Expression and Characterization of Green Bamboo Shoot Invertase in Pichia pastoris
| Autor: | Chang-Wen Hsieh, 謝常文 |
|---|---|
| Rok vydání: | 2004 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 92 In plants, sucrose is the mainly product of photosynthesis and the major transport form of carbohydrate. Invertase (β-fructofuranosidase, EC 3.2.1.26) catalyzes the conversion of sucrose to glucose and fructose for plant cell utilization. Invertae isozymes exit in most of plants. However, the physiological functions of different isozymes have yet to be elucidated. For the purpose of gaining an insight into the role of invertase in green bamboo growth, this research is set to transform the expression vectors containing green bamboo invertase cDNA (Boβfruct2 and Boβfruct3) into yeast Pichia pastoris X-33 for expression of recombinant proteins. After induction with 0.5% methanol for 12 and 24 hours, acid invertase activities could be detected in the media, respectively, and reached the max at 84 hours and 24 hours, respectively. In addition, the invertase activity was very low in the cell extracts, indicating the recombinant proteins were secreted exogenously. The recombinant invertses (rIT2 and rIT3) were partially purified by ammonium sulfate fractionation and affinity chromatography. Native molecular mass of rIT2 and rIT3 are 56 kD and 65 kD, respectively. rIT2 has a pH optimum of 3.0, temperature optimum of 50-60℃, Km of 0.42 mM, and Vmax of 0.10μmole/min･μg. rIT3 has a pH optimum of 4.0, temperature optimum of 40-50℃, Km of 22.9 mM, and Vmax of 0.13μmole/min･μg. rIT2 is stable at 0-30℃ and pH 3-7, and rIT3 is stable at 0-20℃ and pH 3-6. Both enzymes hydrolyzed sucrose and raffinose, but the relative activity for raffinose were lower. The specific antibody of rIT2 has also prepared and it might to be applied to the analysis of expression. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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