| Summary: | 碩士 === 南臺科技大學 === 生物科技系 === 103 === Trehalose is a non-reducing disaccharide, which is widely distributed in nature. Trehalose can provide protection against various stress conditions such as low temperature, high temperature, freezing and drying. Trehalose is composed of two glucose molecules , the binding between which mainly occurs via an alpha,-alpha-1,-1 linkage. In this study, trehalose synthase (PTTS) gene from Picrophilus torridus DSM 9790, was used to produce trehalose from maltose in a one-step reaction, with an aim to reduce the manufacturing costs of trehalose.
We used a "generally recognized as safe" (GRAS) bacterium, Bacillus subtilis, as a host strain. Bacillus subtilis can produce proteins and secrete them into the extracellular space. We took advantage of the B.subtilis to test different Signal Peptide to secreted trehalose synthase.
We also used chitin-binding sites (Chitin Binding Domain; ChBD) to immobilize trehalose synthase, SDS-PAGE showed that the trehalose synthase could specifically bind to the chitin bead via the chitin-binding sites. The optimum pH and temperature for free enzyme and immobilized enzymes was pH 6.0 and 40 °C, respectively. During the reaction, the immobilized enzyme exhibited higher tolerance to temperature and a faster conversion rate than the free enzyme, therefore, we used immobilized enzyme to convert maltose to trehalose, and achieved a conversion rate of 65% of the total concentration.
The immobilized enzyme also retained 50% of its residual activity after the 15th repeated use. In the future, we will attempt to apply the immobilized enzyme reaction for industrial production of trehalose.
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