Expression, partial characterisation and utilization of a GH11 xylanase (Xyn2A) from Trichoderma viride as an additive in monogastric animal feeds
- Mzimkulu-Ncoyi, Nosabatha Happyness
- Authors: Mzimkulu-Ncoyi, Nosabatha Happyness
- Date: 2023-03-29
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/422409 , vital:71940
- Description: Endo-xylanases (shortly called xylanases) are a group of glycoside hydrolase enzymes that target β-D-1,4-linkages in the xylan backbone, leading to the production of xylooligosaccharides (XOS) of varying degree of polymerization (DP). Xylan is an indigestible non-starch polysaccharide present in monogastric animal feeds which in high amounts leads to increased digesta viscosity, slow movement of digesta in the intestines, malabsorption of nutrients among other challenges. The aim of this study was to investigate the effect of xylanase 2A (Xyn2A) from Trichoderma viride on broiler chicken feeds, particularly the hydrolysis of the xylan content, reduction of feed viscosity and the effect of produced XOS on eliciting the growth of gut associated probiotic bacteria. Xyn2AE was successfully induced with 0.8 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and produced in Escherichia coli BL21 (DE3) and Xyn2AC was expressed in tobacco mosaic plants. For the purification of Xyn2AE, an immobilized metal affinity chromatography (IMAC) column and diafiltration using a 3kDa cut-off Amicon filter membranes were used. Xyn2AE and Xyn2AC showed a xylanase active band at a relative weight of 21 kDa. Both enzymes showed high specificity towards soluble wheat arabinoxylan (WAX), with specific activities of 7.61 U/mg for Xyn2AE and 536.5 U/mg for Xyn2AC. Xyn2A kinetic parameters (Vmax and Km) were determined by Michaelis-Menten plots on soluble and insoluble WAX. The Vmax and Km values of Xyn2AC were 1003.01 U/mg and 9.25 mg/mL, 302.89 U/mg and 13.54 mg/mL, respectively. The Vmax and Km values of Xyn2AE for soluble and insoluble WAX were 20.45 U/mg and 12.95 mg/mL, and 8.31 U/mg and 13.15 mg/mL. Xyn2A enzymes displayed optimum activity at pH and temperature parameters of 5.0 and 50°C, respectively, and stability in temperatures ranging between 50 and 80°C and pH 4.0-9.0. Broiler chicken feeds were hydrolysed using Xyn2AE over a 24 h period and analysed using the dinitrosalicylic (DNS) assay, thin layer chromatography (TLC), viscometry and visualized using scanning electron microscope (SEM). The results showed a release of release of XOS xylotriose, xylopentose and xylohexose; enzyme’s ability to decrease the viscosity of the feeds and punched holes of feed surface, which was indicative of xylanase action. XOS produced during hydrolysis was used to study prebiotic effect on selected few bacteria and released short chain fatty acids (SCFAs) were measured. Additionally, SCFAs formation was detected in the presence of XOS as a carbon source for S. thermophilus and L. bulgaricus, whereas B. subtilis formed fewer organic acids in the presence of XOS. The results obtained from this study demonstrated that the supplementation of Xyn2A on broiler feeds has ii a positive effect in decreasing feed viscosity. Furthermore, the results of this investigation will assist the South African poultry farming sector to increase profitability in poultry farming and gain stability in the global trade as far as poultry feed is concerned. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
- Authors: Mzimkulu-Ncoyi, Nosabatha Happyness
- Date: 2023-03-29
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/422409 , vital:71940
- Description: Endo-xylanases (shortly called xylanases) are a group of glycoside hydrolase enzymes that target β-D-1,4-linkages in the xylan backbone, leading to the production of xylooligosaccharides (XOS) of varying degree of polymerization (DP). Xylan is an indigestible non-starch polysaccharide present in monogastric animal feeds which in high amounts leads to increased digesta viscosity, slow movement of digesta in the intestines, malabsorption of nutrients among other challenges. The aim of this study was to investigate the effect of xylanase 2A (Xyn2A) from Trichoderma viride on broiler chicken feeds, particularly the hydrolysis of the xylan content, reduction of feed viscosity and the effect of produced XOS on eliciting the growth of gut associated probiotic bacteria. Xyn2AE was successfully induced with 0.8 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and produced in Escherichia coli BL21 (DE3) and Xyn2AC was expressed in tobacco mosaic plants. For the purification of Xyn2AE, an immobilized metal affinity chromatography (IMAC) column and diafiltration using a 3kDa cut-off Amicon filter membranes were used. Xyn2AE and Xyn2AC showed a xylanase active band at a relative weight of 21 kDa. Both enzymes showed high specificity towards soluble wheat arabinoxylan (WAX), with specific activities of 7.61 U/mg for Xyn2AE and 536.5 U/mg for Xyn2AC. Xyn2A kinetic parameters (Vmax and Km) were determined by Michaelis-Menten plots on soluble and insoluble WAX. The Vmax and Km values of Xyn2AC were 1003.01 U/mg and 9.25 mg/mL, 302.89 U/mg and 13.54 mg/mL, respectively. The Vmax and Km values of Xyn2AE for soluble and insoluble WAX were 20.45 U/mg and 12.95 mg/mL, and 8.31 U/mg and 13.15 mg/mL. Xyn2A enzymes displayed optimum activity at pH and temperature parameters of 5.0 and 50°C, respectively, and stability in temperatures ranging between 50 and 80°C and pH 4.0-9.0. Broiler chicken feeds were hydrolysed using Xyn2AE over a 24 h period and analysed using the dinitrosalicylic (DNS) assay, thin layer chromatography (TLC), viscometry and visualized using scanning electron microscope (SEM). The results showed a release of release of XOS xylotriose, xylopentose and xylohexose; enzyme’s ability to decrease the viscosity of the feeds and punched holes of feed surface, which was indicative of xylanase action. XOS produced during hydrolysis was used to study prebiotic effect on selected few bacteria and released short chain fatty acids (SCFAs) were measured. Additionally, SCFAs formation was detected in the presence of XOS as a carbon source for S. thermophilus and L. bulgaricus, whereas B. subtilis formed fewer organic acids in the presence of XOS. The results obtained from this study demonstrated that the supplementation of Xyn2A on broiler feeds has ii a positive effect in decreasing feed viscosity. Furthermore, the results of this investigation will assist the South African poultry farming sector to increase profitability in poultry farming and gain stability in the global trade as far as poultry feed is concerned. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
- Full Text:
Making the personal political: understanding the impacts of participation in an anti-rape protest for women who have experienced sexual violence
- Authors: Barker, Kim Elise
- Date: 2023-03-29
- Subjects: Sexual assault South Africa , Silent protest , Under-reporting , Sexual abuse victims Attiudes , Narrative inquiry (Research method) , Rape Psychological aspects
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/422590 , vital:71960 , DOI 10.21504/10962/422590
- Description: South Africa is recognised as a country with unusually high levels of rape and sexual violence. The majority of those who experience sexual violence do not acknowledge, disclose or report it, and do not access support. Many factors have been identified which can inhibit acknowledgement and disclosure. While silence is to be honoured as a chosen response, unwanted silencing is of concern. The annual Silent Protest at Rhodes University is a day-long anti-rape protest which draws attention to the attitudes and practices which silence victim-survivors, and enacts solidarity with victim-survivors of sexual violence. I conducted a three year “critically engaged, activist ethnography” with the community of Silent Protestors and organisers. My focus was on the stories that victim-survivors told about the impacts of participating in the protest. Changes in the research context allowed for a participatory action research cycle to be incorporated into the methodology. This offered an opportunity to implement and evaluate some changes suggested by my research contributors. My contributor’s narratives highlighted the ways in which as a society we position those who have been subjected to sexual violence in ways that are limited and limiting and which diminish victim-survivors’ sense of agency and value. This malignant positioning restricts the choices available to victim-survivors. The anticipation of being positioned in negative ways can inhibit victim-survivors from disclosing a violation and accessing care and justice. Participation in the Silent Protest seems to stand against some of the factors which inhibit acknowledgement and disclosure. Through their participation many women took up opportunities to share their story with at least one other person and to receive and offer care. Most described participation as having been beneficial and having helped them move towards living well again. The ways in which the Silent Protest positioned victim-survivors and the choices they were offered in relation to that positioning seemed to open up opportunities for thinking, feeling and acting in ways that they preferred. This suggests that interventions which mobilise both political resistance and mutual care hold promise for developing more accessible and effective services to those affected by sexual violence. , Thesis (PhD) -- Faculty of Humanities, Psychology, 2023
- Full Text:
- Authors: Barker, Kim Elise
- Date: 2023-03-29
- Subjects: Sexual assault South Africa , Silent protest , Under-reporting , Sexual abuse victims Attiudes , Narrative inquiry (Research method) , Rape Psychological aspects
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/422590 , vital:71960 , DOI 10.21504/10962/422590
- Description: South Africa is recognised as a country with unusually high levels of rape and sexual violence. The majority of those who experience sexual violence do not acknowledge, disclose or report it, and do not access support. Many factors have been identified which can inhibit acknowledgement and disclosure. While silence is to be honoured as a chosen response, unwanted silencing is of concern. The annual Silent Protest at Rhodes University is a day-long anti-rape protest which draws attention to the attitudes and practices which silence victim-survivors, and enacts solidarity with victim-survivors of sexual violence. I conducted a three year “critically engaged, activist ethnography” with the community of Silent Protestors and organisers. My focus was on the stories that victim-survivors told about the impacts of participating in the protest. Changes in the research context allowed for a participatory action research cycle to be incorporated into the methodology. This offered an opportunity to implement and evaluate some changes suggested by my research contributors. My contributor’s narratives highlighted the ways in which as a society we position those who have been subjected to sexual violence in ways that are limited and limiting and which diminish victim-survivors’ sense of agency and value. This malignant positioning restricts the choices available to victim-survivors. The anticipation of being positioned in negative ways can inhibit victim-survivors from disclosing a violation and accessing care and justice. Participation in the Silent Protest seems to stand against some of the factors which inhibit acknowledgement and disclosure. Through their participation many women took up opportunities to share their story with at least one other person and to receive and offer care. Most described participation as having been beneficial and having helped them move towards living well again. The ways in which the Silent Protest positioned victim-survivors and the choices they were offered in relation to that positioning seemed to open up opportunities for thinking, feeling and acting in ways that they preferred. This suggests that interventions which mobilise both political resistance and mutual care hold promise for developing more accessible and effective services to those affected by sexual violence. , Thesis (PhD) -- Faculty of Humanities, Psychology, 2023
- Full Text:
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