| Summary: | 碩士 === 國立臺灣海洋大學 === 食品科學系 === 99 === Maltooligosyltrehalose trehalohydrolase (MTHase) mainly hydrolyzes the α-1, 4 linkage adjacent to the α-1, 1 bond of maltooligosyltrehalose to release trehalose, and it can also hydrolyze the α-1, 4 linkage at the reducing end of maltooligosaccharides to release glucose. The relationship between -1、-2、-3 and -4 subsite of MTHase and the substrate is still unknown currently. According to the results of the proposed hydrogen bonds of Sulfolobus sofataricus KM1 MTHase complexed with maltooligosyltrehalose by Thy-Hou Lin’s group in National Tsing Hua university, the purpose of this study is to identify the hydrogen bonds between substrate and -1, -2, -3 subsites of MTHase, and then improving the activity and substrate selectivity of MTHase by adding hydrogen bonds between substate and -2, -3 subsites of MTHase.
The mutant MTHase was established by site-directed mutagenesis which residue is related to substrate-bindings in this study. The plasmids contained mutant treZ genes were obtained by PCR using mutation-designed primers. The wild-type and mutant plasmids were then transformed into Escherichia coli BL21(DE3)-CodonPlus to express wild-type and mutant MTHases, respectively. The specific activities of purified Y155A, Y155F, D156A, H195A, R447A and E450A MTHases were reduced by 99.7%, 77.7%, 76.9%, 99.8%, 88.7% and 22.6%, respectively. The value of Δ(ΔG) of Y155F was 9.66 kJ/mol, the half of this value was between 2.1 and 6.3 kJ/mol, suggesting that there are two uncharged hydrogen bonds between substrate and Y155 of wild-type. Besides, the value of Δ(ΔG) of H195A was 16.2 kJ/mol which was between 14.6 and 18.8 kJ/mol, suggesting that there is one charged hydrogen bonds between substrate and H195 of wild-type.
The specific activity of purified Y430F, G442D, G442E, G442R, G442K and G442Q MTHases were 89.9%, 80.0%, 92.8%, 83.0%, 79.1% and 73.5% of wild-type, respectively. For the kinetic studies of mutant MTHases for trehalose formation from the hydrolysis of G3T, the catalytic efficiencies (kcat/KM) of Y430F, G442D, G442E, G442R, G442K, G442Q MTHases were 1.08, 0.56, 1.19, 1.25, 1.21 and 1.23 times of wild-type MTHase, respectively. Besides, the kinetic studies of mutant MTHase for glucose formation from the hydrolysis of G5, the catalytic efficiencies of Y430F and G442E were 1.66 and 1.64 times of wild-type MTHase, respectively, and G442D, G442R, G442K, G442Q were 70.5%, 83.5%, 73%, 83.4% of wild-type MTHase, respectively. The small change of Δ(ΔG) indicates no additional hydrogen bond formed between substrate and MTHase. The selectivity ratio of G442R, G442K and G442Q were 66.5%, 60.0% and 66.5% of wild-type MTHase respectively, suggesting that they might decrease glucose formation and increase the yield of trehalose. G442E, G442R, and G442K MTHase showed a higher trehalose yield than that of wild-type MTHase, but they had no significant difference.
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