| Summary: | 碩士 === 國立臺灣海洋大學 === 生物科技研究所 === 93 === To test the effect of the physical proximity of two enzymes catalyzing sequential reactions, a bifunctional fusion enzyme, AmyB-DRTreS, was constructed by fusing the Deinococcus radiodurans trehalose synthase (DRTreS) and Thermoanaerobacterium thermosulfurigenes thermophilic β-amylase (AmyB). AmyB-DRTreS catalyzed the sequential reaction in which maltose is formed and then converted to trehalose, leading to the synthesis of trehalose. The fused chimeric gene was over expressed in Escherichia coli, and the recombinant fusion was purified by a raw starch adsorption–desorption method. Its molecular mass was about 120 kDa. The KM values of the AmyB-DRTreS fusion enzyme for the sequential overall reaction from starch to trehalose was 1.4%. Using 3% soluble starch as substrate, the reaction rate (Vi) and final trehalose yield of AmyB-DRTreS were about threefold as compared to those of an equimolar mixture of AmyB and DRTreS. This suggested that the proximity of AmyB to DRTreS in the AmyB-DRTreS fusion enzyme could increase catalytic efficiency. The AmyB-DRTreS fusion enzyme showed optimum catalytic activity at pH 7 and 25°C. The trehalose synthase activities of AmyB-DRTreS fusion enzyme and DRTreS were quite similar, and the β-amylase activities of fusion enzyme and AmyB were also similar, suggesting that the structure of each enzyme moiety in AmyB-DRTreS is unperturbed by intramolecular constraint. The optimal temperature of the trehalose synthase activity of AmyB-DRTreS fusion protein was higher than that of DRTreS. These results clearly demonstrated that the bifunctional fusion enzyme AmyB-DRTreS, which catalyzes sequential reactions, has advantages over a mixture of both enzymes (AmyB and DRTreS).
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