碩士 / 國立中興大學 / 食品暨應用生物科技學系所 / 97 / Broken rice is a low price byproduct from rice milling process. In this study, broken rice was used as a potential carbon source for trehalose synthase production by recombinant Escherichia coli RGB (DE3). Hydrolysate was obtained from enzyme saccharification. Broken rice hydrolysate (BRH) with high glucose content was used as a carbon source in a medium containing other nutrient to produce trehalose synthase by cultivating recombinant E. coli RGB (DE3) barring Picrophilus torridus trehalose synthase gene fragment (E. coli PTTS in RGB).
In a 100-L fermentor, 8kg broken rice flour, 20 L R.O. H2O, and 100 ml amylase (SPEZYME® AA) was reacted at 95℃, 150 rpm for 90 min. After reaction, the contents of glucose, maltose and maltotriose was 17.01, 39.43 and 34.01 mg/ml, respectively. When temperature decreased to 60℃,100 ml glucoamylase (OPTIMAX® 7525 HP) was added for further hydrolysis. After reaction for 120 min, broken rice hydrolysate with 218.14 mg/ml glucose was obtained.
Response surface methodology (RSM) was applied to evaluate the optimal concentration of BRH and peptone to produce trehalose synthase by E. coli RGB (DE3). The results showed that 3.411% BRH and 0.705% peptone support the highest trehalose synthase activity production to a concentration of the medium. Under this medium composition and mixed with 150 mM pH 8 sodium phosphate buffer, 45.07 ± 1.43 U/ml of crude trehalose synthase activity was obtained after 48 hr of cultivation at the inoclum size of O.D.600 2.0 at 27℃and 110 rpm. The result was closed to trehalose synthase activity produced by the same microorganism that cultivated in LB broth.
The characteristics of the purified trehalose synthase produced by E. col RGB (DE3), which cultivated in LB or BRH were the same. The optimum reaction temperature was found to be 45℃ and with good stability at 60℃. The optimum reaction pH value was pH 6.0 and about 80% of enzyme activity was obtained at pH 5.0. This enzyme was found to have good pH stability at pH 6, 7, and 8.
| Identifer | oai:union.ndltd.org:TW/097NCHU5255007 |
| Date | January 2009 |
| Creators | Shih-Han Wang, 王詩涵 |
| Contributors | 方繼 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 91 |
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