Computer simulation of binding site of maltooligosyltrehalose synthase with maltooligosaccharide and effects of mutations of residues located in substrate binding site on the transglycosyl activities

• Main Authors: Chia-jui Lin, 林嘉銳
• Other Authors: TSUEI-YUN FANG
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/11709920497591587935

碩士 === 國立臺灣海洋大學 === 食品科學系 === 94 === The maltooligosyltrehalose synthase (MTSase) mainly catalyzes an intramolecular transglycosyl reaction to form maltooligosyltrehalose from maltooligosaccharides by converting the α-1,4-glucosidic linkage at the reducing end to an α-1,1-glucosidic linkage. In addition to transglycosylation reaction, MTSase also catalysis a hydrolysis reaction to release glucose. The hydrolysis activity of MTSase was higher when using maltotriose as substrate than maltooligosaccharides with a higher degree of polymerization (DP), and this phenomenon may relate to the low binding ability of MTSase to low DP substrates. In order to know the residues located in the substrate binding site, computer simulation was used. The results of computer simulation showed that residues KX, DX, EX, DX and RX are located between subsite -2 and -4, and are hydrogen bonds between enzymes and maltopentaose. In order to confirm the proposed hydrogen bonds between MTSase and maltopentaose, mutants DXA, DXA, and RXA were constructed. The mutant DNA vectors were obtained by PCR amplification. Then the wild-type and mutant DNA were transformed into E. coli Rosetta (DE3) to express wild-type and mutant MTSases, respectively. The specific activities of purified wild-type, mutant DXA, DXA, and RXA MTSases were 84.26 U/mg, 1.10 U/mg, 1.21 U/mg, and 1.19 U/mg, respectively. The kcat / KM values of DXA, DXA, and RXA MTSases were lower than that of wild-type MTSase for 69.63 ~ 76.6 folds. This result suggest that the residues X, X and X were the important residues to the activity of MTSase. All mutant MTSases had large changes in Δ(ΔG), suggesting that there are hydrogen bonds between the substrate and residues X, X and X of wild-type MTSase.