Expression, partial characterisation and utilization of a GH11 xylanase (Xyn2A) from Trichoderma viride as an additive in monogastric animal feeds
- 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
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- Date Issued: 2023-03-29
Biochemical and genetic analysis of the Mycobacterium smegmatis CnoX Chaperedoxin
- Authors: Watkins, Ariana Heloise Jo
- Date: 2023-03-29
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/422403 , vital:71939
- Description: Mycobacterium (M.) tuberculosis (Mtb) encounters numerous physical and chemical stresses associated with host immunity during infection. These include exposure to reactive oxygen, chlorine and nitrogen species, low pH, hypoxia, nutrient starvation, and metal toxicity. Cellular proteins are particularly susceptible to damage by these stresses, and the ability to prevent their irreversible damage is consequently crucial for bacterial growth and survival. Mtb employs a network of proteins that includes chaperones, disaggregases, and proteases to maintain the integrity of its proteome. The chaperedoxin, CnoX, is a recently identified stress-inducible chaperone that combines redox and holdase activities to prevent the over-oxidation and aggregation of proteins in E. coli and other proteobacterial species. In this study, we identified orthologs of the E. coli CnoX (EcCnoX) in Mtb and M. smegmatis (Msm). Bioinformatics analysis of the Mtb and Msm CnoX orthologs (MtCnoX and MsCnoX, respectively) revealed that they possess similar domains, domain architectures and predicted tertiary structures as previously characterised CnoX enzymes, i.e. an N-terminal thioredoxin (Trx) domain fused to a C-terminal TPR-motif containing domain. The EcCnoX, MsCnoX, and MtCnoX enzymes were expressed as recombinant, His-tagged proteins in E. coli and purified to near homogeneity. Biochemical analysis of the recombinant CnoX enzymes revealed that the MsCnoX and MtCnoX both lack thiol-disulphide oxidoreductase (thioredoxin) activity, as evidenced by their inability to catalyse the reduction of the disulphide bonds of insulin in vitro. Both mycobacterial CnoX enzymes displayed activity as chaperones (holdases) during thermal aggregation assays of the model substrate, malate dehydrogenase (MDH). In contrast to previously reported findings for EcCnoX, the holdase activity of the mycobacterial CnoX enzymes was constitutive and did not require exposure to hypochlorous acid (HOCl) for activation. To establish the physiological role of CnoX in Msm, cnoX knockdown (KD) and knockout (KO) mutants were generated using CRISPRi-mediated gene silencing or homologous recombination, respectively. Consistent with previous findings, CnoX activity was not essential for the growth of Msm under conventional growth conditions. Reducing or eliminating CnoX activity in the Msm KD or KO mutants, respectively, did not confer increased sensitivity to HOCl as has been observed for an E. coli cnoX mutant. Reduced CnoX activity in Msm did, however, confer sensitivity to the superoxide generator, plumbagin, and front-line antitubercular drugs rifampicin and isoniazid. The combination of biochemical and physiological data presented suggests that MsCnoX may function as a holdase for substrates following proteotoxic damage induced by certain types of oxidants, a line of investigation that will be pursued in future studies. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
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- Date Issued: 2023-03-29