A novel dimeric exoglucanase (GH5_38): Biochemical and Structural Characterisation towards its Application in Alkyl Cellobioside Synthesis
- Mafa, Mpho S, Dirr, Heinrich, Malgas, Samkelo, Krause, Rui W M, Pletschke, Brett I
- Authors: Mafa, Mpho S , Dirr, Heinrich , Malgas, Samkelo , Krause, Rui W M , Pletschke, Brett I
- Date: 2020
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/193976 , vital:45412 , xlink:href="https://doi.org/10.3390/molecules25030746"
- Description: An exoglucanase (Exg-D) from the glycoside hydrolase family 5 subfamily 38 (GH5_38) was heterologously expressed and structurally and biochemically characterised at a molecular level for its application in alkyl glycoside synthesis. The purified Exg-D existed in both dimeric and monomeric forms in solution, which showed highest activity on mixed-linked β-glucan (88.0 and 86.7 U/mg protein, respectively) and lichenin (24.5 and 23.7 U/mg protein, respectively). They displayed a broad optimum pH range from 5.5 to 7 and a temperature optimum from 40 to 60 °C. Kinetic studies demonstrated that Exg-D had a higher affinity towards β-glucan, with a Km of 7.9 mg/mL and a kcat of 117.2 s−1, compared to lichenin which had a Km of 21.5 mg/mL and a kcat of 70.0 s−1. The circular dichroism profile of Exg-D showed that its secondary structure consisted of 11% α-helices, 36% β-strands and 53% coils. Exg-D performed transglycosylation using p-nitrophenyl cellobioside as a glycosyl donor and several primary alcohols as acceptors to produce methyl-, ethyl- and propyl-cellobiosides. These products were identified and quantified via thin-layer chromatography (TLC) and liquid chromatography–mass spectrometry (LC-MS). We concluded that Exg-D is a novel and promising oligomeric glycoside hydrolase for the one-step synthesis of alkyl glycosides with more than one monosaccharide unit.
- Full Text:
- Date Issued: 2020
- Authors: Mafa, Mpho S , Dirr, Heinrich , Malgas, Samkelo , Krause, Rui W M , Pletschke, Brett I
- Date: 2020
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/193976 , vital:45412 , xlink:href="https://doi.org/10.3390/molecules25030746"
- Description: An exoglucanase (Exg-D) from the glycoside hydrolase family 5 subfamily 38 (GH5_38) was heterologously expressed and structurally and biochemically characterised at a molecular level for its application in alkyl glycoside synthesis. The purified Exg-D existed in both dimeric and monomeric forms in solution, which showed highest activity on mixed-linked β-glucan (88.0 and 86.7 U/mg protein, respectively) and lichenin (24.5 and 23.7 U/mg protein, respectively). They displayed a broad optimum pH range from 5.5 to 7 and a temperature optimum from 40 to 60 °C. Kinetic studies demonstrated that Exg-D had a higher affinity towards β-glucan, with a Km of 7.9 mg/mL and a kcat of 117.2 s−1, compared to lichenin which had a Km of 21.5 mg/mL and a kcat of 70.0 s−1. The circular dichroism profile of Exg-D showed that its secondary structure consisted of 11% α-helices, 36% β-strands and 53% coils. Exg-D performed transglycosylation using p-nitrophenyl cellobioside as a glycosyl donor and several primary alcohols as acceptors to produce methyl-, ethyl- and propyl-cellobiosides. These products were identified and quantified via thin-layer chromatography (TLC) and liquid chromatography–mass spectrometry (LC-MS). We concluded that Exg-D is a novel and promising oligomeric glycoside hydrolase for the one-step synthesis of alkyl glycosides with more than one monosaccharide unit.
- Full Text:
- Date Issued: 2020
Evaluating Feruloyl Esterase—Xylanase Synergism for Hydroxycinnamic Acid and Xylo-Oligosaccharide Production from Untreated, Hydrothermally Pre-Treated and Dilute-Acid Pre-Treated Corn Cobs:
- Mkabayi, Lithalethu, Malgas, Samkelo, Wilhelmi, Brendan S, Pletschke, Brett I
- Authors: Mkabayi, Lithalethu , Malgas, Samkelo , Wilhelmi, Brendan S , Pletschke, Brett I
- Date: 2020
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/149240 , vital:38818 , https://doi.org/10.3390/agronomy10050688
- Description: Agricultural residues are considered the most promising option as a renewable feedstock for biofuel and high valued-added chemical production due to their availability and low cost. The efficient enzymatic hydrolysis of agricultural residues into value-added products such as sugars and hydroxycinnamic acids is a challenge because of the recalcitrant properties of the native biomass. Development of synergistic enzyme cocktails is required to overcome biomass residue recalcitrance, and achieve high yields of potential value-added products. In this study, the synergistic action of two termite metagenome-derived feruloyl esterases (FAE5 and FAE6), and an endo-xylanase (Xyn11) from Thermomyces lanuginosus, was optimized using 0.5% (w/v) insoluble wheat arabinoxylan (a model substrate) and then applied to 1% (w/v) corn cobs for the efficient production of xylo-oligosaccharides (XOS) and hydroxycinnamic acids.
- Full Text:
- Date Issued: 2020
- Authors: Mkabayi, Lithalethu , Malgas, Samkelo , Wilhelmi, Brendan S , Pletschke, Brett I
- Date: 2020
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/149240 , vital:38818 , https://doi.org/10.3390/agronomy10050688
- Description: Agricultural residues are considered the most promising option as a renewable feedstock for biofuel and high valued-added chemical production due to their availability and low cost. The efficient enzymatic hydrolysis of agricultural residues into value-added products such as sugars and hydroxycinnamic acids is a challenge because of the recalcitrant properties of the native biomass. Development of synergistic enzyme cocktails is required to overcome biomass residue recalcitrance, and achieve high yields of potential value-added products. In this study, the synergistic action of two termite metagenome-derived feruloyl esterases (FAE5 and FAE6), and an endo-xylanase (Xyn11) from Thermomyces lanuginosus, was optimized using 0.5% (w/v) insoluble wheat arabinoxylan (a model substrate) and then applied to 1% (w/v) corn cobs for the efficient production of xylo-oligosaccharides (XOS) and hydroxycinnamic acids.
- Full Text:
- Date Issued: 2020
The Effects of Alkaline Pretreatment on Agricultural Biomasses (Corn Cob and Sweet Sorghum Bagasse) and Their Hydrolysis by a Termite-Derived Enzyme Cocktail:
- Mafa, Mpho S, Malgas, Samkelo, Bhattacharya, Abhishek, Rashamuse, Konanani, Pletschke, Brett I
- Authors: Mafa, Mpho S , Malgas, Samkelo , Bhattacharya, Abhishek , Rashamuse, Konanani , Pletschke, Brett I
- Date: 2020
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/160273 , vital:40430 , https://doi.org/10.3390/agronomy10081211
- Description: Sweet sorghum bagasse (SSB) and corncob (CC) have been identified as promising feedstocks for the production of second-generation biofuels and other value-added chemicals. In this study, lime (Ca(OH)2) and NaOH pretreatment efficacy for decreasing recalcitrance from SSB and CC was investigated, and subsequently, the pretreated biomass was subjected to the hydrolytic action of an in-house formulated holocellulolytic enzyme cocktail (HEC-H). Compositional analysis revealed that SSB contained 29.34% lignin, 17.75% cellulose and 16.28% hemicellulose, while CC consisted of 22.51% lignin, 23.58% cellulose and 33.34% hemicellulose. Alkaline pretreatment was more effective in pretreating CC biomass compared to the SSB biomass.
- Full Text:
- Date Issued: 2020
- Authors: Mafa, Mpho S , Malgas, Samkelo , Bhattacharya, Abhishek , Rashamuse, Konanani , Pletschke, Brett I
- Date: 2020
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/160273 , vital:40430 , https://doi.org/10.3390/agronomy10081211
- Description: Sweet sorghum bagasse (SSB) and corncob (CC) have been identified as promising feedstocks for the production of second-generation biofuels and other value-added chemicals. In this study, lime (Ca(OH)2) and NaOH pretreatment efficacy for decreasing recalcitrance from SSB and CC was investigated, and subsequently, the pretreated biomass was subjected to the hydrolytic action of an in-house formulated holocellulolytic enzyme cocktail (HEC-H). Compositional analysis revealed that SSB contained 29.34% lignin, 17.75% cellulose and 16.28% hemicellulose, while CC consisted of 22.51% lignin, 23.58% cellulose and 33.34% hemicellulose. Alkaline pretreatment was more effective in pretreating CC biomass compared to the SSB biomass.
- Full Text:
- Date Issued: 2020
Improved endoglucanase production and mycelial biomass of some ericoid fungi
- Adeoyo, Olusegun Richard, Pletschke, Brett I, Dames, Joanna F
- Authors: Adeoyo, Olusegun Richard , Pletschke, Brett I , Dames, Joanna F
- Date: 2017
- Language: English
- Type: article , text
- Identifier: http://hdl.handle.net/10962/61435 , vital:28026 , https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5209310/
- Description: Fungal species associated with ericaceous plant roots produce a number of enzymes and other bio-active metabolites in order to enhance survival of their host plants in natural environments. This study focussed on endoglucanase production from root associated ericoid mycorrhizal and dark septate endophytic fungal isolates. Out of the five fungal isolates screened, Leohumicola sp. (ChemRU330/PPRI 13195) had the highest relative enzyme activity and was tested along with isolates belonging to Hyloscyphaceae (EdRU083/PPRI 17284) and Leotiomycetes (EdRU002/PPRI 17261) for endoglucanase production under different pH and nutritional conditions that included: carbon sources, nitrogen sources and metal ions, at an optimum temperature of 28 °C. An optimal of pH 5.0 produced enzyme activity of 3.99, 2.18 and 4.31 (U/mg protein) for isolates EdRU083, EdRU002 and Leohumicola sp. respectively. Increased enzyme activities and improved mycelial biomass production were obtained in the presence of supplements such as potassium, sodium, glucose, maltose, cellobiose, tryptone and peptone. While NaFe-EDTA and Co2+ inhibited enzyme activity. The potential role of these fungi as a source of novel enzymes is an ongoing objective of this study.
- Full Text:
- Date Issued: 2017
- Authors: Adeoyo, Olusegun Richard , Pletschke, Brett I , Dames, Joanna F
- Date: 2017
- Language: English
- Type: article , text
- Identifier: http://hdl.handle.net/10962/61435 , vital:28026 , https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5209310/
- Description: Fungal species associated with ericaceous plant roots produce a number of enzymes and other bio-active metabolites in order to enhance survival of their host plants in natural environments. This study focussed on endoglucanase production from root associated ericoid mycorrhizal and dark septate endophytic fungal isolates. Out of the five fungal isolates screened, Leohumicola sp. (ChemRU330/PPRI 13195) had the highest relative enzyme activity and was tested along with isolates belonging to Hyloscyphaceae (EdRU083/PPRI 17284) and Leotiomycetes (EdRU002/PPRI 17261) for endoglucanase production under different pH and nutritional conditions that included: carbon sources, nitrogen sources and metal ions, at an optimum temperature of 28 °C. An optimal of pH 5.0 produced enzyme activity of 3.99, 2.18 and 4.31 (U/mg protein) for isolates EdRU083, EdRU002 and Leohumicola sp. respectively. Increased enzyme activities and improved mycelial biomass production were obtained in the presence of supplements such as potassium, sodium, glucose, maltose, cellobiose, tryptone and peptone. While NaFe-EDTA and Co2+ inhibited enzyme activity. The potential role of these fungi as a source of novel enzymes is an ongoing objective of this study.
- Full Text:
- Date Issued: 2017
Undefined cellulase formulations hinder scientific reproducibility
- Himmel, Michael E, Abbas, Charles A, Baker, John O, Bayer, Edward A, Bomble, Yannick J, Brunecky, Roman, Chen, Xiaowen, Felby, Claus, Jeoh, Tina, Kumar, Rajeev, McCleary, Barry V, Pletschke, Brett I, Tucker, Melvin P, Wyman, Charles E, Decker, Stephen R
- Authors: Himmel, Michael E , Abbas, Charles A , Baker, John O , Bayer, Edward A , Bomble, Yannick J , Brunecky, Roman , Chen, Xiaowen , Felby, Claus , Jeoh, Tina , Kumar, Rajeev , McCleary, Barry V , Pletschke, Brett I , Tucker, Melvin P , Wyman, Charles E , Decker, Stephen R
- Date: 2017
- Language: English
- Type: article , text
- Identifier: http://hdl.handle.net/10962/61402 , vital:28022 , https://doi.org/10.1186/s13068-017-0974-y
- Description: In the shadow of a burgeoning biomass-to-fuels industry, biological conversion of lignocellulose to fermentable sugars in a cost-effective manner is key to the success of second-generation and advanced biofuel production. For the effective comparison of one cellulase preparation to another, cellulase assays are typically carried out with one or more engineered cellulase formulations or natural exoproteomes of known performance serving as positive controls. When these formulations have unknown composition, as is the case with several widely used commercial products, it becomes impossible to compare or reproduce work done today to work done in the future, where, for example, such preparations may not be available. Therefore, being a critical tenet of science publishing, experimental reproducibility is endangered by the continued use of these undisclosed products. We propose the introduction of standard procedures and materials to produce specific and reproducible cellulase formulations. These formulations are to serve as yardsticks to measure improvements and performance of new cellulase formulations.
- Full Text:
- Date Issued: 2017
- Authors: Himmel, Michael E , Abbas, Charles A , Baker, John O , Bayer, Edward A , Bomble, Yannick J , Brunecky, Roman , Chen, Xiaowen , Felby, Claus , Jeoh, Tina , Kumar, Rajeev , McCleary, Barry V , Pletschke, Brett I , Tucker, Melvin P , Wyman, Charles E , Decker, Stephen R
- Date: 2017
- Language: English
- Type: article , text
- Identifier: http://hdl.handle.net/10962/61402 , vital:28022 , https://doi.org/10.1186/s13068-017-0974-y
- Description: In the shadow of a burgeoning biomass-to-fuels industry, biological conversion of lignocellulose to fermentable sugars in a cost-effective manner is key to the success of second-generation and advanced biofuel production. For the effective comparison of one cellulase preparation to another, cellulase assays are typically carried out with one or more engineered cellulase formulations or natural exoproteomes of known performance serving as positive controls. When these formulations have unknown composition, as is the case with several widely used commercial products, it becomes impossible to compare or reproduce work done today to work done in the future, where, for example, such preparations may not be available. Therefore, being a critical tenet of science publishing, experimental reproducibility is endangered by the continued use of these undisclosed products. We propose the introduction of standard procedures and materials to produce specific and reproducible cellulase formulations. These formulations are to serve as yardsticks to measure improvements and performance of new cellulase formulations.
- Full Text:
- Date Issued: 2017
Revisiting cellulase production and redefining current strategies based on major challenges
- Kuhad, Ramesh Chander, Deswal, Deepa, Sharma, Sonia, Bhattacharya, Abhishek, Jain, Kavish Kumar, Kaur, Amandeep, Pletschke, Brett I, Singh, Ajay, Karp, Matti
- Authors: Kuhad, Ramesh Chander , Deswal, Deepa , Sharma, Sonia , Bhattacharya, Abhishek , Jain, Kavish Kumar , Kaur, Amandeep , Pletschke, Brett I , Singh, Ajay , Karp, Matti
- Date: 2016
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/66142 , vital:28909 , https://doi.org/10.1016/j.rser.2015.10.132
- Description: publisher version , Lignocellulosic biomass has been considered as an important and sustainable source of renewable energy. Cellulose constitutes the major component of the lignocellulosic biomass and also offers maximum recalcitrance towards its fullest utilization. The enzymatic breakdown of cellulose is achieved through cellulases. Diverse forms of microbes including fungi, bacteria, actinomycetes and yeast are known to produce cellulases that have found extensive application in various industries. Due to the current global political unrest over oil prices and the threat of global warming following combustion of fossil fuels, the paradigm of research is now focused on biofuel production from plant biomass. Conventional approaches have not been economically feasible for meeting the demands of the industry. This review provides an update regarding the status of present microbial cellulase production technologies and research with special reference to solid state fermentation and different molecular techniques such as mutagenesis, metabolic engineering and heterologous gene expression of cellulases from different microbial domains with improved catalytic and stability properties. Metagenomic and genomic studies for mining of novel cellulase genes in addition to screening of culturable strains using conventional methods have been advanced. In addition the bottlenecks associated with cellulase production and how the future research needs to be directed to provide a comprehensive technology for the production of cellulases with novel traits for application at an industrial level without economic constraints are discussed.
- Full Text: false
- Date Issued: 2016
- Authors: Kuhad, Ramesh Chander , Deswal, Deepa , Sharma, Sonia , Bhattacharya, Abhishek , Jain, Kavish Kumar , Kaur, Amandeep , Pletschke, Brett I , Singh, Ajay , Karp, Matti
- Date: 2016
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/66142 , vital:28909 , https://doi.org/10.1016/j.rser.2015.10.132
- Description: publisher version , Lignocellulosic biomass has been considered as an important and sustainable source of renewable energy. Cellulose constitutes the major component of the lignocellulosic biomass and also offers maximum recalcitrance towards its fullest utilization. The enzymatic breakdown of cellulose is achieved through cellulases. Diverse forms of microbes including fungi, bacteria, actinomycetes and yeast are known to produce cellulases that have found extensive application in various industries. Due to the current global political unrest over oil prices and the threat of global warming following combustion of fossil fuels, the paradigm of research is now focused on biofuel production from plant biomass. Conventional approaches have not been economically feasible for meeting the demands of the industry. This review provides an update regarding the status of present microbial cellulase production technologies and research with special reference to solid state fermentation and different molecular techniques such as mutagenesis, metabolic engineering and heterologous gene expression of cellulases from different microbial domains with improved catalytic and stability properties. Metagenomic and genomic studies for mining of novel cellulase genes in addition to screening of culturable strains using conventional methods have been advanced. In addition the bottlenecks associated with cellulase production and how the future research needs to be directed to provide a comprehensive technology for the production of cellulases with novel traits for application at an industrial level without economic constraints are discussed.
- Full Text: false
- Date Issued: 2016
The inhibitory effects of various substrate pre-treatment by-products and wash liquors on mannanolytic enzymes
- Malgas, Samkelo, Van Dyk, J Susan, Abboo, Sagaran, Pletschke, Brett I
- Authors: Malgas, Samkelo , Van Dyk, J Susan , Abboo, Sagaran , Pletschke, Brett I
- Date: 2016
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/66156 , vital:28911 , https://doi.org/10.1016/j.molcatb.2015.11.014
- Description: publisher version , Biomass pre-treatment is essential for achieving high levels of bioconversion through increased accessibility of hydrolytic enzymes to hydrolysable carbohydrates. However, pre-treatment by-products, such as sugar and lignin degradation products, can negatively affect the performance of hydrolytic (mannanolytic) enzymes. In this study, two monomeric sugars, five sugar degradation products, five lignin derivatives and four liquors from biomass feedstocks pre-treated by different technologies, were evaluated for their inhibitory effects on mannanolytic enzymes (α-galactosidases, β-mannanases and β-mannosidases). Lignin derivatives elicited the greatest inhibitory effect on the mannanolytic enzymes, followed by organic acids and furan derivatives derived from sugar degradation. Lignin derivative inhibition appeared to be as a result of protein–phenolic complexation, leading to protein precipitating out of solution. The functional groups on the phenolic lignin derivatives appeared to be directly related to the ability of the phenolic to interfere with enzyme activity, with the phenolic containing the highest hydroxyl group content exhibiting the greatest inhibition. It was also demonstrated that various pre-treatment technologies render different pre-treatment soluble by-products which interact in various ways with the mannanolytic enzymes. The different types of biomass (i.e. different plant species) were also shown to release different by-products that interacted with the mannanolytic enzymes in a diverse manner even when the biomass was pre-treated using the same technology. Enzyme inhibition by pre-treatment by-products can be alleviated through the removal of these compounds prior to enzymatic hydrolysis to maximize enzyme activity.
- Full Text: false
- Date Issued: 2016
- Authors: Malgas, Samkelo , Van Dyk, J Susan , Abboo, Sagaran , Pletschke, Brett I
- Date: 2016
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/66156 , vital:28911 , https://doi.org/10.1016/j.molcatb.2015.11.014
- Description: publisher version , Biomass pre-treatment is essential for achieving high levels of bioconversion through increased accessibility of hydrolytic enzymes to hydrolysable carbohydrates. However, pre-treatment by-products, such as sugar and lignin degradation products, can negatively affect the performance of hydrolytic (mannanolytic) enzymes. In this study, two monomeric sugars, five sugar degradation products, five lignin derivatives and four liquors from biomass feedstocks pre-treated by different technologies, were evaluated for their inhibitory effects on mannanolytic enzymes (α-galactosidases, β-mannanases and β-mannosidases). Lignin derivatives elicited the greatest inhibitory effect on the mannanolytic enzymes, followed by organic acids and furan derivatives derived from sugar degradation. Lignin derivative inhibition appeared to be as a result of protein–phenolic complexation, leading to protein precipitating out of solution. The functional groups on the phenolic lignin derivatives appeared to be directly related to the ability of the phenolic to interfere with enzyme activity, with the phenolic containing the highest hydroxyl group content exhibiting the greatest inhibition. It was also demonstrated that various pre-treatment technologies render different pre-treatment soluble by-products which interact in various ways with the mannanolytic enzymes. The different types of biomass (i.e. different plant species) were also shown to release different by-products that interacted with the mannanolytic enzymes in a diverse manner even when the biomass was pre-treated using the same technology. Enzyme inhibition by pre-treatment by-products can be alleviated through the removal of these compounds prior to enzymatic hydrolysis to maximize enzyme activity.
- Full Text: false
- Date Issued: 2016
Suitability of total coliform beta-D-galactosidase activity and CFU counts in monitoring faecal contamination of environmental water samples
- Wutor, V C, Togo, C A, Pletschke, Brett I
- Authors: Wutor, V C , Togo, C A , Pletschke, Brett I
- Date: 2009
- Language: English
- Type: Article
- Identifier: vital:6477 , http://hdl.handle.net/10962/d1006165 , http://www.scielo.org.za/scielo.php?pid=S1816-79502009000100010&script=sci_arttext
- Description: Total coliforms are a group of bacteria found in high numbers in mammalian intestines; hence their presence in water indicates the possible contamination with faecal material. Total and faecal coliform counts were monitored over a period of 18 months using mFC, m-Endo and CM1046 media together with enzymatic assays on 215 environmental water samples obtained from the Eastern Cape Province of South Africa. A positive correlation, with an R2 value of 0.9393 was observed between faecal and total coliform colony units employing mFc and m-Endo media, and 0.8818 using CM1046 media. Also, a positive correlation was observed between Escherichia coli colony-forming units and β-D-galactosidase (B-GAL) activity (R2=0.8542). Overall, this study indicated that faecal contamination of environmental water samples could be monitored by measuring total coliform β-galactosidase activity and total coliform colony-forming units.
- Full Text: false
- Date Issued: 2009
- Authors: Wutor, V C , Togo, C A , Pletschke, Brett I
- Date: 2009
- Language: English
- Type: Article
- Identifier: vital:6477 , http://hdl.handle.net/10962/d1006165 , http://www.scielo.org.za/scielo.php?pid=S1816-79502009000100010&script=sci_arttext
- Description: Total coliforms are a group of bacteria found in high numbers in mammalian intestines; hence their presence in water indicates the possible contamination with faecal material. Total and faecal coliform counts were monitored over a period of 18 months using mFC, m-Endo and CM1046 media together with enzymatic assays on 215 environmental water samples obtained from the Eastern Cape Province of South Africa. A positive correlation, with an R2 value of 0.9393 was observed between faecal and total coliform colony units employing mFc and m-Endo media, and 0.8818 using CM1046 media. Also, a positive correlation was observed between Escherichia coli colony-forming units and β-D-galactosidase (B-GAL) activity (R2=0.8542). Overall, this study indicated that faecal contamination of environmental water samples could be monitored by measuring total coliform β-galactosidase activity and total coliform colony-forming units.
- Full Text: false
- Date Issued: 2009
Hydrolytic enzymes in sewage sludge treatment: a mini-review
- Burgess, Jo E, Pletschke, Brett I
- Authors: Burgess, Jo E , Pletschke, Brett I
- Date: 2008
- Language: English
- Type: Article
- Identifier: vital:6478 , http://hdl.handle.net/10962/d1006166 , http://hdl.handle.net/10520/EJC116535
- Description: Biological wastewater treatment processes can be classified as either aerobic or anaerobic. These two biological treatment processes are each characterised by groups of micro-organisms and their associated enzymes. Hydrolytic enzymes secreted by these micro-organisms are vital for the rate-limiting step of hydrolysis in the treatment of highly polymeric substrates present in sewage sludge. In this mini-review, the effects of mass transfer limitation, metabolic intermediates, extracellular polymeric substances (EPS), electron acceptor conditions and pH and temperature on the activity of these enzymes are summarised. The most salient and current perspectives of the significance and the role that hydrolytic enzymes play in sewage sludge treatment are highlighted.
- Full Text:
- Date Issued: 2008
- Authors: Burgess, Jo E , Pletschke, Brett I
- Date: 2008
- Language: English
- Type: Article
- Identifier: vital:6478 , http://hdl.handle.net/10962/d1006166 , http://hdl.handle.net/10520/EJC116535
- Description: Biological wastewater treatment processes can be classified as either aerobic or anaerobic. These two biological treatment processes are each characterised by groups of micro-organisms and their associated enzymes. Hydrolytic enzymes secreted by these micro-organisms are vital for the rate-limiting step of hydrolysis in the treatment of highly polymeric substrates present in sewage sludge. In this mini-review, the effects of mass transfer limitation, metabolic intermediates, extracellular polymeric substances (EPS), electron acceptor conditions and pH and temperature on the activity of these enzymes are summarised. The most salient and current perspectives of the significance and the role that hydrolytic enzymes play in sewage sludge treatment are highlighted.
- Full Text:
- Date Issued: 2008
A novel biosensor for the detection and monitoring of -d-galactosidase of faecal origin in water
- Wutor, V C, Togo, C A, Limson, Janice L, Pletschke, Brett I
- Authors: Wutor, V C , Togo, C A , Limson, Janice L , Pletschke, Brett I
- Date: 2007
- Language: English
- Type: Article
- Identifier: vital:6468 , http://hdl.handle.net/10962/d1005797 , http://dx.doi.org/10.1016/j.enzmictec.2006.10.039
- Description: A voltammetric sensor prepared by the immobilization of metallophthalocyanine complexes onto a glassy carbon electrode has been developed for the detection of β-d-galactosidase (B-GAL) of faecal origin in water. Electrooxidation of chlorophenol red, a breakdown product of the chromogenic substrate chlorophenol red β-d-galactopyranoside, was used as a measure of β-d-galactosidase activity. At metallophthalocyanine modified electrodes, in particular copper(II) phthalocyanine, a decrease in electrode fouling was observed. The sensor was sensitive to fluctuations in pH, not significantly affected by temperature variations and could detect one colony forming unit/100 mL in 15 min. Loss of 40% sensitivity was observed over a period of 30 days. A strong correlation between sensor sensitivity and colony forming units was observed. The sensor is capable of detecting viable but nonculturable bacteria, overcoming this drawback of the use of culture media for detection of coliforms.
- Full Text:
- Date Issued: 2007
- Authors: Wutor, V C , Togo, C A , Limson, Janice L , Pletschke, Brett I
- Date: 2007
- Language: English
- Type: Article
- Identifier: vital:6468 , http://hdl.handle.net/10962/d1005797 , http://dx.doi.org/10.1016/j.enzmictec.2006.10.039
- Description: A voltammetric sensor prepared by the immobilization of metallophthalocyanine complexes onto a glassy carbon electrode has been developed for the detection of β-d-galactosidase (B-GAL) of faecal origin in water. Electrooxidation of chlorophenol red, a breakdown product of the chromogenic substrate chlorophenol red β-d-galactopyranoside, was used as a measure of β-d-galactosidase activity. At metallophthalocyanine modified electrodes, in particular copper(II) phthalocyanine, a decrease in electrode fouling was observed. The sensor was sensitive to fluctuations in pH, not significantly affected by temperature variations and could detect one colony forming unit/100 mL in 15 min. Loss of 40% sensitivity was observed over a period of 30 days. A strong correlation between sensor sensitivity and colony forming units was observed. The sensor is capable of detecting viable but nonculturable bacteria, overcoming this drawback of the use of culture media for detection of coliforms.
- Full Text:
- Date Issued: 2007
Comparison of the direct enzyme assay method with the membrane filtration technique in the quantification and monitoring of microbial indicator organisms - seasonal variations in the activities of coliforms and E.coli, temperature and pH
- Wutor, V C, Togo, C A, Pletschke, Brett I
- Authors: Wutor, V C , Togo, C A , Pletschke, Brett I
- Date: 2007
- Language: English
- Type: Article
- Identifier: vital:6479 , http://hdl.handle.net/10962/d1006241
- Description: The aim of this project was to monitor variations and relationships between coliform and E. coli counts, the activities of their marker enzymes GAL and GUD, and temperature and pH over a period of 12 months in river samples obtained from the Eastern Cape, South Africa. Several polluted water samples were collected for direct coliform β-D-galactosidase (B-GAL) and Escherichia coli β-D-glucuronidase (B-GUD) assays and the membrane filtration technique. While all the samples showed enzyme activities, not all exhibited growth on CM1046 media. Variation in B-GAL activity (40%) was observed between November (highest activity month) and May (lowest activity month). The highest and lowest B-GUD activities were observed in the months of September and May/June, respectively. The sensitivity of the spectrophotometric assay method was indicated by a limit of detection (LOD) of 1 coliform forming unit (CFU)/100 mℓ and 2 CFU/100 mℓ for coliforms and E. coli, respectively. There was a significant (P < 0.05) positive correlation between E. coli counts and GUD activity (R2 = 0.8909). A correlation of R2 = 0.9151 was also observed between total coliforms and B-GAL activity, even though the CFUs were not evenly distributed. Direct enzyme assays were also shown to be more sensitive than the membrane filtration (MF) technique.
- Full Text:
- Date Issued: 2007
- Authors: Wutor, V C , Togo, C A , Pletschke, Brett I
- Date: 2007
- Language: English
- Type: Article
- Identifier: vital:6479 , http://hdl.handle.net/10962/d1006241
- Description: The aim of this project was to monitor variations and relationships between coliform and E. coli counts, the activities of their marker enzymes GAL and GUD, and temperature and pH over a period of 12 months in river samples obtained from the Eastern Cape, South Africa. Several polluted water samples were collected for direct coliform β-D-galactosidase (B-GAL) and Escherichia coli β-D-glucuronidase (B-GUD) assays and the membrane filtration technique. While all the samples showed enzyme activities, not all exhibited growth on CM1046 media. Variation in B-GAL activity (40%) was observed between November (highest activity month) and May (lowest activity month). The highest and lowest B-GUD activities were observed in the months of September and May/June, respectively. The sensitivity of the spectrophotometric assay method was indicated by a limit of detection (LOD) of 1 coliform forming unit (CFU)/100 mℓ and 2 CFU/100 mℓ for coliforms and E. coli, respectively. There was a significant (P < 0.05) positive correlation between E. coli counts and GUD activity (R2 = 0.8909). A correlation of R2 = 0.9151 was also observed between total coliforms and B-GAL activity, even though the CFUs were not evenly distributed. Direct enzyme assays were also shown to be more sensitive than the membrane filtration (MF) technique.
- Full Text:
- Date Issued: 2007
ATP-sulphurylase: An enzymatic marker for biological sulphate reduction
- Oyekola, Oluwaseun O, Pletschke, Brett I
- Authors: Oyekola, Oluwaseun O , Pletschke, Brett I
- Date: 2006
- Language: English
- Type: Article
- Identifier: vital:6461 , http://hdl.handle.net/10962/d1005790 , http://dx.doi.org/10.1016/j.soilbio.2006.05.014
- Description: Adenosine triphosphate-sulphurylase (ATPS) plays a major role in dissimilatory sulphate reduction. In this study, the level of ATPS activity was monitored in a time course study using a biosulphidogenic batch bioreactor system. A coincident decrease in ATPS activity with a decline in sulphate concentration and an increase in sulphide concentration as biosulphidogenesis proceeded was observed. Flask studies further showed sulphate to be stimulatory to ATPS, while sulphide proved to be inhibitory. The effect of ions (Ca^(2+), Cl^(−), Fe^(2+) and Zn^(2+)) on the ATPS activity was also investigated. Most of the ions studied (Ca^(2+), Cl^(−) and Fe^(2+)) were stimulatory at lower concentrations (40–120 mg/l) but proved toxic at higher concentrations (>120 mg/l). In contrast, Zn^(2+) was inhibitory even at low concentrations ( 40 mg/l). ATPS may potentially be used as an enzymatic marker for biological sulphate reduction in sulphate-rich wastewaters and natural environments (anaerobic systems such as soils and sediments found in freshwater and marine systems), providing all residual sulphide and interfering ions are removed using a simple preparative step.
- Full Text:
- Date Issued: 2006
- Authors: Oyekola, Oluwaseun O , Pletschke, Brett I
- Date: 2006
- Language: English
- Type: Article
- Identifier: vital:6461 , http://hdl.handle.net/10962/d1005790 , http://dx.doi.org/10.1016/j.soilbio.2006.05.014
- Description: Adenosine triphosphate-sulphurylase (ATPS) plays a major role in dissimilatory sulphate reduction. In this study, the level of ATPS activity was monitored in a time course study using a biosulphidogenic batch bioreactor system. A coincident decrease in ATPS activity with a decline in sulphate concentration and an increase in sulphide concentration as biosulphidogenesis proceeded was observed. Flask studies further showed sulphate to be stimulatory to ATPS, while sulphide proved to be inhibitory. The effect of ions (Ca^(2+), Cl^(−), Fe^(2+) and Zn^(2+)) on the ATPS activity was also investigated. Most of the ions studied (Ca^(2+), Cl^(−) and Fe^(2+)) were stimulatory at lower concentrations (40–120 mg/l) but proved toxic at higher concentrations (>120 mg/l). In contrast, Zn^(2+) was inhibitory even at low concentrations ( 40 mg/l). ATPS may potentially be used as an enzymatic marker for biological sulphate reduction in sulphate-rich wastewaters and natural environments (anaerobic systems such as soils and sediments found in freshwater and marine systems), providing all residual sulphide and interfering ions are removed using a simple preparative step.
- Full Text:
- Date Issued: 2006
Cellulases (CMCases) and polyphenol oxidases from thermophilic Bacillus spp
- Mayende, Lungisa, Wilhelmi, Brendan S, Pletschke, Brett I
- Authors: Mayende, Lungisa , Wilhelmi, Brendan S , Pletschke, Brett I
- Date: 2006
- Language: English
- Type: Article
- Identifier: vital:6462 , http://hdl.handle.net/10962/d1005791 , http://dx.doi.org/10.1016/j.soilbio.2006.03.019
- Description: In composting, organic matter is degraded by cellulases and ligninolytic enzymes at temperatures typically above 50 °C. This study isolated thermophilic microorganisms from a compost system that were then screened for cellulase and polyphenol oxidase activity. Temperature optima for the cellulases and polyphenol oxidases were determined as 70 and 40 °C, respectively. Maximal cellulase activity was determined as 1.333 mg glucose released ml[superscript −1] min[superscript −1]. Maximal polyphenol oxidase activity attained was 5.111 nmol phenol ml[superscript −1] min[superscript −1]. Cellulases were found to be stable over a period of 1 h. The isolated compost microorganisms were identified as strains of Bacillus using 16S ribosomal DNA sequence analysis.
- Full Text:
- Date Issued: 2006
- Authors: Mayende, Lungisa , Wilhelmi, Brendan S , Pletschke, Brett I
- Date: 2006
- Language: English
- Type: Article
- Identifier: vital:6462 , http://hdl.handle.net/10962/d1005791 , http://dx.doi.org/10.1016/j.soilbio.2006.03.019
- Description: In composting, organic matter is degraded by cellulases and ligninolytic enzymes at temperatures typically above 50 °C. This study isolated thermophilic microorganisms from a compost system that were then screened for cellulase and polyphenol oxidase activity. Temperature optima for the cellulases and polyphenol oxidases were determined as 70 and 40 °C, respectively. Maximal cellulase activity was determined as 1.333 mg glucose released ml[superscript −1] min[superscript −1]. Maximal polyphenol oxidase activity attained was 5.111 nmol phenol ml[superscript −1] min[superscript −1]. Cellulases were found to be stable over a period of 1 h. The isolated compost microorganisms were identified as strains of Bacillus using 16S ribosomal DNA sequence analysis.
- Full Text:
- Date Issued: 2006
Cleaning fouled membranes using sludge enzymes
- Melamane, Xolisa L, Pletschke, Brett I, Leukes, Wintson D, Whiteley, Chris G
- Authors: Melamane, Xolisa L , Pletschke, Brett I , Leukes, Wintson D , Whiteley, Chris G
- Date: 2003
- Language: English
- Type: text
- Identifier: vital:6480 , http://hdl.handle.net/10962/d1006242
- Description: Maintenance of membrane performance requires inevitable cleaning or "defouling" of fouled membranes. Membrane cleaning using sludge enzymes, was investigated by first characterising ostrich abattoir effluent for potential foulants, such as lipids, proteins and polysaccharides. Static fouling of polysulphone membranes using abattoir effluent was also performed. Biochemical analysis was performed using quantitative and qualitative methods for detection of proteins on fouled and defouled membranes. The ability of sulphidogenic proteases to remove proteins adsorbed on polysulphone membranes and capillary ultrafiltration membranes after static fouling, and ability to restore permeate fluxes and transmembrane pressure after dynamic fouling was also investigated. Permeate volumes were analysed for protein and amino acids concentrations. The abattoir effluent contained 553 μg/ml of lipid, 301 μg/ml of protein, 141 μg/ml of total carbohydrate, and 0.63 μg/ml of total reducing sugars. Static fouled membranes removed 23.4percent of proteins. Defouling of dynamically fouled capillary ultrafiltration membranes using sulphidogenic proteases was successful at pH 10, 37°C, within 1 h. Sulphidogenic protease activity was 2.1 U/ml and Flux Recovery (FR percent) was 64 percent.
- Full Text:
- Date Issued: 2003
- Authors: Melamane, Xolisa L , Pletschke, Brett I , Leukes, Wintson D , Whiteley, Chris G
- Date: 2003
- Language: English
- Type: text
- Identifier: vital:6480 , http://hdl.handle.net/10962/d1006242
- Description: Maintenance of membrane performance requires inevitable cleaning or "defouling" of fouled membranes. Membrane cleaning using sludge enzymes, was investigated by first characterising ostrich abattoir effluent for potential foulants, such as lipids, proteins and polysaccharides. Static fouling of polysulphone membranes using abattoir effluent was also performed. Biochemical analysis was performed using quantitative and qualitative methods for detection of proteins on fouled and defouled membranes. The ability of sulphidogenic proteases to remove proteins adsorbed on polysulphone membranes and capillary ultrafiltration membranes after static fouling, and ability to restore permeate fluxes and transmembrane pressure after dynamic fouling was also investigated. Permeate volumes were analysed for protein and amino acids concentrations. The abattoir effluent contained 553 μg/ml of lipid, 301 μg/ml of protein, 141 μg/ml of total carbohydrate, and 0.63 μg/ml of total reducing sugars. Static fouled membranes removed 23.4percent of proteins. Defouling of dynamically fouled capillary ultrafiltration membranes using sulphidogenic proteases was successful at pH 10, 37°C, within 1 h. Sulphidogenic protease activity was 2.1 U/ml and Flux Recovery (FR percent) was 64 percent.
- Full Text:
- Date Issued: 2003
Accelerated sludge solubilisation under sulphate reducing conditions: the effect of hydrolytic enzymes on sludge floc size distribution and EPS composition
- Akhurst, P, Rose, Peter D, Whiteley, Chris G, Pletschke, Brett I
- Authors: Akhurst, P , Rose, Peter D , Whiteley, Chris G , Pletschke, Brett I
- Date: 2002
- Language: English
- Type: Conference paper
- Identifier: vital:6455 , http://hdl.handle.net/10962/d1010430
- Description: Extracellular polymeric substances (EPS) are the construction materials for microbial aggregates such as biofilms, flocs and sludge, and greatly contribute to the structural integrity of sludge flocs in wastewater treatment processes. The loss of integrity of the sewage sludge floc is believed to be due to enhanced hydrolysis of important structural components such as lignin, protein and cellulose in the sludge floc matrix. The mechanism of enhanced sludge floc fracture, due to the action of enzymes hydrolysing these structural components, remains a key element in our understanding of how the floc integrity in systems utilising a sulphate reducing system is compromised. A range of relatively non-specific exogenous enzymes (ß-glucosidase, cellulase, proteases: trypsin, pronase E and chymotrypsin) were added to a sulphidogenic bioreactor- (containing both sulphate reducing bacteria (SRB) and a methanogenic bacterial system) and a (control) methanogenic bioreactor sample, and the effect of these enzymes on sludge floc size (diameter) distribution and EPS composition was investigated. Sludge samples from the bioreactors were examined under bright field and differential interference contrast light microscopy. Proteolytic and glucohydrolytic activity of the enzymes were monitored using standard enzyme assaying techniques, and Bradford, Somogyi-Nelson, and total carbohydrate assays were performed to establish the composition of the EPS (after extraction with 3% (v/v) glutaraldehyde and Sephacryl S-400 size exclusion chromatography). Sludge flocs present in the sulphidogenic environment of the sulphidogenic bioreactor were found to have smaller diameters than their counterparts present in the methanogenic bioreactor. The addition of hydrolytic (i.e. proteolytic and glycohydrolytic) enzymes resulted in an increased rate of matrix hydrolysis, leading to increased rates of floc fracture and deflocculation. The presence of ß-glucosidase, cellulase, and proteases naturally residing within the sludge floc was confirmed. We propose that the addition of commercially available enzymes may be prohibitively costly on a large scale, and that the activity of the enzymes naturally residing within the floc matrix be optimised or enhanced. As the bulk of the EPS was shown to be composed of mainly polysaccharides, we propose that by increasing the activity of the naturally occurring ß-glucosidases residing within the floc matrix, the process of deflocculation may be enhanced. As sulphide has been shown to increase the activity of this very important key enzyme, we propose that this is one of the contributing factors why sludge solubilisation is accelerated under sulphate reducing conditions.
- Full Text:
- Date Issued: 2002
- Authors: Akhurst, P , Rose, Peter D , Whiteley, Chris G , Pletschke, Brett I
- Date: 2002
- Language: English
- Type: Conference paper
- Identifier: vital:6455 , http://hdl.handle.net/10962/d1010430
- Description: Extracellular polymeric substances (EPS) are the construction materials for microbial aggregates such as biofilms, flocs and sludge, and greatly contribute to the structural integrity of sludge flocs in wastewater treatment processes. The loss of integrity of the sewage sludge floc is believed to be due to enhanced hydrolysis of important structural components such as lignin, protein and cellulose in the sludge floc matrix. The mechanism of enhanced sludge floc fracture, due to the action of enzymes hydrolysing these structural components, remains a key element in our understanding of how the floc integrity in systems utilising a sulphate reducing system is compromised. A range of relatively non-specific exogenous enzymes (ß-glucosidase, cellulase, proteases: trypsin, pronase E and chymotrypsin) were added to a sulphidogenic bioreactor- (containing both sulphate reducing bacteria (SRB) and a methanogenic bacterial system) and a (control) methanogenic bioreactor sample, and the effect of these enzymes on sludge floc size (diameter) distribution and EPS composition was investigated. Sludge samples from the bioreactors were examined under bright field and differential interference contrast light microscopy. Proteolytic and glucohydrolytic activity of the enzymes were monitored using standard enzyme assaying techniques, and Bradford, Somogyi-Nelson, and total carbohydrate assays were performed to establish the composition of the EPS (after extraction with 3% (v/v) glutaraldehyde and Sephacryl S-400 size exclusion chromatography). Sludge flocs present in the sulphidogenic environment of the sulphidogenic bioreactor were found to have smaller diameters than their counterparts present in the methanogenic bioreactor. The addition of hydrolytic (i.e. proteolytic and glycohydrolytic) enzymes resulted in an increased rate of matrix hydrolysis, leading to increased rates of floc fracture and deflocculation. The presence of ß-glucosidase, cellulase, and proteases naturally residing within the sludge floc was confirmed. We propose that the addition of commercially available enzymes may be prohibitively costly on a large scale, and that the activity of the enzymes naturally residing within the floc matrix be optimised or enhanced. As the bulk of the EPS was shown to be composed of mainly polysaccharides, we propose that by increasing the activity of the naturally occurring ß-glucosidases residing within the floc matrix, the process of deflocculation may be enhanced. As sulphide has been shown to increase the activity of this very important key enzyme, we propose that this is one of the contributing factors why sludge solubilisation is accelerated under sulphate reducing conditions.
- Full Text:
- Date Issued: 2002
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