Isolation, purification and characterization of a novel glucose oxidase from Penicillium canescens Tt42
- Authors: Simpson, Clinton
- Date: 2006
- Subjects: Penicillium , Glucose , Oxidases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3975 , http://hdl.handle.net/10962/d1004034 , Penicillium , Glucose , Oxidases
- Description: A novel glucose oxidase from Penicillium canescens (Tt42) was isolated, purified and characterised. The P. canescens Tt42 was cultivated using an optimised growth medium from literature, and maximum glucose oxidase activities of 11.5 U/ml and 6.9 U/ml for the intra- and extracellular fractions were obtained. Maximum glucose oxidase production was achieved after 72 hours at 28°C which coincided with glucose depletion. A total of 1104 U (from 60ml) of glucose oxidase was produced with a biomass specific glucose oxidase activity of 1.08 Umg[superscript -1] Four methods of cell disruption were evaluated for release of intracellular glucose oxidase from P. canescens Tt42 cells. These methods were; sonication, French press, Freeze-Thaw and a high pressure cell disrupter (Z-Plus Series) from Constant systems. All the methods were successful in releasing the intracellular glucose oxidase from P. canescens Tt42. The use of the Constant Systems high pressure cell disrupter was preferred, since it was the simplest and most rapid method. Ammonium sulphate precipitation was shown to be effective as an initial purification step for extracellular glucose oxidase from P. canescens Tt42. Comparison of the intra- and extracellular glucose oxidase fractions using isoelectric focusing showed 2 isoenzymes in both fractions. The pI values of the isoenzymes were determined to be 4.30 and 4.67, with the former being dominant. Since both the intra- and extracellular fractions contained the same isoenzymes of glucose oxidase, further purification studies were performed using the extracellular fraction. The glucose oxidase from P. canescens Tt42 was purified using 3 main techniques: ammonium sulphate precipitation (60% - 70% cut), anion exchange chromatography (Super Q 650M) and size exclusion chromatography (Sephadex S200HR). The glucose oxidase was determined to be ±80% pure by size exclusion chromatography. The final purified glucose oxidase was lyophilised, and an overall purification yield of 10.3% was achieved with an 8.6-fold purification. The purified glucose oxidase was confirmed to be catalase free. Glucose oxidase from P. canescens Tt42 was determined to be a dimeric protein (M[subscript r] ±148kDa) likely consisting of 2 equal subunits (M[subscript r] ± 70kDa). The temperature optimum range was shown to be 25-30°C. The optimum pH for the oxidation of β-D-glucose was pH 7. The enzyme was shown to be stable at 25°C for 10 hours, with a half life of approximately 30 minutes at 37°C. The lyophilised enzyme was stable at -20°C for 6 months. The properties of glucose oxidase from Tt42 were comparable to alternative glucose oxidase enzymes from Aspergillus and other Penicillium species. Glucose oxidase from P. canescens Tt42 was shown to have distinct kinetic characteristics. The V[subscript max] and K[subscript m] were shown to be 651 Umg[superscript -1] and 18.4 mM towards β-D-glucose. The catalytic kcat and specificity k[subscript cat]/K[subscript m] constants for glucose oxidase from P. canescens Tt42 were shown to be 791 s[superscript -1] and 40 s[superscript -1]mM[superscript -1] each respectively. The specificity constant (k[subscript cat]/K[subscript m]) of glucose oxidase from P. canescens Tt42 was determined to be 1.3-fold higher than that that of A. niger (Sigma Type VII) and 8.7-fold lower than that of P. amagasakiense (ATCC 28686) from literature.
- Full Text:
- Date Issued: 2006
- Authors: Simpson, Clinton
- Date: 2006
- Subjects: Penicillium , Glucose , Oxidases
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3975 , http://hdl.handle.net/10962/d1004034 , Penicillium , Glucose , Oxidases
- Description: A novel glucose oxidase from Penicillium canescens (Tt42) was isolated, purified and characterised. The P. canescens Tt42 was cultivated using an optimised growth medium from literature, and maximum glucose oxidase activities of 11.5 U/ml and 6.9 U/ml for the intra- and extracellular fractions were obtained. Maximum glucose oxidase production was achieved after 72 hours at 28°C which coincided with glucose depletion. A total of 1104 U (from 60ml) of glucose oxidase was produced with a biomass specific glucose oxidase activity of 1.08 Umg[superscript -1] Four methods of cell disruption were evaluated for release of intracellular glucose oxidase from P. canescens Tt42 cells. These methods were; sonication, French press, Freeze-Thaw and a high pressure cell disrupter (Z-Plus Series) from Constant systems. All the methods were successful in releasing the intracellular glucose oxidase from P. canescens Tt42. The use of the Constant Systems high pressure cell disrupter was preferred, since it was the simplest and most rapid method. Ammonium sulphate precipitation was shown to be effective as an initial purification step for extracellular glucose oxidase from P. canescens Tt42. Comparison of the intra- and extracellular glucose oxidase fractions using isoelectric focusing showed 2 isoenzymes in both fractions. The pI values of the isoenzymes were determined to be 4.30 and 4.67, with the former being dominant. Since both the intra- and extracellular fractions contained the same isoenzymes of glucose oxidase, further purification studies were performed using the extracellular fraction. The glucose oxidase from P. canescens Tt42 was purified using 3 main techniques: ammonium sulphate precipitation (60% - 70% cut), anion exchange chromatography (Super Q 650M) and size exclusion chromatography (Sephadex S200HR). The glucose oxidase was determined to be ±80% pure by size exclusion chromatography. The final purified glucose oxidase was lyophilised, and an overall purification yield of 10.3% was achieved with an 8.6-fold purification. The purified glucose oxidase was confirmed to be catalase free. Glucose oxidase from P. canescens Tt42 was determined to be a dimeric protein (M[subscript r] ±148kDa) likely consisting of 2 equal subunits (M[subscript r] ± 70kDa). The temperature optimum range was shown to be 25-30°C. The optimum pH for the oxidation of β-D-glucose was pH 7. The enzyme was shown to be stable at 25°C for 10 hours, with a half life of approximately 30 minutes at 37°C. The lyophilised enzyme was stable at -20°C for 6 months. The properties of glucose oxidase from Tt42 were comparable to alternative glucose oxidase enzymes from Aspergillus and other Penicillium species. Glucose oxidase from P. canescens Tt42 was shown to have distinct kinetic characteristics. The V[subscript max] and K[subscript m] were shown to be 651 Umg[superscript -1] and 18.4 mM towards β-D-glucose. The catalytic kcat and specificity k[subscript cat]/K[subscript m] constants for glucose oxidase from P. canescens Tt42 were shown to be 791 s[superscript -1] and 40 s[superscript -1]mM[superscript -1] each respectively. The specificity constant (k[subscript cat]/K[subscript m]) of glucose oxidase from P. canescens Tt42 was determined to be 1.3-fold higher than that that of A. niger (Sigma Type VII) and 8.7-fold lower than that of P. amagasakiense (ATCC 28686) from literature.
- Full Text:
- Date Issued: 2006
Thermal and photostability studies of triprolidine hydrochloride and its mixtures with cyclodextrin and glucose
- Authors: Ndlebe, Vuyelwa Jacqueline
- Date: 2004
- Subjects: Antihistamines , Glucose , Cyclodextrins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4387 , http://hdl.handle.net/10962/d1005052 , Antihistamines , Glucose , Cyclodextrins
- Description: Triprolidine hydrochloride (C₁₉H₂₂N₂.HCl.H₂O) (TPH) is a well-known antihistamine drug. It melts between 118°C and 122°C and the amount of water present is 4.5 mass percent. TPH is reported as being photosensitive and must be stored in sealed, light-tight containers. The thermal stabilities of TPH and of 1:1 molar and 1:1 mass ratio physical mixtures of TPH with beta-cyclodextrin (BCD) and with glucose have been examined using DSC, TG and TG-FTIR, complemented by X-ray powder diffraction (XRD) and infrared spectroscopic (IR) studies. Thermal studies of the solid TPH/BCD mixtures indicated that interaction between the components occurs and it is possible that the TPH molecule may be least partially accommodated in the cavity of the BCD host molecule. XRD results support this indication of inclusion. The results for mixtures of TPH/glucose also suggest that there is interaction between the two components. The results of molecular modelling suggest that TPH is most likely to be accommodated in the BCD cavity as a neutral triprolidine molecule with the toluene portion of the molecule entering first. There is also an indication that the Z-isomer should be accommodated slightly more readily than the E-isomer. Photostability studies were done by irradiating thin layers of solid samples of TPH and its mixtures for various times at 40°C using an Atlas Sun test CPS lamp operating at 550 W h m⁻². An analytical method using HPLC was developed and validated to determine the amounts of any photodegradants. DSC, TG, FTIR, XRD and IR were also used examine the irradiated samples. XRD results showed that changes in the TPH crystal structure occurred during irradiation and that these changes increased with the time of irradiation. Irradiation for 20 hours with UV or exposure to sunlight showed the presence of degradants. The results obtained illustrate the general stability of TPH, especially in the solid state. Although the potential for isomerization to the pharmaceutically inactive Z-isomer exists, this transformation would require extreme light conditions. The study has also shown TPH to be compatible with both glucose and BCD, which are potential excipients both in solid and liquid dosage forms. The presents of these excipients in dosage forms will thus not adversely affect the stability and the therapeutic efficacy of TPH. . An analytical method using HPLC was developed and validated to determine the amounts of any photodegradants. DSC, TG, FTIR, XRD and IR were also used examine the irradiated samples. XRD results showed that changes in the TPH crystal structure occurred during irradiation and that these changes increased with the time of irradiation. Irradiation for 20 hours with UV or exposure to sunlight showed the presence of degradants.
- Full Text:
- Date Issued: 2004
- Authors: Ndlebe, Vuyelwa Jacqueline
- Date: 2004
- Subjects: Antihistamines , Glucose , Cyclodextrins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4387 , http://hdl.handle.net/10962/d1005052 , Antihistamines , Glucose , Cyclodextrins
- Description: Triprolidine hydrochloride (C₁₉H₂₂N₂.HCl.H₂O) (TPH) is a well-known antihistamine drug. It melts between 118°C and 122°C and the amount of water present is 4.5 mass percent. TPH is reported as being photosensitive and must be stored in sealed, light-tight containers. The thermal stabilities of TPH and of 1:1 molar and 1:1 mass ratio physical mixtures of TPH with beta-cyclodextrin (BCD) and with glucose have been examined using DSC, TG and TG-FTIR, complemented by X-ray powder diffraction (XRD) and infrared spectroscopic (IR) studies. Thermal studies of the solid TPH/BCD mixtures indicated that interaction between the components occurs and it is possible that the TPH molecule may be least partially accommodated in the cavity of the BCD host molecule. XRD results support this indication of inclusion. The results for mixtures of TPH/glucose also suggest that there is interaction between the two components. The results of molecular modelling suggest that TPH is most likely to be accommodated in the BCD cavity as a neutral triprolidine molecule with the toluene portion of the molecule entering first. There is also an indication that the Z-isomer should be accommodated slightly more readily than the E-isomer. Photostability studies were done by irradiating thin layers of solid samples of TPH and its mixtures for various times at 40°C using an Atlas Sun test CPS lamp operating at 550 W h m⁻². An analytical method using HPLC was developed and validated to determine the amounts of any photodegradants. DSC, TG, FTIR, XRD and IR were also used examine the irradiated samples. XRD results showed that changes in the TPH crystal structure occurred during irradiation and that these changes increased with the time of irradiation. Irradiation for 20 hours with UV or exposure to sunlight showed the presence of degradants. The results obtained illustrate the general stability of TPH, especially in the solid state. Although the potential for isomerization to the pharmaceutically inactive Z-isomer exists, this transformation would require extreme light conditions. The study has also shown TPH to be compatible with both glucose and BCD, which are potential excipients both in solid and liquid dosage forms. The presents of these excipients in dosage forms will thus not adversely affect the stability and the therapeutic efficacy of TPH. . An analytical method using HPLC was developed and validated to determine the amounts of any photodegradants. DSC, TG, FTIR, XRD and IR were also used examine the irradiated samples. XRD results showed that changes in the TPH crystal structure occurred during irradiation and that these changes increased with the time of irradiation. Irradiation for 20 hours with UV or exposure to sunlight showed the presence of degradants.
- Full Text:
- Date Issued: 2004
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