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: Feed additives , Xylanases , Trichoderma viride , Monogastric , Polysaccharides , Plant cell walls , Prebiotics
  • 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

Cloning, expression, partial characterisation and application of a recombinant GH10 xylanase, XT6, from Geobacillus stearothermophilus T6 as an additive to chicken feeds

- Sithole, Tariro

  • 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
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  • Date Issued: 2022-04-06

Nanofiber immobilized cellulases and hemicellulases for fruit waste beneficiation

- Swart, Shanna

The effect of GH family affiliations of mannanolytic enzymes on their synergistic associations during the hydrolysis of mannan-containing substrates

- Malgas, Samkelo

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