The effect of the addition of hydrogen bond between the substrate and substrate-binding site residues on the transglycosyl and hydrolytic activity of maltooligosyltrehalose synthase

• Main Authors: Tsen-Yun Wei, 魏岑芸
• Other Authors: Tsuei-Yun Fang
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/75442618941710997800

碩士 === 國立臺灣海洋大學 === 食品科學系 === 95 === Maltooligosyltrehalose synthase (MTSase) (EC 5.4.99.15) catalyzes an intramolecular transglycosyl reaction to form maltooligosyltrehalose by converting the α-1.4-glucosidic linkage at the reducing end of maltooligosaccharide to an α,α-1,1-glucosidic linkage. In order to study the effect of an additional hydrogen bond between the substrate and substrate-binding site residues on the transglycosyl and hydrolytic activity of maltooligosyltrehalose synthase. The mutant MTSases were constructed by site-directed mutagenesis. The wild-type and mutant MTSases were purified by ion-exchange chromatography and gel filteration chromatography. Among the fifteen mutant MTSases. The kcat/KM value of P402S MTSase was 201% of that of wild-type MTSase. The Δ(ΔG) value was -1.94 (kJ/mol), suggesting that mutant P402S MTSase has an additional hydrogen bond between substrate and enzyme. The selectivity ratio (%) values of P402N, P402Q and P402S MTSases was lower than that of wild-type MTSase. We presumed P402N, P402Q and P402S MTSases might enhance the yield of trehalose. Finally, trehalose production from 10% soluble starch hydrolysis by wild-type and P402S MTSases along with isoamylase and MTHase at 75℃ were 81.47% and 86.87% ,respectively. The enchanced trehalose yield by P402S MTSase agreed with the decreased selectively ratio obstained from enzyme kinetic study .