Purification and characterization of fructosyltransferase for the synthesis of short-chain fructo-oligosaccharides and investigation into thier anti-carcinogenic properties
- Authors: Nemukula, Aluwani
- Date: 2009
- Subjects: Oligosaccharides , Polygalacturonase , Aspergillus , Fructose , Inulin , Cancer -- Prevention , Cancer -- Research , Carcinogens , High performance liquid chromatography
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3927 , http://hdl.handle.net/10962/d1003986 , Oligosaccharides , Polygalacturonase , Aspergillus , Fructose , Inulin , Cancer -- Prevention , Cancer -- Research , Carcinogens , High performance liquid chromatography
- Description: There is a growing attention in the synthesis of fructo-oligosaccharides (FOS) due to their excellent bio-functional and health-promoting properties. The current production processes are limited to chemical hydrolysis reactions of plant extracts, which are often associated with several drawbacks. In this study, fructosyltransferase (FTase) and polygalacturonase (PGase) activities, present in a commercial enzyme preparation (Pectinex® Ultra SP-L) sourced from Aspergillus aculeatus, have been separated and fully purified by anion-exchange and sizeexclusion chromatography. The FTase possesses fructosyl transfer activity for FOS synthesis and the PGase has pectin hydrolytic activity. Fructosyltransferase is a single-band protein with a molecular weight of 85 kDa, whereas PGase is a distinct protein of 40 kDa. The temperature and pH optima of FTase were 60 ºC and 6.0, with a half-life of 8 h; while that for PGase were 40 ºC and 6.0, respectively. FTase was slightly inhibited in the presence of Ni²⁺, Mg²⁺ and urea; but PGase was more susceptible to divalent ions such as Ca²⁺, Mg²⁺ and Mn²⁺. The kinetic parameters (Km and Vmax) of FTase for the hydrolysis of β-(2→1) linkages from sucrose were 752.3 mM and 120.5 μmol.min⁻¹.mL⁻¹, respectively; whereas the same parameters for pectin hydrolysis by PGase were 13.0 mg.mL⁻¹ and 263 μmol.min-1.mL⁻¹, respectively. The purified FTase was able to transfer fructosyl residues from sucrose, synthesizing the corresponding chains of FOS. PGase was relatively stable at 40 ºC (t½ > 3 h), depolymerizing the pectin backbone while releasing the inulins from within the chicory roots. Analysis of various mixtures of FOS by mass spectrometry, HPLC and ¹H-NMR was undertaken. Results indicated that MS with electrospray ionization and ¹H-NMR are capable of providing relative quantitative data of the FOS present in the mixtures. The pharmaceutical effects of various sc-FOS (0.5%, v/v) and SCFA (0.3%, v/v) on certain bacterial enzymes (β-glucuronidase, urease and β-glucosidase) associated with the formation of carcinogens were also studied. These enzyme activities were not directly influenced by the sc-FOS, but were found to be remarkably decreased by SCFA, pointing toward the prebiotic effect of FOS in intestinal microflora modulation.
- Full Text:
- Date Issued: 2009
- Authors: Nemukula, Aluwani
- Date: 2009
- Subjects: Oligosaccharides , Polygalacturonase , Aspergillus , Fructose , Inulin , Cancer -- Prevention , Cancer -- Research , Carcinogens , High performance liquid chromatography
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3927 , http://hdl.handle.net/10962/d1003986 , Oligosaccharides , Polygalacturonase , Aspergillus , Fructose , Inulin , Cancer -- Prevention , Cancer -- Research , Carcinogens , High performance liquid chromatography
- Description: There is a growing attention in the synthesis of fructo-oligosaccharides (FOS) due to their excellent bio-functional and health-promoting properties. The current production processes are limited to chemical hydrolysis reactions of plant extracts, which are often associated with several drawbacks. In this study, fructosyltransferase (FTase) and polygalacturonase (PGase) activities, present in a commercial enzyme preparation (Pectinex® Ultra SP-L) sourced from Aspergillus aculeatus, have been separated and fully purified by anion-exchange and sizeexclusion chromatography. The FTase possesses fructosyl transfer activity for FOS synthesis and the PGase has pectin hydrolytic activity. Fructosyltransferase is a single-band protein with a molecular weight of 85 kDa, whereas PGase is a distinct protein of 40 kDa. The temperature and pH optima of FTase were 60 ºC and 6.0, with a half-life of 8 h; while that for PGase were 40 ºC and 6.0, respectively. FTase was slightly inhibited in the presence of Ni²⁺, Mg²⁺ and urea; but PGase was more susceptible to divalent ions such as Ca²⁺, Mg²⁺ and Mn²⁺. The kinetic parameters (Km and Vmax) of FTase for the hydrolysis of β-(2→1) linkages from sucrose were 752.3 mM and 120.5 μmol.min⁻¹.mL⁻¹, respectively; whereas the same parameters for pectin hydrolysis by PGase were 13.0 mg.mL⁻¹ and 263 μmol.min-1.mL⁻¹, respectively. The purified FTase was able to transfer fructosyl residues from sucrose, synthesizing the corresponding chains of FOS. PGase was relatively stable at 40 ºC (t½ > 3 h), depolymerizing the pectin backbone while releasing the inulins from within the chicory roots. Analysis of various mixtures of FOS by mass spectrometry, HPLC and ¹H-NMR was undertaken. Results indicated that MS with electrospray ionization and ¹H-NMR are capable of providing relative quantitative data of the FOS present in the mixtures. The pharmaceutical effects of various sc-FOS (0.5%, v/v) and SCFA (0.3%, v/v) on certain bacterial enzymes (β-glucuronidase, urease and β-glucosidase) associated with the formation of carcinogens were also studied. These enzyme activities were not directly influenced by the sc-FOS, but were found to be remarkably decreased by SCFA, pointing toward the prebiotic effect of FOS in intestinal microflora modulation.
- Full Text:
- Date Issued: 2009
Effects of vitamin A on tumour and untransformed cells
- Authors: De Villiers, Diane Lynette
- Date: 1988
- Subjects: Vitamin A , Vitamin A in the body , Cancer -- Prevention
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3881 , http://hdl.handle.net/10962/d1001615
- Description: Vitamin A and its chemical analogues (retinoids) are known to play a role in the maintenance and differentiation of epithelial tissue. Retinoids have been shown to inhibit carcinogenesis in a number of tissues in experimental animals and to inhibit the growth of various untransformed and cancer cell lines in vitro. This study investigated the effect of retinyl acetate supplemented at concentrations of 1 μM, 5 μM, 10 μM and 100 μM to in vitro cultured untransformed LLCMK cells, and transformed BL-6 melanoma and human hepatoma cell lines. A small but non-significant effect of vitamin A addition on the growth of the untransformed cells was observed, while substantial inhibition of proliferation of the two tumour cell lines was found. At the cytotoxic level of 100 μM supplemented vitamin A, all three cell lines showed marked inhibition of growth. This led to an electron microscopy study to examine the ultrastructural effect of the vitamin A addition. At the low non-toxic levels of vitamin A addition (1 - 10 μM), no ultrastructural changes were observed in the untransformed cells. However, at a level of 5 μM and 10 μM vitamin A addition in the tumour cells, an increase in the size of suspected lipid droplets was observed. At the cytotoxic level of 100 μM supplemented vitamin A, large lipid droplets were very apparent, as was much cellular degeneration. This effect was more marked in the tumour cells than in the untransformed cells. The lipid nature of the droplets was confirmed by using the lipid stain, Sudan IV. In order to investigate the effect of added vitamin A at the cell surface level, an ELISA system was used to quantify the level of the cell surface glycoprotein, fibronectin, in the culture media. Vitamin A plays an important role in the production of mature fibronectin by participating in the glycosylation of the molecule. This study showed no major effect of added vitamin A on the release of fibronectin into the culture media. This did not, however, exclude the possibility that the vitamin A was involved in the production and enhanced binding of fibronectin to the cell surface, and was possibly also exerting an effect on the availability of fibronectin receptors. Further studies would, however, be required to substantiate such effects of vitamin A supplementation. No single mechanism of action of vitamin A on tumour cell growth inhibition was identified, but the possibility that at least two mechanisms exist, was suggested
- Full Text:
- Date Issued: 1988
- Authors: De Villiers, Diane Lynette
- Date: 1988
- Subjects: Vitamin A , Vitamin A in the body , Cancer -- Prevention
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3881 , http://hdl.handle.net/10962/d1001615
- Description: Vitamin A and its chemical analogues (retinoids) are known to play a role in the maintenance and differentiation of epithelial tissue. Retinoids have been shown to inhibit carcinogenesis in a number of tissues in experimental animals and to inhibit the growth of various untransformed and cancer cell lines in vitro. This study investigated the effect of retinyl acetate supplemented at concentrations of 1 μM, 5 μM, 10 μM and 100 μM to in vitro cultured untransformed LLCMK cells, and transformed BL-6 melanoma and human hepatoma cell lines. A small but non-significant effect of vitamin A addition on the growth of the untransformed cells was observed, while substantial inhibition of proliferation of the two tumour cell lines was found. At the cytotoxic level of 100 μM supplemented vitamin A, all three cell lines showed marked inhibition of growth. This led to an electron microscopy study to examine the ultrastructural effect of the vitamin A addition. At the low non-toxic levels of vitamin A addition (1 - 10 μM), no ultrastructural changes were observed in the untransformed cells. However, at a level of 5 μM and 10 μM vitamin A addition in the tumour cells, an increase in the size of suspected lipid droplets was observed. At the cytotoxic level of 100 μM supplemented vitamin A, large lipid droplets were very apparent, as was much cellular degeneration. This effect was more marked in the tumour cells than in the untransformed cells. The lipid nature of the droplets was confirmed by using the lipid stain, Sudan IV. In order to investigate the effect of added vitamin A at the cell surface level, an ELISA system was used to quantify the level of the cell surface glycoprotein, fibronectin, in the culture media. Vitamin A plays an important role in the production of mature fibronectin by participating in the glycosylation of the molecule. This study showed no major effect of added vitamin A on the release of fibronectin into the culture media. This did not, however, exclude the possibility that the vitamin A was involved in the production and enhanced binding of fibronectin to the cell surface, and was possibly also exerting an effect on the availability of fibronectin receptors. Further studies would, however, be required to substantiate such effects of vitamin A supplementation. No single mechanism of action of vitamin A on tumour cell growth inhibition was identified, but the possibility that at least two mechanisms exist, was suggested
- Full Text:
- Date Issued: 1988
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