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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/35051

Title: Geodermatophilus obscurus DSM 43160 來源之 L-核糖異構酶之基因選殖、表現、純化及特性探討
Cloning, Expression, Purification, Characterization of L-Ribose Isomerase from Geodermatophilus obscurus DSM 43160
Authors: Ming-Yuan Yu
游銘遠
Contributors: NTOU:Department of Food Science
國立臺灣海洋大學:食品科學系
Keywords: L-核糖異構酶
L-Ribose Isomerase;Geodermatophilus obscurus DSM 43160
Date: 2012
Issue Date: 2013-10-07T02:49:54Z
Abstract: L-核糖異構酶 (L-ribose isomerase, L-RI) 為一種可用來生產 L-核糖的酵素,目前已知的L-RI 只有一個來源,是由 Acinectobacter sp. DL-28 中分離出來的,透過 BLAST (Basic Local Alignment Search Tool) 分析 Acinectobacter sp. DL-28 來源之 L-RI 胺基酸序列,可找到一個 Geodermatophilus obscurus DSM 43160 來源之假設性蛋白 (putative protein) 與 Acinectobacter sp. DL-28 來源之 L-RI 有高度相似性,因此判定此蛋白質應為 L-RI,將 Go-ri 分別構築至 pET-21b、及 pET-15b 載體上,形成三種型式之重組載體 pET-21b-Go-ri w/C-Histag、pET15-b-Go-ri w/NC-Histag 及 pET-15b-Go-ri w/N-Histag,這些重組載體可分別表現帶有 C-端、N-,C-兩端及 N-端 His-tag 的 Go-RI 酵素,在純化過程中雖然於 SDS-PAGE 膠片上有兩條亮帶,但透過 MALDI-TOF 質譜儀及 Native-PAGE 分析,判斷這兩個條帶應都是 Go-RI,以 Go-RI w/N-Histag 進行酵素特性分析,最適作用溫度為 30 ℃,最適作用 pH 為 pH 9.0 (Glycine-NaOH buffer),在 pH 6.5~10.0 下保存 24 小時仍具有 80 % 以上的相對活性,於 10~40℃ 之間保溫 2 小時仍具有 85 % 以上的相對活性,加入汞離子會完全抑制酵素活性,加入其他金屬離子不會增加酵素活性,加入 EDTA 對酵素活性沒有影響,因此 Go-RI 並不需要金屬離子,而在基質特異性部分,Go-RI 對 D-來蘇糖 (D-lyxose)、L-核糖 (L-ribose) 具有酵素活性,對 L-核糖之 Km 為 51.6 mM、Vmax 為 39.2 µmol/min/mg 、Kcat 為 0.106 sec-1。
L-ribose isomerase (L-RI) catalyzes the aldose-ketose isomerization between L-ribose and L-ribulose. In this study, the Geodermatophilus obscurus DSM 43160 L-ri gene (Go-ri) was PCR-cloned into pET-21b and pET-15b, and then expressed in Escherichia coli. Three different recombinant plasmids had been constructed. Each recombinant plasmid expressed Go-RI with a C-terminal His-tag, Go-RI with both N-terminal and C-terminal His-tag, and Go-RI with a N-terminal His-tag, respectively. Although these three types of Go-RI have two bands in SDS-PAGE after purified by nickel affinity chromatography and ion exchange chromatography, the MALDI-TOF MS and Native-PAGE results suggest that both two bands in SDS-PAGE are Go-RI. The purified Go-RI w/N-Histag was used to characterize the enzyme properties. The optimum activity of Go-RI was obtained at 30 ℃ and pH 9.0 (Glycine-NaOH buffer). Go-RI remanined more than 80 % residual activity after treatment with 10~40 ℃ for two hours and more than 85 % residual activity after incubated in pH 6.5~10.0 for 24 hours at 4 ℃. No metal ions could increase Go-RI activity, but Hg2+ inhibited Go-RI activity completely. Adding EDTA could not inhibit Go-RI activity suggest that metal ions are not required for Go-RI activity. In substrate specificity test, Go-RI had enzyme activity when using L-ribose or D-lyxose as substrate. The Km for L-ribose was 51.6 mM, Vmax was 39.2µmol/min/mg and Kcat was 0.106 sec-1.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0019932026
http://ntour.ntou.edu.tw/handle/987654321/35051
Appears in Collections:[食品科學系] 博碩士論文

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