Cloning, expression, partial characterisation and application of a recombinant GH10 xylanase, XT6, from Geobacillus stearothermophilus T6 as an additive to chicken feeds
- Authors: Sithole, Tariro
- Date: 2022-04-06
- Subjects: Chicken feed industry , Chickens Feeding and feeds , Bacillus (Bacteria) , Xylanases , Polysaccharides , Geobacillus stearothermophilus
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/292693 , vital:57007
- Description: Monogastric animal farming has largely been sustained by feeding animals with grain feedstocks containing non-starch polysaccharides (NSPs) and anti-nutritive factors, which cause adverse effects, such as increased digesta viscosity and entrapment of nutrients, which leads to the inaccessibility of nutrients. These effects have been linked to a reduction in nutrient digestion and absorption, which results in a decreased feed conversion ratio, energy metabolism and animal growth. Monogastric animals do not produce enzymes that can hydrolyse these NSPs. The application of exogenous enzymes as supplements to animal feeds has been implemented to reduce viscosity and increase nutrient absorption in poultry and pigs over the past few decades. The aim of this study was to clone, express, partially characterise and apply a glycoside hydrolase (GH) family 10 xylanase (XT6), derived from Geobacillus stearothermophilus T6, as an additive to locally produced chicken feeds. The xt6 gene (1,236 bp) was subcloned and expressed in Escherichia coli DH5α and BL21(DE3) cells, respectively. Upon expression, XT6 had a molecular weight of 42 kDa and was partially purified by Ni-NTA chromatography and ultrafiltration. The purification step resulted in a yield of 66.7% with a 16.8-fold increase in purification. XT6 exhibited maximal activity when incubated at a pH and temperature of pH 6.0 and 70°C, respectively, with a high thermostability over a broad range of pH (2–9) and temperature (30–90 °C). The specific activities of XT6 on extracted soluble and insoluble wheat flour arabinoxylans were 110.9 U/mg and 63.98 U/mg, respectively. Kinetic data showed that XT6 displayed a higher catalytic activity and affinity (Vmax = 231.60 μmol/min/mg and KM = 2.759 mg/ml) for soluble wheat arabinoxylan, compared to insoluble wheat arabinoxylan (Vmax = 99.02 μmol/min/mg and KM = 5.058 mg/ml). High-performance liquid chromatography (HPLC) analysis showed that the enzyme hydrolysed wheat flour, arabinoxylan and chicken feeds, producing a range of xylooligosaccharides (XOS), with xylotetraose and xylopentaose being the predominant XOS species. Hydrolysis of both soluble and insoluble wheat flour arabinoxylans by XT6 led to a significant reduction in substrate viscosity. The effects of simulated gastrointestinal fluid contents, such as proteases, bile salts and mucins, on XT6 stability were also studied. Exposure of XT6 to pepsin did not significantly reduce its activity; however, the inhibitory effect of trypsin and mucin on XT6 was much greater. The presence of gut-derived bile salts had no iii | P a g e significant effect on XT6 activity. Finally, it was shown that the XOS produced from the hydrolysis of chicken feeds (starter and grower feeds) by XT6 significantly enhanced the growth of the probiotic bacteria B. subtilis, while there was no significant improvement in the growth of S. thermophilus and L. bulgaricus. In conclusion, the recombinantly produced XT6 demonstrated efficient hydrolysis of starter and grower feeds, and produced XOS that showed prebiotic activity on selected probiotic bacteria. In addition, the pH, temperature and simulated gastric juice content stability of XT6 renders it an attractive candidate as an additive for chicken feeds. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Authors: Sithole, Tariro
- Date: 2022-04-06
- Subjects: Chicken feed industry , Chickens Feeding and feeds , Bacillus (Bacteria) , Xylanases , Polysaccharides , Geobacillus stearothermophilus
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/292693 , vital:57007
- Description: Monogastric animal farming has largely been sustained by feeding animals with grain feedstocks containing non-starch polysaccharides (NSPs) and anti-nutritive factors, which cause adverse effects, such as increased digesta viscosity and entrapment of nutrients, which leads to the inaccessibility of nutrients. These effects have been linked to a reduction in nutrient digestion and absorption, which results in a decreased feed conversion ratio, energy metabolism and animal growth. Monogastric animals do not produce enzymes that can hydrolyse these NSPs. The application of exogenous enzymes as supplements to animal feeds has been implemented to reduce viscosity and increase nutrient absorption in poultry and pigs over the past few decades. The aim of this study was to clone, express, partially characterise and apply a glycoside hydrolase (GH) family 10 xylanase (XT6), derived from Geobacillus stearothermophilus T6, as an additive to locally produced chicken feeds. The xt6 gene (1,236 bp) was subcloned and expressed in Escherichia coli DH5α and BL21(DE3) cells, respectively. Upon expression, XT6 had a molecular weight of 42 kDa and was partially purified by Ni-NTA chromatography and ultrafiltration. The purification step resulted in a yield of 66.7% with a 16.8-fold increase in purification. XT6 exhibited maximal activity when incubated at a pH and temperature of pH 6.0 and 70°C, respectively, with a high thermostability over a broad range of pH (2–9) and temperature (30–90 °C). The specific activities of XT6 on extracted soluble and insoluble wheat flour arabinoxylans were 110.9 U/mg and 63.98 U/mg, respectively. Kinetic data showed that XT6 displayed a higher catalytic activity and affinity (Vmax = 231.60 μmol/min/mg and KM = 2.759 mg/ml) for soluble wheat arabinoxylan, compared to insoluble wheat arabinoxylan (Vmax = 99.02 μmol/min/mg and KM = 5.058 mg/ml). High-performance liquid chromatography (HPLC) analysis showed that the enzyme hydrolysed wheat flour, arabinoxylan and chicken feeds, producing a range of xylooligosaccharides (XOS), with xylotetraose and xylopentaose being the predominant XOS species. Hydrolysis of both soluble and insoluble wheat flour arabinoxylans by XT6 led to a significant reduction in substrate viscosity. The effects of simulated gastrointestinal fluid contents, such as proteases, bile salts and mucins, on XT6 stability were also studied. Exposure of XT6 to pepsin did not significantly reduce its activity; however, the inhibitory effect of trypsin and mucin on XT6 was much greater. The presence of gut-derived bile salts had no iii | P a g e significant effect on XT6 activity. Finally, it was shown that the XOS produced from the hydrolysis of chicken feeds (starter and grower feeds) by XT6 significantly enhanced the growth of the probiotic bacteria B. subtilis, while there was no significant improvement in the growth of S. thermophilus and L. bulgaricus. In conclusion, the recombinantly produced XT6 demonstrated efficient hydrolysis of starter and grower feeds, and produced XOS that showed prebiotic activity on selected probiotic bacteria. In addition, the pH, temperature and simulated gastric juice content stability of XT6 renders it an attractive candidate as an additive for chicken feeds. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
Trace element and sulphur isotope variations of sulphides in the Koperberg Suite, O’okiep Copper District, Namaqualand, South Africa: implications for formation of sulphides and the role of crustal sulphur assimilation
- Authors: Marima, Edmore
- Date: 2022-04-06
- Subjects: Sulfur Isotopes , Magmatism South Africa Namaqualand , Sulfides , Koperberg Suite (South Africa) , Copper sulfide , Sulfur Absorption and adsorption
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/291117 , vital:56820
- Description: The major economic copper sulphide deposits hosted in the late Mesoproterozoic intrusions of the Koperberg Suite in the O’okiep Copper District immediately overlie sulphur-bearing paragneisses of the Khurisberg Subgroup in an otherwise low-sulphur granitic basement. The dominant sulphide assemblage (chalcopyrite and bornite) hosted in the Koperberg Suite is also atypical of the intermediate solid solution (iss) assemblage (chalcopyrite and pyrrhotite) observed in most Cu-Ni magmatic sulphide deposits. This study presents sulphur isotope and in-situ trace element analysis of sulphides from the Koperberg Suite and the Khurisberg Subgroup with the view of placing constraints on the role of sulphide-bearing supracrustal metasedimentary of the Khurisberg Subgroup as a source of additional sulphur in the genesis of these deposits, and ore-forming (sulphide formation) processes which result in trace element variations registered by sulphides hosted in the Koperberg Suite. The high concentrations (up to 2100 ppm) of monosulphide solid solution (mss)-incompatible trace elements (e.g., Te, Se, Bi, Ag, Pb), and the depletion in Ni and Co (<40 ppm) of sulphides hosted in the Koperberg Suite are instead consistent with the derivation of such sulphides from a Cu-rich sulphide melt which segregated from a Ni-rich sulphide melt prior to magma emplacement in the middle crust, in agreement with one of the petrogenetic models for the Koperberg Suite proposed in the existing literature. The low S/Se ratios ( ̴650-10300) of sulphides hosted in the Koperberg Suite and the high S/Se ratios ( ̴18800-56000) registered by the main sulphide phase (pyrite) in the Khurisberg Subgroup argues against crustal contamination of the Koperberg Suite magmas by the Khurisberg Subgroup. The S/Se and Cu/S ratios of coexisting bornite and chalcopyrite hosted in the Koperberg Suite are positively correlated with the bornite modal abundance in the Koperberg Suite. Such trends are interpreted to be consistent with progressive oxidation of sulphide melt, a process which results in the crystallisation of iss-bornite assemblage and/or replacement of iss with bornite due to the enrichment of Cu and depletion in S of the sulphide melt. The oxidation of sulphide melt is likely to have been effectuated by the fractional crystallisation of mss in a prior sulphide melt segregation event and/or the fractional crystallisation of Fe2+-dominated silicate phases. Fractionation of the Cu-rich melt sulphide melt (segregated from mss) also tends to enrich the residual sulphide melts in Se. Thus, the chalcopyrite-dominated assemblage with S/Se ratios of ̴1300-10200 observed in the less basic rocks in the Koperberg Suite (leucodiorites and leuconorites) is interpreted to have formed from the least evolved sulphide melt, whereas the bornite-dominated assemblage with S/Se ratios of ̴650-5500 observed in the more mafic members of the Koperberg Suite (orthopyroxenites and norites) is interpreted to have formed from the most evolved sulphide melt. The ẟ34S isotopic signatures in sulphides of the Koperberg Suite (-1.4 to +1.91‰) and the proposed contaminant, the Khurisberg Subgroup (-1.2 to +3.5‰), overlap with the those of the Koperberg Suite below the Khurisberg Subgroup (+0.74‰) and typical mantle-derived magmatic rocks (0 ± iv 2‰). Hence, the sulphur isotope variations are inconclusive as an indicator of possible crustal sulphur assimilation into the intruding mantle magma. However, considering the trace element systematics and the sulphur isotope data, the Koperberg magmas likely attained sulphur saturation at deeper crustal levels. , Thesis (MSc) -- Faculty of Science, Geology, 2022
- Full Text:
- Authors: Marima, Edmore
- Date: 2022-04-06
- Subjects: Sulfur Isotopes , Magmatism South Africa Namaqualand , Sulfides , Koperberg Suite (South Africa) , Copper sulfide , Sulfur Absorption and adsorption
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/291117 , vital:56820
- Description: The major economic copper sulphide deposits hosted in the late Mesoproterozoic intrusions of the Koperberg Suite in the O’okiep Copper District immediately overlie sulphur-bearing paragneisses of the Khurisberg Subgroup in an otherwise low-sulphur granitic basement. The dominant sulphide assemblage (chalcopyrite and bornite) hosted in the Koperberg Suite is also atypical of the intermediate solid solution (iss) assemblage (chalcopyrite and pyrrhotite) observed in most Cu-Ni magmatic sulphide deposits. This study presents sulphur isotope and in-situ trace element analysis of sulphides from the Koperberg Suite and the Khurisberg Subgroup with the view of placing constraints on the role of sulphide-bearing supracrustal metasedimentary of the Khurisberg Subgroup as a source of additional sulphur in the genesis of these deposits, and ore-forming (sulphide formation) processes which result in trace element variations registered by sulphides hosted in the Koperberg Suite. The high concentrations (up to 2100 ppm) of monosulphide solid solution (mss)-incompatible trace elements (e.g., Te, Se, Bi, Ag, Pb), and the depletion in Ni and Co (<40 ppm) of sulphides hosted in the Koperberg Suite are instead consistent with the derivation of such sulphides from a Cu-rich sulphide melt which segregated from a Ni-rich sulphide melt prior to magma emplacement in the middle crust, in agreement with one of the petrogenetic models for the Koperberg Suite proposed in the existing literature. The low S/Se ratios ( ̴650-10300) of sulphides hosted in the Koperberg Suite and the high S/Se ratios ( ̴18800-56000) registered by the main sulphide phase (pyrite) in the Khurisberg Subgroup argues against crustal contamination of the Koperberg Suite magmas by the Khurisberg Subgroup. The S/Se and Cu/S ratios of coexisting bornite and chalcopyrite hosted in the Koperberg Suite are positively correlated with the bornite modal abundance in the Koperberg Suite. Such trends are interpreted to be consistent with progressive oxidation of sulphide melt, a process which results in the crystallisation of iss-bornite assemblage and/or replacement of iss with bornite due to the enrichment of Cu and depletion in S of the sulphide melt. The oxidation of sulphide melt is likely to have been effectuated by the fractional crystallisation of mss in a prior sulphide melt segregation event and/or the fractional crystallisation of Fe2+-dominated silicate phases. Fractionation of the Cu-rich melt sulphide melt (segregated from mss) also tends to enrich the residual sulphide melts in Se. Thus, the chalcopyrite-dominated assemblage with S/Se ratios of ̴1300-10200 observed in the less basic rocks in the Koperberg Suite (leucodiorites and leuconorites) is interpreted to have formed from the least evolved sulphide melt, whereas the bornite-dominated assemblage with S/Se ratios of ̴650-5500 observed in the more mafic members of the Koperberg Suite (orthopyroxenites and norites) is interpreted to have formed from the most evolved sulphide melt. The ẟ34S isotopic signatures in sulphides of the Koperberg Suite (-1.4 to +1.91‰) and the proposed contaminant, the Khurisberg Subgroup (-1.2 to +3.5‰), overlap with the those of the Koperberg Suite below the Khurisberg Subgroup (+0.74‰) and typical mantle-derived magmatic rocks (0 ± iv 2‰). Hence, the sulphur isotope variations are inconclusive as an indicator of possible crustal sulphur assimilation into the intruding mantle magma. However, considering the trace element systematics and the sulphur isotope data, the Koperberg magmas likely attained sulphur saturation at deeper crustal levels. , Thesis (MSc) -- Faculty of Science, Geology, 2022
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