碩士 / 國立臺灣海洋大學 / 食品科學系 / 97 / Trehalose (α, α-trehalose) is a naturally occurring sugar containing two D-glucose units in an α, α-1, 1 linkage. Trehalose functions in many organisms as an energy source or a protectant against the effects of freezing or dehydration. It also possesses physical and/or chemical properties that are different from other sugars, which may make trehalose an attractive ingredient in food, health, beauty and pharmaceutical products.
The structures of maltooligosyltrehalose trehalohydrolase (MTHase) in complex with maltose and trehalose from Deinococcus radiodurans reveal the trehalose could be bound specifically in the +1 and +2 binding subsites. Therefore, we would like to increase the hydrogen bond between this substrate and the +1 and +2 subsites by mutagenesis.
Predicted residues which may form hydrogen bond with substrate after mutation, many mutations may form hydrogen bond at one residual using site saturation mutagenesis, including residual A256, D288 and Y328. Only one mutation may form hydrogen bond using site-directed mutagenesis , including residual S353 and Q381. The mutant plasmids were obtained by megaprimed QuickChange site-directed mutagenesis method (megaprimed QCM method), and were transformed to E. coli BL21(DE3)-CodonPlus-RIL. Through the screening, the mutants Y328W, Y328L, S353A, and S353G were selected from site saturation mutagenesis. The specific activities of wild-type, mutant Y328W and S353G MTHases were 455, 485, and 255, respectively, and other mutant including Y328L, S353A, S353R, Q381Y were lower than wild-type by 50%.
Wild-type and Y328W MTHases showed the optimal activity at 85℃ and pH 5.0, while S353G MTHase was at 70℃ and pH 5.5. The wild-type and Y328W MTHases had the similar catalytic efficiencies (kcat / KM) on G3T, and the catalytic efficiency on G5 for Y328W MTHase was lower than that of wild-type by 9.4%. The catalytic efficiencies of S353G MTHase were lower than those of wild-type by 28.1% and 5.8% on G3T and G5, respectively. The selectivity ratios of wild-type, Y328W and S353G were 1.23, 1.06, and 1.67, respectively, indicating Y328W MTHase may be more preferable than wild-type to produce trehalose.
| Identifer | oai:union.ndltd.org:TW/097NTOU5253056 |
| Date | January 2009 |
| Creators | Han-Yang Lin, 林瀚洋 |
| 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 | 86 |
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