The effects of mutations of active site residues on the activity and substrate selectivity of trehalose forming enzyme from Sulfolobus solfataricus ATCC35092

• Main Authors: Tong-Yuan Shih, 石東原
• Other Authors: Tsuei-Yun Fang
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/59767006057034684175

碩士 === 國立臺灣海洋大學 === 食品科學系 === 93 === Abstract Trehalose forming enzyme (TFE) mainly hydrolyzes the α-1, 4 linkage adjacent to the α-1, 1 bond of trehalosyl maltooligosaccharides to release trehalose, and it also hydrolyzes the α-1, 4 linkage at the reducing end of maltooligosaccharides to release glucose leading to the decrease of the trehalose yield. In this study, site-directed mutagenesis was used to construct mutations at residues related to substrate in order to understand the effects of that active site residues on TFE hydrolytic activitues and substrate binding. We would like to find the relationship between the kinetic parameters and trehalose yields in TFEs. The specific activities of D255A, E286A, and D380A TFEs had about 0.008-0.11％ that of wild-type TFE, indicating D255, E286 and D380 are important resideues related to catalytic activity. A259S, Y328F, F355Y, and R356K TFEs had similar catalytic efficiencies (Kcat/Km) on the degree of polymerization (DP) 4-7 trehalosyl maltooligosaccharides like those of wild-type TFE. Trehalosyl maltotetraose and trehalosyl maltopentaose were the most preferred subatrate, the worst substrate was trehalosyl maltose. Wild-type and mutant TFEs have the highest catalytic efficiencies on maltopentaose and maltohexaose, while they had the lowest catalytic efficiencies on maltotetraose. The Km values were decreased as the DP values of substrates increased for both trehalosyl maltooligosaccharides and maltooligosaccharides. The large changes in Δ(ΔG) values for trehalosyl maltotetraose hydrolysis by W218A and W218F TFEs suggested that there are a loss of hydrogen bond between the substrate and TFE. Trehalose yields from starch hydrolysis were 87.27±0.26％, 87.42 ± 0.21％, 85.84 ± 0.27％, 86.22 ± 0.61％, and 86.36 ± 0.43％ for wild-type, A259S, Y328F, F355Y, and R356K TFEs, respectively. The ratio of initial rates of glucose formation to trehalose formation from starch hydrolysis and selectivity ratios from kinetic analysis had inverse correlation with trehalose yields. The higher trehalose yields from 10％ soluble starch hydrolysis were by wild-type and mutant TFEs having the lower selectivity ratio.