Mutation of maltooligosyltrehalose synthase genes and the expressions and activity assays of resulting mutant enzymes

• Main Authors: Man-Ling Tham, 譚曼凌
• Other Authors: Tsuei-Yun Fang
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/79343628498089082604

碩士 === 國立臺灣海洋大學 === 食品科學系 === 99 === The maltooligosyltrehalose synthase (MTSase) has glucosyltransferase activity and converts the first α-1,4-glycosidic linkage at the reducing end into α-1,1 linkage to produce maltooligosyltrehalose, and the maltooligosyltrehalose trehalohydrolase (MTHase) catalyses the hydrolytic release of trehalose from maltooligosyltrehalose. In order to improve the expression and activities of MTSase from S. solfataricus ATCC 33909, silent mutation had been used. Five silent-mutated were produced by mutating the SStreY genes sequence to be similar to chimeric gene of SA21-G107W/G131E, and displacing a partial sequence of SStreY gene to be codon which has higher usage frequencies in E. coli. The appropriate IPTG concentrations of silent-mutated MTSases in E. coli BL21(DE3)-CodonPlus-RIL were 0 mM for SS1 and 0.05 mM for SS2~SS5 respectively. According to the SDS-PAGE analysis, the expressions of MTSases of SS2 and SS5 were increased when cultivated at 20℃ for 20 hours, however, the expression of SS4 and SS5 were increased after induced by 0.05mM IPTG. After cultivated the silent-mutated at 20℃ for 20 hours, the MTSases expressions of SS3 and SS4 were decreased when the genes were expressed in E. coli BL21(DE3)-CodonPlus-RIL, but the activities of silent-mutated MTSases were significantly different to that of wild-type. When the genes were expressed in E. coli BL21(DE3)-CodonPlus-RIL, SS2 and SS4 are lower than wild type, SS1 and SS5, but the activities of SS3, SS4 and SS5 had higher than that of wild-type after induced by 0.05mM IPTG. The expression at 0.05mM IPTG for silent-mutated MTSases of SS4 and SS5 are increased when the gene were expressed in E. coli Rosetta(DE3), but the activities of SS1~SS5 were significantly different to that of wild-type. When SS4 and SS5 were expressed in E. coli Rosetta(DE3) without IPTG, the activities of SS4 and SS5 crude extract were higher than that of wild-type after treatment by French Press. After 2 hours heat treatment, the activities of SS5 was not significantly different to that of wild type excepted SS4. The SS5 mRNA secondary structure is different to that of wild-type, because SS5 had change 27 nucleic acids, which might destroy the secondary structure of wild-type, and might make the structure looser.