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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/49103

Title: Actinotalea fermentans ATCC 43279 來源重組 L-核糖異構酶之特性與固定化並以蛋白質工程改變其熱穩定性
Authors: Wu, Tai-Jeng
吳泰徵
Contributors: NTOU:Department of Food Science
國立臺灣海洋大學:食品科學系
Keywords: L-核糖;L-核糖異構酶;固定化菌體
L-ribose;L-ribose isomerase;MAESTRO;immobilized cells
Date: 2017
Issue Date: 2018-08-22T06:43:17Z
Abstract: Actinotalea fermentans ATCC 43279 含有一可能為 L-核糖異構酶之假設性蛋白,合成此蛋白之基因並構築至 pET-21b 載體後以 Escherichia coli 大量表現。此重組型 L-核糖異構酶 (Af-RI) 的最適作用溫度為 45ºC,最適作用 pH 值為 8.0 (Tricine-NaOH buffer);不需要額外添加金屬離子即有活性,但汞離子會使酵素完全失活;於 50ºC 之活性半衰期約為 74 分鐘;於 45ºC 反應至平衡時 L-核酮糖對 L-核糖之比例約為 30:70;對 L-核糖之 KM 值為 31.3 mM,Vmax 值為 232 U/mg,kcat 值為 6700 min-1,催化效率 (kcat/KM) 為 214 min-1·mM-1。與各來源之 L-RI 相比,Af-RI 具有最高的 Vmax 值、kcat 值、催化效率及酵素活性表現量,顯示 Af-RI 有生產 L-核糖之工業應用潛力。使用 MAESTRO 伺服器預測可能增加穩定性之突變,並建構一連接 SUMO (small ubiquitin-related modifier) 蛋白之 Af-RI,以期提昇 Af-RI 之熱穩定性,初步活性篩選的結果顯示,G39F、E61W、A68V、A68W 及 A165R 之突變型 Af-RI 的粗酵素液皆無活性;仍具活性之酵素進行熱穩定性篩選,於 50ºC 保溫 20 分鐘後,原生型 Af-RI、G176W、A233W、G43C/A238W 之突變型 Af-RI 及 SUMO-Af-RI 的殘餘活性分別為 19.0%、0%、16.2%、12.7% 及 9.1%,顯示各突變型 Af-RI 和 SUMO-Af-RI 的熱穩定性皆不如原生型 Af-RI。以不產內毒素之 E. coli ClearColi BL21 (DE3) 作為表現宿主,並藉由海藻酸鈣之包埋法製備含 Af-RI 的固定化菌體,其最適條件為使用 0.05% Triton X-100;5% 海藻酸鈉;50 g/L 之菌體;200 mM 氯化鈣;於氯化鈣溶液中固化 4 小時。以最適條件製備之固定化菌體的最適作用溫度為 50ºC;最適作用 pH 值為 8.0 (Tricine-NaOH buffer);於 50ºC 之活性半衰期約為 1386 分鐘,約為未固定酵素的 19 倍。
A putative L-ribose isomerase gene of A. fermentans ATCC 43279 was chemically synthesized, subcloned into pET-21b vector, and then overexpressed in Escherichia coli. The purified recombinant L-ribose isomerase (Af-RI) demonstrated its optimal activity at 45ºC and pH 8 (in Tricine-NaOH buffer). Metal ions are not required for L-ribose isomerase (L-RI) activity, but Hg2+ inhibits its activity completely. The enzyme has a half-life of 74 min at 50ºC and the equilibrium ratio of 30:70 between L-ribulose and L-ribose at 45ºC. The Vmax, kcat, KM, and catalytic efficiency of Af-RI against L-ribose are 232 U/mg, 6700 min-1, 31.3 mM, and 214 min-1·mM-1, respectively. The high expression yield of the Af-RI and its highest Vmax, kcat, and catalytic efficiency among the characterized recombinant L-RIs suggest that this recombinant enzyme shows a potential application to produce L-ribose in industry. To enhance the thermostability of Af-RI by mutagenesis, several mutations predicted by using the MAESTRO sever was constructed, and a recombinant Af-RI with a fused SUMO (small ubiquitin-related modifier) protein was also constructed. The crude extracts containing G39F, E61W, A68V, A68W and A165R mutant Af-RIs are no activity. The residual activities of the crude extracts containing wild-type, G176W, A233W, G43C/A238C Af-RIs and SUMO-Af-RI after incubating at 50ºC for 20 min are 19.0, 0, 16.2, 12.7 and 9.1%, respectively. It means that the thermostabilities of all mutant Af-RIs and SUMO-Af-RI aren’t better than that of wild-type Af-RI. Af-RI was overexpressed in strain E. coli ClearColi BL21 (DE3) which doesn’t produce endotoxin. The immobilized cells harboring Af-RI was prepared by entrapment with calcium alginate.The optimal conditions of immobilization are 0.05% Triton X-100, 5% alginate, 50 g/L cells, 200 mM CaCl2 solution, hardening in CaCl2 solution with 4 h. The immobilized cells harboring Af-RI showed its optimal activity at 50ºC and pH 8 (in Tricine-NaOH buffer) and have a half-life of 1386 min at 50ºC. The half-life of immobilized cells at 50ºC is about nineteen times longer than that of free enzyme.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0010432035.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/49103
Appears in Collections:[食品科學系] 博碩士論文

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