Prodution of Microbial D-hydantoinase in Transgenic rice plants
| Autor: | Kuang-Chih lee, 李寬志 |
|---|---|
| Rok vydání: | 2000 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 88 D-hydantoinase (DHT) catalyzes a reversible ring-opening hydrolysis of D, L-p-hydroxyphenyl-hydantoins (D, L-p-HPH). DHT has been used as biocatalyst for the stereospecific production of optically active non-proteinogenic amino acids, which are valuable synthons for the synthesis of antibiotics. DHT has been successfully exploited in different enzymatic systems in the production of pharmaceuticals. However there is a problem in the application of DHT in these operations, since the solubility of the substrate (D, L-p-HPH) requires a temperature over 50 ºC. At the temperature of 50 ºC, activity of the regular DHT is significantly decreased. Therefore, it is important to isolate a thermal stable DHT for this process. Although DHT can be easily expressed in microorganisms, mostly forms inclusion body, and usually does not retain enzyme activity. In this study, the possibility to produce DHT in transgenic rice plants was evaluated. Two full-length of DHT genes (including regular and thermal stable DHTs) were isolated and cloned in plant expression vectors under the control of constitutive (CaMV35S and rice actin) or seed specific (Ose721 and Ose705) promoters, respectively. These expression vectors were introduced into rice plants through agrobacterium-mediated transformation method. The existence of DHT gene in antibiotic resistant plants was confirmed by southern blot assays. Expression of DHT gene in various tissues was analyzed through western analyses. For gene driven by constitutive promoters, DHT was detected in both leaves and seeds, while gene driven by Ose721 and Ose705 promoters, DHT was observed only in seeds. Our preliminary results indicated that the DHTs produced in transgenic rice retain their enzymatic activities. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
| Externí odkaz: |
|