| Summary: | 碩士 === 國立臺灣海洋大學 === 食品科學系 === 92 === The cloned treX gene from Sulfolobus solfataricus ATCC 35092 was transformed to Escherichia coli. The recombinant treX gene was expressed well under the T7 expression system compared to those of T5, Tac, and ara expression systems. The subunit molecular weight of GDE estimated by SDS-PAGE was about 83 kDa. Since this gene was fused in frame with the His-tag coding sequence on pET-15b-treX expression vector, we purified recombinant GDE by metal chelating chromatography after heat treatment at 80oC. In order to express wild-type GDE, we transformed pET-15b-DH-treX, which containing no His-tag coding sequence, to E. coli BL21 (DE3)-CodonPlus-RIL. The wild-type GDE was purified sequentially by using heat treatment, ultrafiltration, and gel filtration. The recombinant his-tagged GDE was purified by using heat treatment, and metal-affinity chromatography. The obtained recombinant GDEs in both forms showed similar enzymatic properties. They all had an apparent optimal pH of 5 and an optimal temperature at 75oC. The enzymes were quite stable at the temperature up to 80oC for 2-h incubation. These GDEs were activated by K+、Na+、Ca2+、Mg2+、Zn2+、Cu2+、Ni2+ ions. The Cu2+ ion increased the activities of wild-type and recombinant GDEs up to 225% and 185%, respectively.
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