: 2009
- Subjects: Lignocellulose Lignocellulose -- Biotechnology Lignocellulose -- Biodegradation Plant biotechnology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3936 , http://hdl.handle.net/10962/d1003995
- Description: The biological degradation of lignocellulose into fermentable sugars for the production of liquid transportation fuels is feasible and sustainable, but equires a variety of enzymes working in synergy as lignocellulose is a complex and recalcitrant substrate. The cellulosome is a multi-enzyme complex (MEC) with a variety of cellulolytic and hemicellulolytic enzymes that appears to facilitate an enhanced synergy and efficiency, as compared to free enzymes, for the degradation of recalcitrant substrates such as lignocellulose and plant cell walls. Most of the studies on cellulosomes have focused on a few organisms; C. thermocellum, C. cellulovorans and C. cellulolyticum, and there is only limited knowledge vailable on similar complexes in other organisms. Some MECs have been identified in aerobic bacteria such as Bacillus circulans and Paenibacillus curdlanolyticus, but the nature of these MECs have not been fully elucidated. This study investigated the cellulolytic and emi-cellulolytic system of Bacillus licheniformis SVD1 with specific reference to the presence of a MEC, which has never been reported in the literature for B. licheniformis. A MEC of approximately 2,000 kDa in size, based on size exclusion chromatography using Sepharose 4B, was purified from a culture of B. licheniformis. When investigating the presence of enzyme activity in the total crude fraction as well as the MEC of a birchwood xylan culture, B. licheniformis was found to display a variety of enzyme activities on a range of substrates, although xylanases were by far the predominant enzyme activity present in both the crude and MEC fractions. Based on zymogram analysis there were three CMCases, seven xylanases, three mannanases and two pectinases in the crude fraction, while the MEC had two CMCases, seven xylanases, two mannanases and one pectinase. The pectinases in the crude could be identified as a pectin methyl esterase and a lyase, while the methyl esterase was absent in the MEC. Seventeen protein species could be detected in the MEC but only nine of these displayed activity on the substrates tested. The possible presence of a β-xylosidase in the crude fraction was deduced from thin layer chromatography (TLC) which demonstrated the production of xylose by the crude fraction. It was furthermore established that B. licheniformis SVD1 was able to regulate levels of enzyme expression based on the substrate the organism was cultured on. It was found that complexed xylanase activity had a pH optimum of between pH 6.0 and 7.0 and a temperature optimum of 55oC. Complexed xylanase activity was found to be slightly inhibited by CaCl2 and inhibited to a greater extent by EDTA. Complexed xylanase activity was further shown to be activated in the presence of xylose and xylobiose, both compounds which are products of enzymatic degradation. Ethanol was found to inhibit complexed xylanase activity. The kinetic parameters for complexed xylanase activity were measured and the Km value was calculated as 2.84 mg/ml while the maximal velocity (Vmax) was calculated as 0.146 U (μmol/min/ml). Binding studies, transmission electron microscopy (TEM) and a bioinformatic analysis was conducted to investigate whether the MEC in B. licheniformis SVD1 was a putative cellulosome. The MEC was found to be unable to bind to Avicel, but was able to bind to insoluble birchwood xylan, indicating the absence of a CBM3a domain common to cellulosomal scaffoldin proteins. TEM micrographs revealed the presence of cell surface structures on cells of B. licheniformis SVD1 cultured on cellobiose and birchwood xylan. However, it could not be established whether these cell surface structures could be ascribed to the presence of the MECs on the cell surface. Bioinformatic analysis was conducted on the available genome sequence of a different strain of B. licheniformis, namely DSM 13 and ATCC 14580. No sequence homology was found with cohesin and dockerin sequences from various cellulosomal species, indicating that these strains most likely do not encode for a cellulosome. This study described and characterised a MEC that was a functional enzyme complex and did not appear to be a mere aggregation of proteins. It displayed a variety of hemi-cellulolytic activities and the available evidence suggests that it is not a cellulosome, but should rather be termed a xylanosome. Further investigation should be carried out to determine the structural basis of this MEC.
- Full Text:
- Authors: Van Dyk, Jacoba Susanna
- Date: 2009
- Subjects: Lignocellulose Lignocellulose -- Biotechnology Lignocellulose -- Biodegradation Plant biotechnology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3936 , http://hdl.handle.net/10962/d1003995
- Description: The biological degradation of lignocellulose into fermentable sugars for the production of liquid transportation fuels is feasible and sustainable, but equires a variety of enzymes working in synergy as lignocellulose is a complex and recalcitrant substrate. The cellulosome is a multi-enzyme complex (MEC) with a variety of cellulolytic and hemicellulolytic enzymes that appears to facilitate an enhanced synergy and efficiency, as compared to free enzymes, for the degradation of recalcitrant substrates such as lignocellulose and plant cell walls. Most of the studies on cellulosomes have focused on a few organisms; C. thermocellum, C. cellulovorans and C. cellulolyticum, and there is only limited knowledge vailable on similar complexes in other organisms. Some MECs have been identified in aerobic bacteria such as Bacillus circulans and Paenibacillus curdlanolyticus, but the nature of these MECs have not been fully elucidated. This study investigated the cellulolytic and emi-cellulolytic system of Bacillus licheniformis SVD1 with specific reference to the presence of a MEC, which has never been reported in the literature for B. licheniformis. A MEC of approximately 2,000 kDa in size, based on size exclusion chromatography using Sepharose 4B, was purified from a culture of B. licheniformis. When investigating the presence of enzyme activity in the total crude fraction as well as the MEC of a birchwood xylan culture, B. licheniformis was found to display a variety of enzyme activities on a range of substrates, although xylanases were by far the predominant enzyme activity present in both the crude and MEC fractions. Based on zymogram analysis there were three CMCases, seven xylanases, three mannanases and two pectinases in the crude fraction, while the MEC had two CMCases, seven xylanases, two mannanases and one pectinase. The pectinases in the crude could be identified as a pectin methyl esterase and a lyase, while the methyl esterase was absent in the MEC. Seventeen protein species could be detected in the MEC but only nine of these displayed activity on the substrates tested. The possible presence of a β-xylosidase in the crude fraction was deduced from thin layer chromatography (TLC) which demonstrated the production of xylose by the crude fraction. It was furthermore established that B. licheniformis SVD1 was able to regulate levels of enzyme expression based on the substrate the organism was cultured on. It was found that complexed xylanase activity had a pH optimum of between pH 6.0 and 7.0 and a temperature optimum of 55oC. Complexed xylanase activity was found to be slightly inhibited by CaCl2 and inhibited to a greater extent by EDTA. Complexed xylanase activity was further shown to be activated in the presence of xylose and xylobiose, both compounds which are products of enzymatic degradation. Ethanol was found to inhibit complexed xylanase activity. The kinetic parameters for complexed xylanase activity were measured and the Km value was calculated as 2.84 mg/ml while the maximal velocity (Vmax) was calculated as 0.146 U (μmol/min/ml). Binding studies, transmission electron microscopy (TEM) and a bioinformatic analysis was conducted to investigate whether the MEC in B. licheniformis SVD1 was a putative cellulosome. The MEC was found to be unable to bind to Avicel, but was able to bind to insoluble birchwood xylan, indicating the absence of a CBM3a domain common to cellulosomal scaffoldin proteins. TEM micrographs revealed the presence of cell surface structures on cells of B. licheniformis SVD1 cultured on cellobiose and birchwood xylan. However, it could not be established whether these cell surface structures could be ascribed to the presence of the MECs on the cell surface. Bioinformatic analysis was conducted on the available genome sequence of a different strain of B. licheniformis, namely DSM 13 and ATCC 14580. No sequence homology was found with cohesin and dockerin sequences from various cellulosomal species, indicating that these strains most likely do not encode for a cellulosome. This study described and characterised a MEC that was a functional enzyme complex and did not appear to be a mere aggregation of proteins. It displayed a variety of hemi-cellulolytic activities and the available evidence suggests that it is not a cellulosome, but should rather be termed a xylanosome. Further investigation should be carried out to determine the structural basis of this MEC.
- Full Text:
Characterisation of the plasmodium falciparum Hsp40 chaperones and their partnerships with Hsp70
- Authors: Botha, Melissa
- Date: 2009
- Subjects: Heat shock proteins Plasmodium falciparum Protein folding Molecular chaperones Malaria
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3938 , http://hdl.handle.net/10962/d1003997
- Description: Central to this research, 40 kDa Heat shock proteins (Hsp40s) are known to partner (or cochaperone) 70 kDa Heat shock proteins (Hsp70s), facilitating the selection and transfer of protein substrate to Hsp70 and the stimulation of the protein folding ability of Hsp70. Members of the diverse Hsp70-Hsp40 protein complement of Plasmodium falciparum have been implicated in the cytoprotection of this malaria parasite, and are thought to facilitate the protein folding, assembly and translocation tasks required by the parasite to commandeer the infected human erythrocyte subsequent to invasion. In particular, the parasite has evolved an expanded and specialised 43- member suite of Hsp40 proteins, 19 of which bear an identifiable export motif for secretion into the infected erythrocyte cytoplasm where they potentially interact with human Hsp70. Although type I Hsp40 proteins are representative of typical regulators of Hsp70 activity, only two of these proteins are apparent in the parasite’s Hsp40 complement. These include a characteristic type I Hsp40 termed PfHsp40, and a larger, atypical type I Hsp40 termed Pfj1. Both Hsp40 proteins are predicted to be parasite-resident and are most likely to facilitate the co-chaperone regulation of the highly abundant and stress-inducible Hsp70 homolog, PfHsp70-I. In this work, the co-chaperone functionality of PfHsp40 and Pfj1 was elucidated using in vivo and in vitro assays. Purified recombinant PfHsp40 was shown to stimulate the ATPase activity of PfHsp70-I in in vitro single turnover and steady state ATPase assays, and co-operate with PfHsp70-I in in vitro aggregation suppression assays. In these in vitro assays, heterologous partnerships could be demonstrated between PfHsp70-I and the human Hsp40, Hsj1a, and human Hsp70 and PfHsp40, suggesting a common mode of Hsp70-Hsp40 interaction in the parasite and host organism. The functionality of the signature Hsp40 domain, the Jdomain, of Pfj1 was demonstrated by its ability to replace the equivalent domain of the A. tumefaciens Hsp40, Agt DnaJ, in interactions with the prokaryotic Hsp70, DnaK, in the thermosensitive dnaJ cbpA E. coli OD259 deletion strain. An H33Q mutation introduced into the invariant and crucial HPD tripeptide motif abrogated the functionality of the J-domain in the in vivo complementation system. These findings provide the first evidence for the conservation of the prototypical mode of J-domain based interaction of Hsp40 with Hsp70 in P. falciparum. Immunofluorescence staining revealed the localisation of PfHsp40 to the parasite cytoplasm, and Pfj1 to the parasite cytoplasm and nucleus in cultured intraerythrocytic stage P. falciparum parasites. PfHsp70-I was also shown to localise to the parasite cytoplasm and nucleus in these stages, consistent with the literature. Overall we propose that PfHsp40 and Pfj1 co-localise with and regulate the chaperone activity of PfHsp70-I in P. falciparum. This is the first study to identify and provide evidence for a functional Hsp70-Hsp40 partnership in P. falciparum, and provides a platform for future studies to elucidate the importance of these chaperone partnerships in the establishment and survival of the parasite in the intraerythrocytic-stages of development.
- Full Text:
- Authors: Botha, Melissa
- Date: 2009
- Subjects: Heat shock proteins Plasmodium falciparum Protein folding Molecular chaperones Malaria
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3938 , http://hdl.handle.net/10962/d1003997
- Description: Central to this research, 40 kDa Heat shock proteins (Hsp40s) are known to partner (or cochaperone) 70 kDa Heat shock proteins (Hsp70s), facilitating the selection and transfer of protein substrate to Hsp70 and the stimulation of the protein folding ability of Hsp70. Members of the diverse Hsp70-Hsp40 protein complement of Plasmodium falciparum have been implicated in the cytoprotection of this malaria parasite, and are thought to facilitate the protein folding, assembly and translocation tasks required by the parasite to commandeer the infected human erythrocyte subsequent to invasion. In particular, the parasite has evolved an expanded and specialised 43- member suite of Hsp40 proteins, 19 of which bear an identifiable export motif for secretion into the infected erythrocyte cytoplasm where they potentially interact with human Hsp70. Although type I Hsp40 proteins are representative of typical regulators of Hsp70 activity, only two of these proteins are apparent in the parasite’s Hsp40 complement. These include a characteristic type I Hsp40 termed PfHsp40, and a larger, atypical type I Hsp40 termed Pfj1. Both Hsp40 proteins are predicted to be parasite-resident and are most likely to facilitate the co-chaperone regulation of the highly abundant and stress-inducible Hsp70 homolog, PfHsp70-I. In this work, the co-chaperone functionality of PfHsp40 and Pfj1 was elucidated using in vivo and in vitro assays. Purified recombinant PfHsp40 was shown to stimulate the ATPase activity of PfHsp70-I in in vitro single turnover and steady state ATPase assays, and co-operate with PfHsp70-I in in vitro aggregation suppression assays. In these in vitro assays, heterologous partnerships could be demonstrated between PfHsp70-I and the human Hsp40, Hsj1a, and human Hsp70 and PfHsp40, suggesting a common mode of Hsp70-Hsp40 interaction in the parasite and host organism. The functionality of the signature Hsp40 domain, the Jdomain, of Pfj1 was demonstrated by its ability to replace the equivalent domain of the A. tumefaciens Hsp40, Agt DnaJ, in interactions with the prokaryotic Hsp70, DnaK, in the thermosensitive dnaJ cbpA E. coli OD259 deletion strain. An H33Q mutation introduced into the invariant and crucial HPD tripeptide motif abrogated the functionality of the J-domain in the in vivo complementation system. These findings provide the first evidence for the conservation of the prototypical mode of J-domain based interaction of Hsp40 with Hsp70 in P. falciparum. Immunofluorescence staining revealed the localisation of PfHsp40 to the parasite cytoplasm, and Pfj1 to the parasite cytoplasm and nucleus in cultured intraerythrocytic stage P. falciparum parasites. PfHsp70-I was also shown to localise to the parasite cytoplasm and nucleus in these stages, consistent with the literature. Overall we propose that PfHsp40 and Pfj1 co-localise with and regulate the chaperone activity of PfHsp70-I in P. falciparum. This is the first study to identify and provide evidence for a functional Hsp70-Hsp40 partnership in P. falciparum, and provides a platform for future studies to elucidate the importance of these chaperone partnerships in the establishment and survival of the parasite in the intraerythrocytic-stages of development.
- Full Text:
Characterisation of Trypanosomal Type III and Type IV Hsp40 proteins
- Louw, Cassandra Alexandrovna
- Authors: Louw, Cassandra Alexandrovna
- Date: 2009
- Subjects: Trypanosoma Heat shock proteins African trypanosomiasis Epidemic encephalitis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3926 , http://hdl.handle.net/10962/d1003985
- Description: The heat shock protein-70 (Hsp70) family of molecular chaperones are ubiquitous highly conserved proteins that are critical for the viability of cellular homeostasis. The ATPase activity of Hsp70 proteins is critical to their function as the affinity of a given Hsp70 for non-native substrate is modulated by ATP binding and hydrolysis. When bound to ATP, Hsp70s possess a low affinity for a given substrate protein, while the hydrolysis of ATP to ADP causes a conformational change that results in a high affinity for substrate proteins. The basal ATPase activity of Hsp70s is too low to facilitate their function in vivo, and co-chaperones are essential to modulate the efficient protein folding by Hsp70. Heat shock protein-40 (Hsp40) heat shock proteins are essential for the in vivo function of Hsp70s by stimulating the ATPase activity of these proteins and facilitating transfer of substrates. The Type III class of Hsp40 proteins have not been well characterised due to their poor levels of conservation at the primary sequence level. This is due to the fact that Type III Hsp40s only contain a J-domain and a poorly conserved C-terminal region. The newly identified Type IV class of Hsp40s, contain an abrogated HPD tripeptide motif in the J-domain and have also not been extensively studied. Trypanosoma brucei (T. brucei) is a unicellular flagellated protozoan parasite. It is the causative agent of Human African Trypansomiasis (HAT) which results in thousands of deaths and devastating agricultural losses in many parts of Africa. T. brucei undergoes a complex lifecycle that is characterised by the transition from an insect vector to a mammalian host in markedly different conditions of temperature, pH, nutrient availability and respiratory requirements. It has been proposed that molecular chaperones may enhance the survival of these parasites due to their cytoprotective effect in combating cellular stress. Due to the fact that T. brucei infection is invariably fatal if left untreated, and that no novel treatment regimens have been developed recently, the identification of potential novel drug targets among proteins essential to the parasite’s survival in the host organism is an attractive aspect of T. brucei research. Because Type III Hsp40s are poorly conserved with respect to Hsp40s found in the human host, the identification of any of these proteins found to be essential to T. brucei survival in humans could potentially make attractive novel drug targets. An in depth in silico investigation into the Type III Hsp40 complement as well as partner Hsp70 proteins in T.brucei was performed. T. brucei possesses 65 Hsp40 proteins, of which 47 were classed as Type III and 6 of which were identified as being putative Type IV Hsp40s. A small but significant number (5) of Type III TbHsp40s contained tetratricopeptide (TPR) domains in addition to the J-domain. The J-domains of the Type III TbHsp40 complement were found to be conserved with respect to those of canonical Hsp40 proteins, although the mutation of certain residues that play a key role in Hsp40-Hsp70 interaction was noted. Potential partnerships of these proteins in the parasite was also investigated. The coding regions of three previously uncharacterised TbHsp40s were successfully amplified from T. brucei TREU927 genomic DNA and cloned into an expression vector. Tbj1, a Tcj1 ortholog, was selected for further study and successfully expressed and biochemically characterised. Tbj1 expressed in E. coli was found to be insoluble, but large amounts were recovered with the aid of a denaturing purification followed by refolding elution strategies, and the bulk of the protein recovered was in compact monomeric form as determined by size-exclusion chromatography fast protein liquid chromatography (SEC-FPLC). The addition of Tbj1 to a thermally aggregated substrate resulted in increased levels of aggregation, although Tbj1 was able to assist two Hsp70 proteins in the suppression of aggregation. Tbj1 proved unable to stimulate the ATPase activity of these same Hsp70s, and could not rescue temperature sensitive cells when replacing E.coli DnaJ and CbpA. It was concluded that Tbj1 does not possess independent chaperone activity, but could display Hsp40 co-chaperone properties under certain circumstances. This could allude to a specialised function in the T. brucei parasite. The lack of human orthologues to Tbj1 could result in the attractiveness of this protein as a novel drug target.
- Full Text:
- Authors: Louw, Cassandra Alexandrovna
- Date: 2009
- Subjects: Trypanosoma Heat shock proteins African trypanosomiasis Epidemic encephalitis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3926 , http://hdl.handle.net/10962/d1003985
- Description: The heat shock protein-70 (Hsp70) family of molecular chaperones are ubiquitous highly conserved proteins that are critical for the viability of cellular homeostasis. The ATPase activity of Hsp70 proteins is critical to their function as the affinity of a given Hsp70 for non-native substrate is modulated by ATP binding and hydrolysis. When bound to ATP, Hsp70s possess a low affinity for a given substrate protein, while the hydrolysis of ATP to ADP causes a conformational change that results in a high affinity for substrate proteins. The basal ATPase activity of Hsp70s is too low to facilitate their function in vivo, and co-chaperones are essential to modulate the efficient protein folding by Hsp70. Heat shock protein-40 (Hsp40) heat shock proteins are essential for the in vivo function of Hsp70s by stimulating the ATPase activity of these proteins and facilitating transfer of substrates. The Type III class of Hsp40 proteins have not been well characterised due to their poor levels of conservation at the primary sequence level. This is due to the fact that Type III Hsp40s only contain a J-domain and a poorly conserved C-terminal region. The newly identified Type IV class of Hsp40s, contain an abrogated HPD tripeptide motif in the J-domain and have also not been extensively studied. Trypanosoma brucei (T. brucei) is a unicellular flagellated protozoan parasite. It is the causative agent of Human African Trypansomiasis (HAT) which results in thousands of deaths and devastating agricultural losses in many parts of Africa. T. brucei undergoes a complex lifecycle that is characterised by the transition from an insect vector to a mammalian host in markedly different conditions of temperature, pH, nutrient availability and respiratory requirements. It has been proposed that molecular chaperones may enhance the survival of these parasites due to their cytoprotective effect in combating cellular stress. Due to the fact that T. brucei infection is invariably fatal if left untreated, and that no novel treatment regimens have been developed recently, the identification of potential novel drug targets among proteins essential to the parasite’s survival in the host organism is an attractive aspect of T. brucei research. Because Type III Hsp40s are poorly conserved with respect to Hsp40s found in the human host, the identification of any of these proteins found to be essential to T. brucei survival in humans could potentially make attractive novel drug targets. An in depth in silico investigation into the Type III Hsp40 complement as well as partner Hsp70 proteins in T.brucei was performed. T. brucei possesses 65 Hsp40 proteins, of which 47 were classed as Type III and 6 of which were identified as being putative Type IV Hsp40s. A small but significant number (5) of Type III TbHsp40s contained tetratricopeptide (TPR) domains in addition to the J-domain. The J-domains of the Type III TbHsp40 complement were found to be conserved with respect to those of canonical Hsp40 proteins, although the mutation of certain residues that play a key role in Hsp40-Hsp70 interaction was noted. Potential partnerships of these proteins in the parasite was also investigated. The coding regions of three previously uncharacterised TbHsp40s were successfully amplified from T. brucei TREU927 genomic DNA and cloned into an expression vector. Tbj1, a Tcj1 ortholog, was selected for further study and successfully expressed and biochemically characterised. Tbj1 expressed in E. coli was found to be insoluble, but large amounts were recovered with the aid of a denaturing purification followed by refolding elution strategies, and the bulk of the protein recovered was in compact monomeric form as determined by size-exclusion chromatography fast protein liquid chromatography (SEC-FPLC). The addition of Tbj1 to a thermally aggregated substrate resulted in increased levels of aggregation, although Tbj1 was able to assist two Hsp70 proteins in the suppression of aggregation. Tbj1 proved unable to stimulate the ATPase activity of these same Hsp70s, and could not rescue temperature sensitive cells when replacing E.coli DnaJ and CbpA. It was concluded that Tbj1 does not possess independent chaperone activity, but could display Hsp40 co-chaperone properties under certain circumstances. This could allude to a specialised function in the T. brucei parasite. The lack of human orthologues to Tbj1 could result in the attractiveness of this protein as a novel drug target.
- Full Text:
Establishing experimental systems for studying the replication biology of Providence virus
- Authors: Walter, Cheryl Tracy
- Date: 2009
- Subjects: Insects -- Viruses Insects -- Diseases Insects -- Parasites Host-virus relationships RNA viruses DNA Insects as carriers of disease
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3928 , http://hdl.handle.net/10962/d1003987
- Description: Providence virus (PrV) is a member of the Tetraviridae, a family of small, positive sense, single-stranded RNA viruses, which characteristically infect the midgut tissue of heliothine larvae. PrV is the only known tetravirus that replicates in cultured insect cells. The virus comprises a monopartite genome resembling members of the genus Betatetravirus with the capsid precursor protein undergoing autoproteolytic cleavage at its C-terminus consistent with other tetravirus capsid precursor proteins. Analysis of viral cDNA predicted the presence of three potential overlapping gene products (from 5` to 3`): (1) p130, a protein of unrecognized nucleotide or amino acid homology with a 2A-like processing site at its N-terminus; (2) p104, the replicase ORF, which was found to be phylogenetically related to tombus-and umbraviruses replicases. The presence of a read-through stop signal in the p104 ORF was proposed to produce and amino terminal product with a predicted MW of 40 kDa (p40) and (3) the capsid protein precursor (81 kDa) which has two 2A-like processing sites at its N-terminus. Metabolic radiolabelling of viral translation products in persistently infected MG8 cells and in vitro translation of the individual ORFs were performed in order to analyse the expression of PrV gene products. p130 was translated with no evidence of 2A-like processing. Two products of 40 kDa and 104 kDa were translated from the p104 ORF, indicating that the read-through stop signal was likely to be functional. Finally, the capsid protein precursor ORF produced a major translation product of 68 kDa corresponding to the capsid protein precursor as well a peptide of 15 kDa that was attributed to the activity of the second 2A-like site at the N-terminus of the p81 ORF. The subcellular distribution of viral RNA (vRNA) and p40 in MG8 cells was investigated using immunofluorescence and biochemical fractionation. The results showed that p40/p104 and vRNA accumulated in polarized, punctate structures in some but not all MG8 cells and in some cases, co-localization was observed. This thesis concludes that PrV is a novel tetravirus with significant similarities plant carmolike viruses that should be re-classified at the family level.
- Full Text:
- Authors: Walter, Cheryl Tracy
- Date: 2009
- Subjects: Insects -- Viruses Insects -- Diseases Insects -- Parasites Host-virus relationships RNA viruses DNA Insects as carriers of disease
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3928 , http://hdl.handle.net/10962/d1003987
- Description: Providence virus (PrV) is a member of the Tetraviridae, a family of small, positive sense, single-stranded RNA viruses, which characteristically infect the midgut tissue of heliothine larvae. PrV is the only known tetravirus that replicates in cultured insect cells. The virus comprises a monopartite genome resembling members of the genus Betatetravirus with the capsid precursor protein undergoing autoproteolytic cleavage at its C-terminus consistent with other tetravirus capsid precursor proteins. Analysis of viral cDNA predicted the presence of three potential overlapping gene products (from 5` to 3`): (1) p130, a protein of unrecognized nucleotide or amino acid homology with a 2A-like processing site at its N-terminus; (2) p104, the replicase ORF, which was found to be phylogenetically related to tombus-and umbraviruses replicases. The presence of a read-through stop signal in the p104 ORF was proposed to produce and amino terminal product with a predicted MW of 40 kDa (p40) and (3) the capsid protein precursor (81 kDa) which has two 2A-like processing sites at its N-terminus. Metabolic radiolabelling of viral translation products in persistently infected MG8 cells and in vitro translation of the individual ORFs were performed in order to analyse the expression of PrV gene products. p130 was translated with no evidence of 2A-like processing. Two products of 40 kDa and 104 kDa were translated from the p104 ORF, indicating that the read-through stop signal was likely to be functional. Finally, the capsid protein precursor ORF produced a major translation product of 68 kDa corresponding to the capsid protein precursor as well a peptide of 15 kDa that was attributed to the activity of the second 2A-like site at the N-terminus of the p81 ORF. The subcellular distribution of viral RNA (vRNA) and p40 in MG8 cells was investigated using immunofluorescence and biochemical fractionation. The results showed that p40/p104 and vRNA accumulated in polarized, punctate structures in some but not all MG8 cells and in some cases, co-localization was observed. This thesis concludes that PrV is a novel tetravirus with significant similarities plant carmolike viruses that should be re-classified at the family level.
- Full Text:
Investigating the enzymatic mechanism of platinum nanoparticle synthesis in sulfate-reducing bacteria
- Authors: Riddin, Tamsyn Louise
- Date: 2009
- Subjects: Platinum , Nanoparticles , Sulfate-reducing bacteria
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3965 , http://hdl.handle.net/10962/d1004024 , Platinum , Nanoparticles , Sulfate-reducing bacteria
- Description: Efforts to discover an efficient yet environmentally friendly mode of metal nanoparticle (NP) synthesis are increasing rapidly. A ‘green’ route that avoids the high costs, toxic wastes and complicated protocols associated with chemical synthesis methods is therefore highly sought after. A biologically based protocol will provide the possibility of gaining control over the mechanism merely by manipulating the experimental conditions of the system. Given that the properties of nanoparticles are highly dependant on the morphology of the particles themselves, this mechanistic control will provide significant industrial advantages with regards to tailoring specific properties of the nanoparticles produced. The key objectives of this study were to: a) determine whether a consortium of sulfate-reducing bacteria was capable of platinum nanoparticle synthesis, b) elucidate the bioreductive, enzymatic mechanism responsible, and c) attempt to control the morphologies of the particles produced. A consortium of sulfate-reducing bacteria (SRB), isolated from sewage sludge, was used in these investigations due to the advantages a consortium provides in comparison to pure cultures. The syntrophic relationships established within the constituent species not only prevent the growth of contaminant microbes, but increases the oxygen-tolerance of the system as a whole. The sulfate-reducing consortium was shown to possess an aerobic mechanism for Pt(IV) reduction which, though different from the anaerobic bioreductive mechanism previously identified in literature, did not require an exogenous electron donor. It was demonstrated that the Pt(IV) ion becomes reduced to Pt(0) via a two-cycle mechanism involving Pt(II) as the intermediate. Further investigation elucidated the reduction of Pt(IV) to Pt(II) to be dependant on a novel Pt(IV) reductase which becomes upregulated in the presence of Cu(II), while the reduction of Pt(II) to Pt(0) occurred by means of a periplasmic hydrogenase. To our knowledge, this is the first time a coupled mechanism for Pt(IV) reduction by micro-organisms has been proposed. A cell-free, crude protein solution from the consortium produced both geometric and irregular platinum nanoparticles. The wavelength of 334 nm was chosen as a nonquantitative indicator of Pt(0) nanoparticle formation over time. The optimum conditions for nanoparticle synthesis were pH 9.0, 65 ˚C and 0.75 mM Pt(IV) as H2PtCl6 salt. In the absence of a buffer a Pt(IV) concentration > 1 mM resulted in the precipitation of protein-nanoparticle bioconjugates, due to unfavourable acidic conditions. This demonstrated that the nanoparticles were binding to and becoming stabilised by general protein in the cell-free solution. Upon addition of a sodium-bicarbonate buffer, a general increase in Pt(IV) reduction to Pt(II) was observed. The addition of the buffer also resulted in an unexplained change in particle morphology and for this reason was not used in subsequent investigations. Polyvinylpyrrolidone (PVP) was shown to compromise the reduction rate of the Pt(IV) ion by SRB cells. The presence of extracellular NP’s was suggested by the colour of the supernatant turning brown and the A334 increasing over time. Attempts to visualise the particles by transmission electron microscopy (TEM) resulted in an unexpected phenomenon where nanoparticles could be observed to form dynamically upon irradiation by the electron beam. Extended irradiation by the electron beam also resulted in structural changes of the particles occurring during observation. An increase in temperature was shown to increase the reduction rate which in turn resulted in particles decreasing in size. The starting pH was shown to have a significant effect on the reduction rate and particle morphology although specific trends could not be identified. In conclusion, the cell-soluble extract from the sulfate-reducing consortium investigated, is capable of Pt(0) nanoparticle synthesis. Precise control over the particle morphology was not attained although the mechanism was further clarified and optimal conditions for nanoparticle synthesis were determined.
- Full Text:
- Authors: Riddin, Tamsyn Louise
- Date: 2009
- Subjects: Platinum , Nanoparticles , Sulfate-reducing bacteria
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3965 , http://hdl.handle.net/10962/d1004024 , Platinum , Nanoparticles , Sulfate-reducing bacteria
- Description: Efforts to discover an efficient yet environmentally friendly mode of metal nanoparticle (NP) synthesis are increasing rapidly. A ‘green’ route that avoids the high costs, toxic wastes and complicated protocols associated with chemical synthesis methods is therefore highly sought after. A biologically based protocol will provide the possibility of gaining control over the mechanism merely by manipulating the experimental conditions of the system. Given that the properties of nanoparticles are highly dependant on the morphology of the particles themselves, this mechanistic control will provide significant industrial advantages with regards to tailoring specific properties of the nanoparticles produced. The key objectives of this study were to: a) determine whether a consortium of sulfate-reducing bacteria was capable of platinum nanoparticle synthesis, b) elucidate the bioreductive, enzymatic mechanism responsible, and c) attempt to control the morphologies of the particles produced. A consortium of sulfate-reducing bacteria (SRB), isolated from sewage sludge, was used in these investigations due to the advantages a consortium provides in comparison to pure cultures. The syntrophic relationships established within the constituent species not only prevent the growth of contaminant microbes, but increases the oxygen-tolerance of the system as a whole. The sulfate-reducing consortium was shown to possess an aerobic mechanism for Pt(IV) reduction which, though different from the anaerobic bioreductive mechanism previously identified in literature, did not require an exogenous electron donor. It was demonstrated that the Pt(IV) ion becomes reduced to Pt(0) via a two-cycle mechanism involving Pt(II) as the intermediate. Further investigation elucidated the reduction of Pt(IV) to Pt(II) to be dependant on a novel Pt(IV) reductase which becomes upregulated in the presence of Cu(II), while the reduction of Pt(II) to Pt(0) occurred by means of a periplasmic hydrogenase. To our knowledge, this is the first time a coupled mechanism for Pt(IV) reduction by micro-organisms has been proposed. A cell-free, crude protein solution from the consortium produced both geometric and irregular platinum nanoparticles. The wavelength of 334 nm was chosen as a nonquantitative indicator of Pt(0) nanoparticle formation over time. The optimum conditions for nanoparticle synthesis were pH 9.0, 65 ˚C and 0.75 mM Pt(IV) as H2PtCl6 salt. In the absence of a buffer a Pt(IV) concentration > 1 mM resulted in the precipitation of protein-nanoparticle bioconjugates, due to unfavourable acidic conditions. This demonstrated that the nanoparticles were binding to and becoming stabilised by general protein in the cell-free solution. Upon addition of a sodium-bicarbonate buffer, a general increase in Pt(IV) reduction to Pt(II) was observed. The addition of the buffer also resulted in an unexplained change in particle morphology and for this reason was not used in subsequent investigations. Polyvinylpyrrolidone (PVP) was shown to compromise the reduction rate of the Pt(IV) ion by SRB cells. The presence of extracellular NP’s was suggested by the colour of the supernatant turning brown and the A334 increasing over time. Attempts to visualise the particles by transmission electron microscopy (TEM) resulted in an unexpected phenomenon where nanoparticles could be observed to form dynamically upon irradiation by the electron beam. Extended irradiation by the electron beam also resulted in structural changes of the particles occurring during observation. An increase in temperature was shown to increase the reduction rate which in turn resulted in particles decreasing in size. The starting pH was shown to have a significant effect on the reduction rate and particle morphology although specific trends could not be identified. In conclusion, the cell-soluble extract from the sulfate-reducing consortium investigated, is capable of Pt(0) nanoparticle synthesis. Precise control over the particle morphology was not attained although the mechanism was further clarified and optimal conditions for nanoparticle synthesis were determined.
- Full Text:
Molecular and biochemical analysis of the diet of the black rhinoceros
- Authors: Kgopa, Ananias Hodi
- Date: 2009 , 2013-07-15
- Subjects: Black rhinoceros -- South Africa -- Eastern Cape , Browse (Animal food) -- South Africa -- Eastern Cape -- Analysis , Black rhinoceros -- Manure -- Analysis , Phenols , Antioxidants , Plant ecology -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4064 , http://hdl.handle.net/10962/d1004721 , Black rhinoceros -- South Africa -- Eastern Cape , Browse (Animal food) -- South Africa -- Eastern Cape -- Analysis , Black rhinoceros -- Manure -- Analysis , Phenols , Antioxidants , Plant ecology -- South Africa -- Eastern Cape
- Description: The black rhinoceros, Diceros bicornis, is listed as critically endangered. The black rhinoceros population in the Great Fish River Reserve (GFRR) has increased steadily to a current estimate of one hundred animals since the re-introduction of four animals in 1986. In an effort to contribute to the effective conservation and management of this species, dietary composition was studied in the medium Portulcaria thicket vegetation of the GFRR. This study used a molecular approach to determine the diet of the black rhinoceros of the GFRR by sequencing the ribulose bisphosphate carboxylase large subunit (rbcL) gene in plants and dung. Twenty-three plant species were collected from the reserve, and 802 bp of the rbcL gene were sequenced. These plant sequences were used as a reference database for the identification of plant sequences generated from black rhinoceros dung. Initial studies investigated the amplification, cloning and sequencing of DNA extracted from the dung samples which indicated the viability of the molecular approach. Thereafter, dung generated rbcL DNA was analyzed by GS FLX sequencing. Of the plant sequences identified by comparison to the GenBank database, Carissa bispinosa was the most prevalent. The study further characterized the antioxidant activities and phenolic content of plants eaten by the black rhinoceros using four different assays. Phyllanthus verrucosus, Putterlickia pyracantha, Maytenus capitata, Euclea undulata and Ozoroa mucrunata consistently had high antioxidant activities when assayed against 2,2-azinobis (3-ethyl benzothiazolium-6-sulfonic acid) (ABTSʹ⁺), 2,2-diphenyl-1-picrylhydrazyl (DPPHʹ), and ferric reducing antioxidant potentials (FRAP) and phenolic content when evaluated using the Folin-Ciocalteu assay. The majority of plants investigated showed low antioxidant potentials and low phenolic content. The extent to which antioxidants influenced the browse selection by the black rhinoceros remains inconclusive.
- Full Text:
- Authors: Kgopa, Ananias Hodi
- Date: 2009 , 2013-07-15
- Subjects: Black rhinoceros -- South Africa -- Eastern Cape , Browse (Animal food) -- South Africa -- Eastern Cape -- Analysis , Black rhinoceros -- Manure -- Analysis , Phenols , Antioxidants , Plant ecology -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4064 , http://hdl.handle.net/10962/d1004721 , Black rhinoceros -- South Africa -- Eastern Cape , Browse (Animal food) -- South Africa -- Eastern Cape -- Analysis , Black rhinoceros -- Manure -- Analysis , Phenols , Antioxidants , Plant ecology -- South Africa -- Eastern Cape
- Description: The black rhinoceros, Diceros bicornis, is listed as critically endangered. The black rhinoceros population in the Great Fish River Reserve (GFRR) has increased steadily to a current estimate of one hundred animals since the re-introduction of four animals in 1986. In an effort to contribute to the effective conservation and management of this species, dietary composition was studied in the medium Portulcaria thicket vegetation of the GFRR. This study used a molecular approach to determine the diet of the black rhinoceros of the GFRR by sequencing the ribulose bisphosphate carboxylase large subunit (rbcL) gene in plants and dung. Twenty-three plant species were collected from the reserve, and 802 bp of the rbcL gene were sequenced. These plant sequences were used as a reference database for the identification of plant sequences generated from black rhinoceros dung. Initial studies investigated the amplification, cloning and sequencing of DNA extracted from the dung samples which indicated the viability of the molecular approach. Thereafter, dung generated rbcL DNA was analyzed by GS FLX sequencing. Of the plant sequences identified by comparison to the GenBank database, Carissa bispinosa was the most prevalent. The study further characterized the antioxidant activities and phenolic content of plants eaten by the black rhinoceros using four different assays. Phyllanthus verrucosus, Putterlickia pyracantha, Maytenus capitata, Euclea undulata and Ozoroa mucrunata consistently had high antioxidant activities when assayed against 2,2-azinobis (3-ethyl benzothiazolium-6-sulfonic acid) (ABTSʹ⁺), 2,2-diphenyl-1-picrylhydrazyl (DPPHʹ), and ferric reducing antioxidant potentials (FRAP) and phenolic content when evaluated using the Folin-Ciocalteu assay. The majority of plants investigated showed low antioxidant potentials and low phenolic content. The extent to which antioxidants influenced the browse selection by the black rhinoceros remains inconclusive.
- Full Text:
Phenolic compounds in water and the implications for rapid detection of indicator micro-organisms using ß-D-Galactosidase and ß-D-Glucuronidase
- Authors: Abboo, Sagaran
- Date: 2009
- Subjects: Water -- Purification -- Biological treatment , Pollutants -- Biodegradation , Phenol , Organic water pollutants , Water quality biological assessment , Water -- Pollution
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3978 , http://hdl.handle.net/10962/d1004037 , Water -- Purification -- Biological treatment , Pollutants -- Biodegradation , Phenol , Organic water pollutants , Water quality biological assessment , Water -- Pollution
- Description: Faecal contamination in water is detected using appropriate microbial models such as total coliforms, faecal coliforms and E. coli. Βeta-D-Galactosidase (β-GAL) and Beta-D-glucuronidase (β-GUD) are two marker enzymes that are used to test for the presence of total coliforms and E. coli in water samples, respectively. Various assay methods have been developed using chromogenic and fluorogenic substrates. In this study, the chromogenic substrates chlorophenol red β-D-galactopyranoside (CPRG) for β-GAL and p-nitrophenyl-β-D-galactopyranoside (PNPG) for β-GUD were used. Potential problems associated with this approach include interference from other organisms present in the environment (e.g. plants, algae and other bacteria), as well as the presence of certain chemicals, such as phenolic compounds in water. Phenolic compounds are present in the aquatic environment due to their extensive industrial applications. The USA Enviromental Protection Agency (EPA) lists 11 Priority Pollutant Phenols (PPP) due to their high level of toxicity. This study investigated the interfering effects of the eleven PPP found in water on the enzyme activities of both the β-GAL and β-GUD enzyme assays. The presence of these PPP in the β-GAL and β-GUD enzyme assays showed that over and underestimation of activity may occur due to inhibition or activation of these enzymes. Three types of inhibition to enzyme activities were identified from double reciprocal Lineweaver-Burk plots. The inhibition constants (Ki) were determined for all inhibitory phenolic compounds from appropriate secondary plots. Furthermore, this study presented a validated reverse phase high performance liquid chromatography (RP-HPLC) method, developed for the simultaneous detection, separation and determination of all eleven phenolic compounds found in the environment. This method demonstrated good linearity, reproducibility, accuracy and sensitivity. Environmental water samples were collected from rivers, streams, industrial sites and wastewater treatment plant effluent. These samples were extracted and concentrated using a solid phase extraction (SPE) procedure prior to analysis employing the newly developed HPLC method in this study. Seasonal variations on the presence of the PPP in the environment were observed at certain collection sites. The concentrations found were between 0.033 μg/ml for 2,4-dinitrophenol in a running stream to 0.890 mg/ml for pentachlorophenol from an tannery industrial site. These concentrations of phenolic compounds found in these environments were able to interfere with the β-GAL and β-GUD enzyme assays.
- Full Text:
- Authors: Abboo, Sagaran
- Date: 2009
- Subjects: Water -- Purification -- Biological treatment , Pollutants -- Biodegradation , Phenol , Organic water pollutants , Water quality biological assessment , Water -- Pollution
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3978 , http://hdl.handle.net/10962/d1004037 , Water -- Purification -- Biological treatment , Pollutants -- Biodegradation , Phenol , Organic water pollutants , Water quality biological assessment , Water -- Pollution
- Description: Faecal contamination in water is detected using appropriate microbial models such as total coliforms, faecal coliforms and E. coli. Βeta-D-Galactosidase (β-GAL) and Beta-D-glucuronidase (β-GUD) are two marker enzymes that are used to test for the presence of total coliforms and E. coli in water samples, respectively. Various assay methods have been developed using chromogenic and fluorogenic substrates. In this study, the chromogenic substrates chlorophenol red β-D-galactopyranoside (CPRG) for β-GAL and p-nitrophenyl-β-D-galactopyranoside (PNPG) for β-GUD were used. Potential problems associated with this approach include interference from other organisms present in the environment (e.g. plants, algae and other bacteria), as well as the presence of certain chemicals, such as phenolic compounds in water. Phenolic compounds are present in the aquatic environment due to their extensive industrial applications. The USA Enviromental Protection Agency (EPA) lists 11 Priority Pollutant Phenols (PPP) due to their high level of toxicity. This study investigated the interfering effects of the eleven PPP found in water on the enzyme activities of both the β-GAL and β-GUD enzyme assays. The presence of these PPP in the β-GAL and β-GUD enzyme assays showed that over and underestimation of activity may occur due to inhibition or activation of these enzymes. Three types of inhibition to enzyme activities were identified from double reciprocal Lineweaver-Burk plots. The inhibition constants (Ki) were determined for all inhibitory phenolic compounds from appropriate secondary plots. Furthermore, this study presented a validated reverse phase high performance liquid chromatography (RP-HPLC) method, developed for the simultaneous detection, separation and determination of all eleven phenolic compounds found in the environment. This method demonstrated good linearity, reproducibility, accuracy and sensitivity. Environmental water samples were collected from rivers, streams, industrial sites and wastewater treatment plant effluent. These samples were extracted and concentrated using a solid phase extraction (SPE) procedure prior to analysis employing the newly developed HPLC method in this study. Seasonal variations on the presence of the PPP in the environment were observed at certain collection sites. The concentrations found were between 0.033 μg/ml for 2,4-dinitrophenol in a running stream to 0.890 mg/ml for pentachlorophenol from an tannery industrial site. These concentrations of phenolic compounds found in these environments were able to interfere with the β-GAL and β-GUD enzyme assays.
- Full Text:
Polymers, catalysts and nanostructures a hybrid approach to biomolecule detection
- Authors: Frith, Kelly-Anne
- Date: 2009
- Subjects: Polymers , Nanostructured materials , Biomolecules , Tryptophan , Melatonin , Electrodes , Electrochemistry , Tryptophan oxygenase
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3980 , http://hdl.handle.net/10962/d1004039 , Polymers , Nanostructured materials , Biomolecules , Tryptophan , Melatonin , Electrodes , Electrochemistry , Tryptophan oxygenase
- Description: The main goals in electroanalytical sensing are towards improved sensitivity and selectivity, or specificity, of an analyte. There are several approaches to achieving these goals with the main approach being modification of an electrode surface with synthetic or natural catalysts (enzymes), polymers and also utilisation of nanostructured materials. At present, there is a strong movement towards hybrid sensing which couple different properties of two or more surface modification approaches. In this thesis, a range of these surface modifications were explored for analysis and detection of two main analytes: the amino acid, tryptophan (Trp); and, the neurotransmitter, dopamine (DA). Specifically, this thesis aimed to utilise these methods to enhance the sensitivity and selectivity for Trp over an interferent, the indoleamine, melatonin (Mel); and, DA over the vitamin, ascorbic acid (AA). For Trp detection, immobilisation of an enzyme, Tryptophanase (Trpase) resulted in poor selectivity for the analyte. However, enhanced sensitivity and selectivity was achieved through pH manipulation of the electrolyte medium at a Nafion®-modified electrode surface for both Trp and Mel. At pH 3.0, the Mel and Trp anodic peak potentials were sufficiently resolved allowing for an LOD of 1.60 and 1.62 nM,respectively, and permitting the accurate analysis of Trp in a dietary supplement containing Mel. Multi-walled carbon nanotubes (MWCNTs) suspended in Nafion® exhibited further increases in the signal responses of these analytes at pH 3.0 and 7.4 with minimal change in the resolution of the anodic peaks. A lower sensitivity was, therefore, observed at the Nafion® and MWCNT modified electrode compared to the Nafion®-modified electrode at pH 3.0 with LODs of 0.59 and 0.80 nM exhibited for Trp and Mel, respectively. Enhanced selectivity for Trp in the presence of Mel can be achieved with MWCNTs in the presence of metallotetrasulphonated phthalocyanines (MTSPcs) particularly at pH 3.0, owing to cation exchange effects. However, the lack of sensitivity towards Trp, and even Mel, at this CoTSPc and MWCNT modified electrode remains a drawback. For DA, detection at the MWCNT and Nafion® surface resulted in improved sensitivity over that of both the bare electrode (613.0 nM) and the Nafion® modified electrode (1045.1 nM) with a calculated LOD of 133.9 nM at this layer. Furthermore, improvements in the selectivity of DA were achieved at the Nafion® and MWCNT modified electrode as exclusion of AA (150 μM) was achieved. At the MWCNT and CoTSPc surface, AA was excluded up to 130 μM with sensitivity for DA extending as low as 14.3 nM, far greater than observed for Trp and Mel. These concentrations are well within physiological concentration ranges and represent the most significant solution yet in terms of AA exclusion and enhanced sensitivity for DA. An examination of the surface layering by impedance spectroscopy and atomic force microscopy indicates that the success of the hybrid sensor utilising CoTSPc and MWCNTs lay in improved dispersion of MWCNTs and improved electron transfer kinetics, facilitated by the net charge of the materials present. This thesis, thus, showed the utility of a judicious selection of synthetic and biological catalysts, polymers and carbon nanomaterials towards a hybrid approach to the electrochemical sensing of Trp, Mel, DA and AA with focus on sensitivity and selectivity of these analytes.
- Full Text:
- Authors: Frith, Kelly-Anne
- Date: 2009
- Subjects: Polymers , Nanostructured materials , Biomolecules , Tryptophan , Melatonin , Electrodes , Electrochemistry , Tryptophan oxygenase
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3980 , http://hdl.handle.net/10962/d1004039 , Polymers , Nanostructured materials , Biomolecules , Tryptophan , Melatonin , Electrodes , Electrochemistry , Tryptophan oxygenase
- Description: The main goals in electroanalytical sensing are towards improved sensitivity and selectivity, or specificity, of an analyte. There are several approaches to achieving these goals with the main approach being modification of an electrode surface with synthetic or natural catalysts (enzymes), polymers and also utilisation of nanostructured materials. At present, there is a strong movement towards hybrid sensing which couple different properties of two or more surface modification approaches. In this thesis, a range of these surface modifications were explored for analysis and detection of two main analytes: the amino acid, tryptophan (Trp); and, the neurotransmitter, dopamine (DA). Specifically, this thesis aimed to utilise these methods to enhance the sensitivity and selectivity for Trp over an interferent, the indoleamine, melatonin (Mel); and, DA over the vitamin, ascorbic acid (AA). For Trp detection, immobilisation of an enzyme, Tryptophanase (Trpase) resulted in poor selectivity for the analyte. However, enhanced sensitivity and selectivity was achieved through pH manipulation of the electrolyte medium at a Nafion®-modified electrode surface for both Trp and Mel. At pH 3.0, the Mel and Trp anodic peak potentials were sufficiently resolved allowing for an LOD of 1.60 and 1.62 nM,respectively, and permitting the accurate analysis of Trp in a dietary supplement containing Mel. Multi-walled carbon nanotubes (MWCNTs) suspended in Nafion® exhibited further increases in the signal responses of these analytes at pH 3.0 and 7.4 with minimal change in the resolution of the anodic peaks. A lower sensitivity was, therefore, observed at the Nafion® and MWCNT modified electrode compared to the Nafion®-modified electrode at pH 3.0 with LODs of 0.59 and 0.80 nM exhibited for Trp and Mel, respectively. Enhanced selectivity for Trp in the presence of Mel can be achieved with MWCNTs in the presence of metallotetrasulphonated phthalocyanines (MTSPcs) particularly at pH 3.0, owing to cation exchange effects. However, the lack of sensitivity towards Trp, and even Mel, at this CoTSPc and MWCNT modified electrode remains a drawback. For DA, detection at the MWCNT and Nafion® surface resulted in improved sensitivity over that of both the bare electrode (613.0 nM) and the Nafion® modified electrode (1045.1 nM) with a calculated LOD of 133.9 nM at this layer. Furthermore, improvements in the selectivity of DA were achieved at the Nafion® and MWCNT modified electrode as exclusion of AA (150 μM) was achieved. At the MWCNT and CoTSPc surface, AA was excluded up to 130 μM with sensitivity for DA extending as low as 14.3 nM, far greater than observed for Trp and Mel. These concentrations are well within physiological concentration ranges and represent the most significant solution yet in terms of AA exclusion and enhanced sensitivity for DA. An examination of the surface layering by impedance spectroscopy and atomic force microscopy indicates that the success of the hybrid sensor utilising CoTSPc and MWCNTs lay in improved dispersion of MWCNTs and improved electron transfer kinetics, facilitated by the net charge of the materials present. This thesis, thus, showed the utility of a judicious selection of synthetic and biological catalysts, polymers and carbon nanomaterials towards a hybrid approach to the electrochemical sensing of Trp, Mel, DA and AA with focus on sensitivity and selectivity of these analytes.
- Full Text:
Purification and characterization of fructosyltransferase for the synthesis of short-chain fructo-oligosaccharides and investigation into thier anti-carcinogenic properties
- Authors: Nemukula, Aluwani
- Date: 2009
- Subjects: Oligosaccharides , Polygalacturonase , Aspergillus , Fructose , Inulin , Cancer -- Prevention , Cancer -- Research , Carcinogens , High performance liquid chromatography
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3927 , http://hdl.handle.net/10962/d1003986 , Oligosaccharides , Polygalacturonase , Aspergillus , Fructose , Inulin , Cancer -- Prevention , Cancer -- Research , Carcinogens , High performance liquid chromatography
- Description: There is a growing attention in the synthesis of fructo-oligosaccharides (FOS) due to their excellent bio-functional and health-promoting properties. The current production processes are limited to chemical hydrolysis reactions of plant extracts, which are often associated with several drawbacks. In this study, fructosyltransferase (FTase) and polygalacturonase (PGase) activities, present in a commercial enzyme preparation (Pectinex® Ultra SP-L) sourced from Aspergillus aculeatus, have been separated and fully purified by anion-exchange and sizeexclusion chromatography. The FTase possesses fructosyl transfer activity for FOS synthesis and the PGase has pectin hydrolytic activity. Fructosyltransferase is a single-band protein with a molecular weight of 85 kDa, whereas PGase is a distinct protein of 40 kDa. The temperature and pH optima of FTase were 60 ºC and 6.0, with a half-life of 8 h; while that for PGase were 40 ºC and 6.0, respectively. FTase was slightly inhibited in the presence of Ni²⁺, Mg²⁺ and urea; but PGase was more susceptible to divalent ions such as Ca²⁺, Mg²⁺ and Mn²⁺. The kinetic parameters (Km and Vmax) of FTase for the hydrolysis of β-(2→1) linkages from sucrose were 752.3 mM and 120.5 μmol.min⁻¹.mL⁻¹, respectively; whereas the same parameters for pectin hydrolysis by PGase were 13.0 mg.mL⁻¹ and 263 μmol.min-1.mL⁻¹, respectively. The purified FTase was able to transfer fructosyl residues from sucrose, synthesizing the corresponding chains of FOS. PGase was relatively stable at 40 ºC (t½ > 3 h), depolymerizing the pectin backbone while releasing the inulins from within the chicory roots. Analysis of various mixtures of FOS by mass spectrometry, HPLC and ¹H-NMR was undertaken. Results indicated that MS with electrospray ionization and ¹H-NMR are capable of providing relative quantitative data of the FOS present in the mixtures. The pharmaceutical effects of various sc-FOS (0.5%, v/v) and SCFA (0.3%, v/v) on certain bacterial enzymes (β-glucuronidase, urease and β-glucosidase) associated with the formation of carcinogens were also studied. These enzyme activities were not directly influenced by the sc-FOS, but were found to be remarkably decreased by SCFA, pointing toward the prebiotic effect of FOS in intestinal microflora modulation.
- Full Text:
- Authors: Nemukula, Aluwani
- Date: 2009
- Subjects: Oligosaccharides , Polygalacturonase , Aspergillus , Fructose , Inulin , Cancer -- Prevention , Cancer -- Research , Carcinogens , High performance liquid chromatography
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3927 , http://hdl.handle.net/10962/d1003986 , Oligosaccharides , Polygalacturonase , Aspergillus , Fructose , Inulin , Cancer -- Prevention , Cancer -- Research , Carcinogens , High performance liquid chromatography
- Description: There is a growing attention in the synthesis of fructo-oligosaccharides (FOS) due to their excellent bio-functional and health-promoting properties. The current production processes are limited to chemical hydrolysis reactions of plant extracts, which are often associated with several drawbacks. In this study, fructosyltransferase (FTase) and polygalacturonase (PGase) activities, present in a commercial enzyme preparation (Pectinex® Ultra SP-L) sourced from Aspergillus aculeatus, have been separated and fully purified by anion-exchange and sizeexclusion chromatography. The FTase possesses fructosyl transfer activity for FOS synthesis and the PGase has pectin hydrolytic activity. Fructosyltransferase is a single-band protein with a molecular weight of 85 kDa, whereas PGase is a distinct protein of 40 kDa. The temperature and pH optima of FTase were 60 ºC and 6.0, with a half-life of 8 h; while that for PGase were 40 ºC and 6.0, respectively. FTase was slightly inhibited in the presence of Ni²⁺, Mg²⁺ and urea; but PGase was more susceptible to divalent ions such as Ca²⁺, Mg²⁺ and Mn²⁺. The kinetic parameters (Km and Vmax) of FTase for the hydrolysis of β-(2→1) linkages from sucrose were 752.3 mM and 120.5 μmol.min⁻¹.mL⁻¹, respectively; whereas the same parameters for pectin hydrolysis by PGase were 13.0 mg.mL⁻¹ and 263 μmol.min-1.mL⁻¹, respectively. The purified FTase was able to transfer fructosyl residues from sucrose, synthesizing the corresponding chains of FOS. PGase was relatively stable at 40 ºC (t½ > 3 h), depolymerizing the pectin backbone while releasing the inulins from within the chicory roots. Analysis of various mixtures of FOS by mass spectrometry, HPLC and ¹H-NMR was undertaken. Results indicated that MS with electrospray ionization and ¹H-NMR are capable of providing relative quantitative data of the FOS present in the mixtures. The pharmaceutical effects of various sc-FOS (0.5%, v/v) and SCFA (0.3%, v/v) on certain bacterial enzymes (β-glucuronidase, urease and β-glucosidase) associated with the formation of carcinogens were also studied. These enzyme activities were not directly influenced by the sc-FOS, but were found to be remarkably decreased by SCFA, pointing toward the prebiotic effect of FOS in intestinal microflora modulation.
- Full Text:
Understanding the complexity of metabolic regulatory systems an investigation into the regulation of hydantoin-hydrolysis in Pseudomonas putida RU-KM3s
- Authors: De la Mare, Jo-Anne
- Date: 2009
- Subjects: Pseudomonas , Hydantoin , Hydrolysis , Enzymes -- Regulation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3993 , http://hdl.handle.net/10962/d1004053 , Pseudomonas , Hydantoin , Hydrolysis , Enzymes -- Regulation
- Description: It has been well-established that Pseudomonas species possess extremely versatile metabolic systems allowing them to utilise a wide range of nutrient sources and, furthermore, that the regulation of these enzyme systems involves highly evolved and sophisticated regulatory machinery. This study examined the complexity of metabolic regulation in Pseudomonas using the hydantoin-hydrolysing system of the environmental isolate, Pseudomonas putida RU-KM3s. In this system, the genes encoding dihydropyrimidinase and β-ureidopropionase (dhp and bup) are arranged divergently on the chromosome, separated by a 616 bp intergenic region involved in the transcriptional regulation of these genes. The focus was on the transcriptional regulation of dhp expression. DHP activity was found to be sensitive to several environmental signals including growth phase, carbon catabolite repression (CCR), substrate induction and quorum sensing (QS). Bioinformatic analysis of the intergenic region upstream of dhp revealed a number of putative binding sites for transcriptional regulators, including recognition sequences for the alternate sigma factors σ54 and σ38, as well as for the global regulators Anr (for anaerobic regulator) and Vfr (for virulence factor regulator). The targeted disruption of the genes encoding the transcriptional regulators, Vfr and the major CCR protein, Crc, resulted in a partial relief from repression for the vfr- mutant under quorum sensing conditions and a general decrease in activity in the crc- mutant. This data suggested that both Vfr and Crc were involved in regulating DHP activity. Mutational analysis of the dhp promoter revealed that at least two sites were involved in regulating transcriptional activity, one which mediated activation and the other repression. These sites were designated as a putative Anr box, situated 232 bp from the start codon of dhp, and a CRP-like binding site, at a position 213 bp upstream of dhp. Taken together, this data shows the involvement of several global regulatory factors in controlling the expression of dhp. A complex synergistic model was proposed for the transcriptional regulation of dhp, involving alternate sigma factors in addition to both global and specific regulators and responding to a number of environmental signals associated with growth phase, including nutrient availability, cell density and oxygen status.
- Full Text:
- Authors: De la Mare, Jo-Anne
- Date: 2009
- Subjects: Pseudomonas , Hydantoin , Hydrolysis , Enzymes -- Regulation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3993 , http://hdl.handle.net/10962/d1004053 , Pseudomonas , Hydantoin , Hydrolysis , Enzymes -- Regulation
- Description: It has been well-established that Pseudomonas species possess extremely versatile metabolic systems allowing them to utilise a wide range of nutrient sources and, furthermore, that the regulation of these enzyme systems involves highly evolved and sophisticated regulatory machinery. This study examined the complexity of metabolic regulation in Pseudomonas using the hydantoin-hydrolysing system of the environmental isolate, Pseudomonas putida RU-KM3s. In this system, the genes encoding dihydropyrimidinase and β-ureidopropionase (dhp and bup) are arranged divergently on the chromosome, separated by a 616 bp intergenic region involved in the transcriptional regulation of these genes. The focus was on the transcriptional regulation of dhp expression. DHP activity was found to be sensitive to several environmental signals including growth phase, carbon catabolite repression (CCR), substrate induction and quorum sensing (QS). Bioinformatic analysis of the intergenic region upstream of dhp revealed a number of putative binding sites for transcriptional regulators, including recognition sequences for the alternate sigma factors σ54 and σ38, as well as for the global regulators Anr (for anaerobic regulator) and Vfr (for virulence factor regulator). The targeted disruption of the genes encoding the transcriptional regulators, Vfr and the major CCR protein, Crc, resulted in a partial relief from repression for the vfr- mutant under quorum sensing conditions and a general decrease in activity in the crc- mutant. This data suggested that both Vfr and Crc were involved in regulating DHP activity. Mutational analysis of the dhp promoter revealed that at least two sites were involved in regulating transcriptional activity, one which mediated activation and the other repression. These sites were designated as a putative Anr box, situated 232 bp from the start codon of dhp, and a CRP-like binding site, at a position 213 bp upstream of dhp. Taken together, this data shows the involvement of several global regulatory factors in controlling the expression of dhp. A complex synergistic model was proposed for the transcriptional regulation of dhp, involving alternate sigma factors in addition to both global and specific regulators and responding to a number of environmental signals associated with growth phase, including nutrient availability, cell density and oxygen status.
- Full Text:
- «
- ‹
- 1
- ›
- »