臺灣博碩士論文加值系統

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.

目錄 i
圖目錄 iii
表目錄 v
附錄目錄 v
壹、 前言 1
貳、 文獻整理 2
一、 稀有糖類 (rare sugars) 2
二、 核糖 3
三、 L-核糖生產法 4
1. 甘露醇-1-脫氫酶 5
2. 核糖醇脫氫酶 6
3. L-阿拉伯糖異構酶 6
4. 甘露糖-6-磷酸異構酶 7
5. L-核糖異構酶 9
四、 Geodermatophilus obscurus DSM 43160 菌株簡介 10
叁、 實驗設計與流程 11
一、 Geodermatophilus obscurus DSM 43160 之 L-ribose isomerase 基因選殖 11
二、重新構築質體使 Go-RI 接上不同形式的 His-tag 12
三、Go-RI 之表現與純化 13
四、Go-RI 之特性分析 13
肆、 材料與方法 13
一、 實驗材料 13
1. 菌株與載體 13
2. 標準品 14
3. 酵素 14
4. 市售套組 15
5. 培養基材料 15
6. 化學藥品 16
7. 實驗設備 18
二、 實驗方法 20
1. Geodermatophilus obscurus DSM 43160 之 L-ribose isomerase 基因選殖 20
2. 重新構築質體使 Go-RI 接上不同形式的 His-tag 29
3. Go-RI 之蛋白質表現與純化 34
4. Go-RI 之特性分析 44
伍、結果與討論 47
一. Geodermatophilus obscurus DSM 43160 來源之 L-ri 基因選殖 47
二、Go-RI w/C-Histag 之表現與純化 48
三. 重新構築質體使 Go-RI 接上不同形式的 His-tag 49
四. Go-RI 之蛋白質表現與純化 50
五. Go-RI 之特性分析 53
陸、結論 58
柒、 參考文獻 59

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