Expression of recombinant trehalose-producing related enzymes in Bacillus subtilis and trehalose production from starch

• Main Authors: Liu, Ying-Chan, 劉英嬋
• Other Authors: Fang, Tsuei-Yun
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/m3v3ed

碩士 === 國立臺灣海洋大學 === 食品科學系 === 102 === The gram-negative bacterium Escherichia coli and gram-positive Bacillus subtilis are the most commonly used organisms for the productions of heterologous recombinant proteins. B. subtilis has a general advantage over E. coli that it has no lipopolysaccharides. These lipopolysaccharides are well-known as endotoxins, which can induce a systemic inflammatory response in humans or other mammals. In order to produce trehalose with higher safety for food and medical purposes at high temperatures, this study has used B. subtilis to express thermostable trehalose-producing related enzymes. The trehalose-producing related genes, including treX、treY、treZ of Sulfolobus solfataticus and treY of S. acidocaldarius, were subcloned respectively into pHT254 vector by using PCR and other gene recombination techniques. Then the recombinant plasmids were respectively transferred into B. subtilis by using electroporation, and the expressed enzymes were obtained simply by cell disruption and heat treatment. The trehalose-producing related enzymes, including isoamylase, MTSase (including SsMTSase and SaMTSase) and MTHase, were used to produce trehalose from different sources of starch. The results show that both MTSases can use raw potato starch as substrate to obtain better trehalose yields, which are 79.0% and 74.4% for SsMTSase and SaMTSase, respectively. The trehalose productions from raw potato starch at different temperatures have shown that the better yields of 82.2% and 79.0% for SsMTSase and SaMTSase, respectively, can be obtained at 80oC. When three different amounts of enzymes (isoamylase / MTSase / MTHase) were added to produce trehalose, the trehalose yields were 82.3% / 77.7% (SsMTSase /SaMTSase), 82.1% / 78.8% and 82.2% / 79.0%, for 200 U / 10 U / 10 U, 400 U / 20 U / 20 U and 800 U / 40U / 40U, respectively. The yields were not significant affected by the amounts of enzymes added. Finally, trehalose productions from raw potato starch were carried out at 80oC and the amounts of enzymes isoamylase / MTSase / MTHase = 200 U / 10 U / 10 U were added. In the reaction volume of 80 mL, trehalose yields of 80.0% and 73.5% were obtained for SsMTSase and SaMTSase, respectively, and there were no significant difference in trehalose yields between reaction volumes of 1.5 and 80 mL.