碩士 / 國立臺灣海洋大學 / 食品科學系 / 101 / Cyanophycin is a natural source protein-like polymer which has a molecular mass of 25 to 100 kDa，it can be used for the production of polyaspartic acid﹔Polyaspartic acid is used in a number of industrial﹐biomedical﹐and medical applications. Synthesis of cyanophycin is only catalyzed by cyanophycin synthetase﹐the activity of cyanophycin synthetase is generally depends on the presence of ATP﹐K+﹐Mg2+﹐and thiol reagent.
In this study﹐cyanophycin synthetase (cphA) was cloned into pRham N-His SUMO Kan Vector﹐and a recombinant plasmid pRham-cphA was be constructed. The plasmid with a N-terminal His-tag was transformed into Escherichia coli NEB turbo﹐then rhamnose (final concentration 0.1％) was added﹐and the cultivation was incubated for 24 hours. Cyanophycin synthetase was purified by nickel affinity chromatography﹐and SDS-PAGE analysis revealed that recombinant protein had approximately 98 kDa molecular mass.
The optimum activity of recombinant cyanophycin synthetase was obtained at 55 ℃ and pH 9.0 (Tris-HCl buffer). Recombinant cyanophycin synthetase remained more than 80％ residual activity after treatment with 20~50 ℃ for two hours and more than 70％ residual activity after incubated in pH 7.0~9.5 for 24 hours at 25 ℃. Recombinant cyanophycin synthetase had enzyme activity when Mg2+ were added. In substrate specificity test﹐recombinant cyanophycin synthetase had enzyme activity when using L-arginine and L-aspartic acid as substrate.
| Identifer | oai:union.ndltd.org:TW/101NTOU5253036 |
| Date | January 2013 |
| Creators | Ming-Feng Hong, 洪銘鋒 |
| Contributors | Tsuei-Yun Fang, 方翠筠 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 61 |
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