Bio-prospecting a Soil Metagenomic Library for Carbohydrate Active Esterases
- Authors: Shezi, Ntombifuthi
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4172 , http://hdl.handle.net/10962/d1021266
- Description: Lignocellulosic biomass is a promising renewable resource on earth. Plant biomass contains fermentable sugars and other moieties that can be converted to biofuels or other chemicals. Enzymatic hydrolysis of these biopolymers is significant in the liberation of sugars for fermentation into desired products. Owing to its complex structure, synergistic action of enzymes is required for its degradation. Enzymes that are involved in biomass degradation include cellulases, hemicellulases and the accessory enzymes acetyl xylan esterases and ferulic acid esterases. Ferulic acid esterases (FAEs, EC 3.1.1.73), represent a subclass of carboxylester hydrolases (EC 3.1.1.-) that catalyse the release of hydroxycinnamic acids (such as ferulic acid, p-coumaric, ferulic, sinapic and caffeic acid) that are generally found esterified to polysaccharides, such as arabinoxylans. Hydroxycinnamic acids have widespread potential applications due to their antimicrobial, photoprotectant and antioxidant properties, as well as their use as flavour precursors. Therefore, this interesting group of FAEs has a potentially wide variety of applications in agriculture, food and pharmaceutical industries. In the search for novel biocatalysts, metagenomics is considered as an alternative approach to conventional microbe screening, therefore, searching for novel biocatalysts from a soil metagenome that harbours a unique diversity of biocatalyst is significant. The aim of this study was to extract DNA from soil associated with cattle manure and construct a soil metagenomic library using a fosmid based plasmid vector and subsequently functionally screen for ferulic acid esterases using ethyl ferulate as a model substrate. A total of 59 recombinant fosmids conferring ferulic acid esterase phenotypes were identified (Hit rate 1:3122) and the two fosmids that consistently showed high FAE activities were selected for further study. Following nucleotide sequencing and translational analysis, two fae encoding open reading frames (FAE9 and FAE27) of approximately 274 and 322 aa, respectively, were identified. The amino acid sequence of the two ORFs contained a classical conserved esterase/lipase G-x-S-x-G sequence motif. The two genes (fae9 and fae27) were successfully expressed in Escherichia coli BL21 (DE3) and the purified enzymes exhibited respective temperature optima of 50 °C and 40 °C, and respective pH optima of 6.0 and 7.0. Further biochemical characterisation showed that FAE9 and FAE27 have high substrate specificity, following the fact that EFA is the preferred substrate for FAE9 (kcat/Km value of 128 s−1.mM-1) and also the preferred substrate for FAE27 (kcat/Km value of 137 s−1.mM-1). This work proves that soil is a valuable environmental source for novel esterase screening through functional based metagenomic approach. Therefore, this method may be used to screen for other valuable enzymes from environmental sources using inexpensive natural sources to encourage the screening of specific enzymes. Biochemistry of the two isolated enzymes makes these enzymes to be useful in industrial applications due to broad substrate activity that could replace the specialised enzymes to complete plant biomass degradation.
- Full Text:
- Date Issued: 2016
- Authors: Shezi, Ntombifuthi
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4172 , http://hdl.handle.net/10962/d1021266
- Description: Lignocellulosic biomass is a promising renewable resource on earth. Plant biomass contains fermentable sugars and other moieties that can be converted to biofuels or other chemicals. Enzymatic hydrolysis of these biopolymers is significant in the liberation of sugars for fermentation into desired products. Owing to its complex structure, synergistic action of enzymes is required for its degradation. Enzymes that are involved in biomass degradation include cellulases, hemicellulases and the accessory enzymes acetyl xylan esterases and ferulic acid esterases. Ferulic acid esterases (FAEs, EC 3.1.1.73), represent a subclass of carboxylester hydrolases (EC 3.1.1.-) that catalyse the release of hydroxycinnamic acids (such as ferulic acid, p-coumaric, ferulic, sinapic and caffeic acid) that are generally found esterified to polysaccharides, such as arabinoxylans. Hydroxycinnamic acids have widespread potential applications due to their antimicrobial, photoprotectant and antioxidant properties, as well as their use as flavour precursors. Therefore, this interesting group of FAEs has a potentially wide variety of applications in agriculture, food and pharmaceutical industries. In the search for novel biocatalysts, metagenomics is considered as an alternative approach to conventional microbe screening, therefore, searching for novel biocatalysts from a soil metagenome that harbours a unique diversity of biocatalyst is significant. The aim of this study was to extract DNA from soil associated with cattle manure and construct a soil metagenomic library using a fosmid based plasmid vector and subsequently functionally screen for ferulic acid esterases using ethyl ferulate as a model substrate. A total of 59 recombinant fosmids conferring ferulic acid esterase phenotypes were identified (Hit rate 1:3122) and the two fosmids that consistently showed high FAE activities were selected for further study. Following nucleotide sequencing and translational analysis, two fae encoding open reading frames (FAE9 and FAE27) of approximately 274 and 322 aa, respectively, were identified. The amino acid sequence of the two ORFs contained a classical conserved esterase/lipase G-x-S-x-G sequence motif. The two genes (fae9 and fae27) were successfully expressed in Escherichia coli BL21 (DE3) and the purified enzymes exhibited respective temperature optima of 50 °C and 40 °C, and respective pH optima of 6.0 and 7.0. Further biochemical characterisation showed that FAE9 and FAE27 have high substrate specificity, following the fact that EFA is the preferred substrate for FAE9 (kcat/Km value of 128 s−1.mM-1) and also the preferred substrate for FAE27 (kcat/Km value of 137 s−1.mM-1). This work proves that soil is a valuable environmental source for novel esterase screening through functional based metagenomic approach. Therefore, this method may be used to screen for other valuable enzymes from environmental sources using inexpensive natural sources to encourage the screening of specific enzymes. Biochemistry of the two isolated enzymes makes these enzymes to be useful in industrial applications due to broad substrate activity that could replace the specialised enzymes to complete plant biomass degradation.
- Full Text:
- Date Issued: 2016
Genetic and biological characterisation of a novel South African Cydia pomonella granulovirus (CpGV-SA) isolate
- Motsoeneng, Boitumelo Madika
- Authors: Motsoeneng, Boitumelo Madika
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:20503 , http://hdl.handle.net/10962/d1021266
- Description: The codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), is the primary pest of pome fruit cultivated worldwide. The control of this insect pest has been dependent on the frequent use of broad-spectrum chemical pesticides, which has led to the development of resistance in pest populations and negative effects on human health and the environment. The Betabaculovirus of C. pomonella has successfully been applied as a biological control agent in integrated pest management (IPM) programmes for the suppression of pest populations worldwide. Previously, all Cydia pomonella granulovirus (CpGV) biopesticides were based on a Mexican isolate (CpGV-M) and although these products are highly efficient at controlling C. pomonella, resistance cases have been reported across Europe. The identification of novel CpGV isolates as additional or alternative control agents to manage resistance is therefore necessary. This study aimed to genetically and biologically characterise a novel South African C. pomonella granulovirus isolate and to test its virulence against neonate larvae. Based on the morphology of the occlusion bodies observed using transmission electron microscopy, granuloviruses were recovered from diseased and dead larvae collected from an orchard in South Africa where no virus applications had been made. DNA was extracted and the identification of the isolated granulovirus was achieved through the PCR amplification and sequencing of the lef-8, lef-9, granulin and egt genes. Submission of the gene sequences to BLAST revealed high percentage identities to sequences from various CpGV isolates, resulting in the naming of the isolate in this study as the South African Cydia pomonella granulovirus (CpGV-SA) isolate. Phylogenetic analysis based on the single nucleotide polymorphisms (SNPs) detected in the lef-8, lef-9 and granulin nucleotide sequences grouped the South African isolate with CpGV-E2 (genome type B) and CpGV-S (genome type E). The CpGV-SA isolate was further genetically characterised by restriction endonuclease analysis and complete sequencing of the genomic DNA. Differences were observed for the BamHI, EcoRI, PstI and XhoI profiles of CpGV-SA in comparison to the respective profiles generated for CpGV-M extracted from a biopesticide, Carpovirusine® (Arysta Lifescience, France). Several genetic variations between the complete genome sequence of CpGV-SA and the reference isolate, CpGV-M1, as well as a recent genome submission of CpGV-M, both representing genome type A were observed. The complete genome analysis confirmed that CpGV-SA is genetically different from the Mexican CpGV isolate, used in thedevelopment of most biopesticides. In silico restriction profiles of the genome sequence obtained for CpGV-SA and genome sequences of genetically different CpGV isolates originating from Mexico (M1 and M), England (E2), Canada (S) and Iran (I12 and I07), available on the NCBI’s GenBank database confirmed that CpGV-SA is of mixed genotypes. Furthermore, the South African isolate shared the single common difference found in the pe38 gene of resistance overcoming isolates, which was the absence of an internal 24 nucleotide repeat present in CpGV-M1. In addition to the common difference, SNPs detected in the pe38 gene grouped the isolate with the CpGV-S isolate, suggesting that the CpGV-SA isolate is predominantly of genome type E. To determine the biological activity of CpGV-SA against neonate C. pomonella larvae, surface bioassays were conducted alongside CpGV-M (Carpovirusine®) bioassays. The LC50 and LC90 values for the South African isolate were 1.6 × 103 and 1.2 × 105 OBs/ml respectively. The LT50 was determined to be 135 hours. These values were similar to the values obtained for CpGV-M (Carpovirusine®). The results in this study suggest that a novel South African CpGV isolate of mixed genotypes, potentially able to overcome resistance in C. pomonella, with biological activity similar to CpGV-M (Carpovirusine®) and important for the control of C. pomonella was recovered. The CpGV-SA isolate could therefore potentially be developed into a biopesticide for use in resistance management strategies against C. pomonella populations in South Africa.
- Full Text:
- Date Issued: 2016
- Authors: Motsoeneng, Boitumelo Madika
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:20503 , http://hdl.handle.net/10962/d1021266
- Description: The codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), is the primary pest of pome fruit cultivated worldwide. The control of this insect pest has been dependent on the frequent use of broad-spectrum chemical pesticides, which has led to the development of resistance in pest populations and negative effects on human health and the environment. The Betabaculovirus of C. pomonella has successfully been applied as a biological control agent in integrated pest management (IPM) programmes for the suppression of pest populations worldwide. Previously, all Cydia pomonella granulovirus (CpGV) biopesticides were based on a Mexican isolate (CpGV-M) and although these products are highly efficient at controlling C. pomonella, resistance cases have been reported across Europe. The identification of novel CpGV isolates as additional or alternative control agents to manage resistance is therefore necessary. This study aimed to genetically and biologically characterise a novel South African C. pomonella granulovirus isolate and to test its virulence against neonate larvae. Based on the morphology of the occlusion bodies observed using transmission electron microscopy, granuloviruses were recovered from diseased and dead larvae collected from an orchard in South Africa where no virus applications had been made. DNA was extracted and the identification of the isolated granulovirus was achieved through the PCR amplification and sequencing of the lef-8, lef-9, granulin and egt genes. Submission of the gene sequences to BLAST revealed high percentage identities to sequences from various CpGV isolates, resulting in the naming of the isolate in this study as the South African Cydia pomonella granulovirus (CpGV-SA) isolate. Phylogenetic analysis based on the single nucleotide polymorphisms (SNPs) detected in the lef-8, lef-9 and granulin nucleotide sequences grouped the South African isolate with CpGV-E2 (genome type B) and CpGV-S (genome type E). The CpGV-SA isolate was further genetically characterised by restriction endonuclease analysis and complete sequencing of the genomic DNA. Differences were observed for the BamHI, EcoRI, PstI and XhoI profiles of CpGV-SA in comparison to the respective profiles generated for CpGV-M extracted from a biopesticide, Carpovirusine® (Arysta Lifescience, France). Several genetic variations between the complete genome sequence of CpGV-SA and the reference isolate, CpGV-M1, as well as a recent genome submission of CpGV-M, both representing genome type A were observed. The complete genome analysis confirmed that CpGV-SA is genetically different from the Mexican CpGV isolate, used in thedevelopment of most biopesticides. In silico restriction profiles of the genome sequence obtained for CpGV-SA and genome sequences of genetically different CpGV isolates originating from Mexico (M1 and M), England (E2), Canada (S) and Iran (I12 and I07), available on the NCBI’s GenBank database confirmed that CpGV-SA is of mixed genotypes. Furthermore, the South African isolate shared the single common difference found in the pe38 gene of resistance overcoming isolates, which was the absence of an internal 24 nucleotide repeat present in CpGV-M1. In addition to the common difference, SNPs detected in the pe38 gene grouped the isolate with the CpGV-S isolate, suggesting that the CpGV-SA isolate is predominantly of genome type E. To determine the biological activity of CpGV-SA against neonate C. pomonella larvae, surface bioassays were conducted alongside CpGV-M (Carpovirusine®) bioassays. The LC50 and LC90 values for the South African isolate were 1.6 × 103 and 1.2 × 105 OBs/ml respectively. The LT50 was determined to be 135 hours. These values were similar to the values obtained for CpGV-M (Carpovirusine®). The results in this study suggest that a novel South African CpGV isolate of mixed genotypes, potentially able to overcome resistance in C. pomonella, with biological activity similar to CpGV-M (Carpovirusine®) and important for the control of C. pomonella was recovered. The CpGV-SA isolate could therefore potentially be developed into a biopesticide for use in resistance management strategies against C. pomonella populations in South Africa.
- Full Text:
- Date Issued: 2016
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