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
Isolation, purification and characterization of inulin and fructooligosaccharides from chicorium intybus and inulinase from aspergillus niger
- Mavumengwana, Vuyo Bhongelethu
- Authors: Mavumengwana, Vuyo Bhongelethu
- Date: 2005
- Subjects: Aspergillus , Inulin , Chicory -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3954 , http://hdl.handle.net/10962/d1004013 , Aspergillus , Inulin , Chicory -- South Africa
- Description: Inulin is a non-digestible carbohydrate fructan polymer consisting mainly of β (1→2) fructosyl fructose links. Enzymatic hydrolysis of inulin by inulinase results in the production of low D.P (degree of polymerization) oligosaccharides also called fructooligosaccharides(FOS). Isolation of inulin from chicory root (Chicorium intybus) was achieved by first, extraction using deionized water (600C), followed by carbonation (0.1 M Ca(OH)2 and CO2 gas). This was filtered in order to remove the non sugars, thereafter, treated successfully with polyamide 6 powder. A cation exchanger and an anion exchanger were used to further exclude other components such as tannins and pigments. The extracted inulin was quantified using the Somogyi-Nelson colourimetric assay. Chicory root (207 g, 30 % being water) yielded 30 g of the raw extract. A 100 mg of the raw extract was assayed and found to contain 11 % yield of inulin which was 80.2 % in purity and 4 % free fructose. Analysis of the crude and purified inulin extracts on the MALDI TOF spectrometry showed the samples to have a DP of 2 to 22 and 2 to 27 respectively. Maximum inulinase production from Aspergillus niger grown on inulin was observed after 60 hours. The enzyme activity was found to be 1.168 U/ml with a temperature and pH optimum of 30 °C and 7.7 respectively. The enzyme proved to be unstable as it progressively lost its total activity during attempts at purification.
- Full Text:
- Date Issued: 2005
- Authors: Mavumengwana, Vuyo Bhongelethu
- Date: 2005
- Subjects: Aspergillus , Inulin , Chicory -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3954 , http://hdl.handle.net/10962/d1004013 , Aspergillus , Inulin , Chicory -- South Africa
- Description: Inulin is a non-digestible carbohydrate fructan polymer consisting mainly of β (1→2) fructosyl fructose links. Enzymatic hydrolysis of inulin by inulinase results in the production of low D.P (degree of polymerization) oligosaccharides also called fructooligosaccharides(FOS). Isolation of inulin from chicory root (Chicorium intybus) was achieved by first, extraction using deionized water (600C), followed by carbonation (0.1 M Ca(OH)2 and CO2 gas). This was filtered in order to remove the non sugars, thereafter, treated successfully with polyamide 6 powder. A cation exchanger and an anion exchanger were used to further exclude other components such as tannins and pigments. The extracted inulin was quantified using the Somogyi-Nelson colourimetric assay. Chicory root (207 g, 30 % being water) yielded 30 g of the raw extract. A 100 mg of the raw extract was assayed and found to contain 11 % yield of inulin which was 80.2 % in purity and 4 % free fructose. Analysis of the crude and purified inulin extracts on the MALDI TOF spectrometry showed the samples to have a DP of 2 to 22 and 2 to 27 respectively. Maximum inulinase production from Aspergillus niger grown on inulin was observed after 60 hours. The enzyme activity was found to be 1.168 U/ml with a temperature and pH optimum of 30 °C and 7.7 respectively. The enzyme proved to be unstable as it progressively lost its total activity during attempts at purification.
- Full Text:
- Date Issued: 2005
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