水稻蔗糖合成酶 RSuS2 在酵母菌 Pichia pastoris 中的表現及生化性質檢定

Expression and Characterization of Rice Sucrose Synthase RSuS2 in Pichia pastoris

陳姿利(Tz-Li Chen)

指導教授 ： 王愛玉

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.02251

摘要

蔗糖合成酶催化蔗糖與 UDP 轉換為果糖及 UDPG 之可逆反應。在水稻中有三種 RSus 基因 (RSus1, RSus2, Rus3)，RSus1 以及RSus2 在水稻中各組織中皆有表現，而 RSus3 則主要表現於水稻種子中。本論文由 Pichia pastoris 表現之 RSuS2，探討其酵素功能。
將 RSus2 cDNA 送入酵母菌 P. pastoris 中表現，轉形株以甲醇誘導 60 小時後，可以得到最大的 RSuS2 蛋白質量以及 SuS 活性。將重組 RSuS2 進行純化並進行生化性質分析。重組 RSuS2 對於蔗糖、UDP、果糖及UDPG之 Km 值分別為 104.8 mM、0.099 mM、6.65 mM 及 0.146 mM。Ca2+ 以及 Mg2+ 會促進合成方向的活性。代謝中間產物 fructose 6-phosphate 會抑制分解方向的活性，而 fructose 2,6 bisphosphate 則會活化分解方向的活性。此外，重組 RSuS2 具有膜結合型式的蛋白質存在，並且純化後的重組 RSuS2 分解方向活性可以被界面活性劑 Triton X-100 活化，合成方向的活性則可以被磷脂質 (phospholipid) 活化。

關鍵字

水稻蔗糖合成酶

並列摘要

Sucrose synthase (SuS) catalyzes the reversible conversion of sucrose and UDP into fructose and UDPG. There are three Sus isogenes in rice (Oryza sativa) (RSus1, RSus2 and RSus3). Among these three isogenes, RSus1 and RSus2 are ubiquitously expressed in various tissues, while RSus3 is expressed predominantly in rice seed. In this research, recombinant RSuS2 expressed and purified from Pichia pastoris was used to study on the enzyme function of RSuS2.
The RSus2 cDNA was introduced into yeast Pichia pastoris for expression. The transformed cells accumulated high level of RSuS2 protein and SuS activity after 60 hours of methanol induction. The recombinant RSuS2 was purified and characterized on its general properties. The Michaelis constant (Km) of the recombinant RSuS2 for sucrose, UDP, fructose, and UDPG were 104.8 mM, 0.099 mM, 6.65 mM, and 0.146 mM, respectively. The synthetic activity could be stimulated by Mg2+ and Ca2+. Moreover, the cleavage activity of RSuS2 was inhibited by fructose 6-phosphate, but was activated by fructose 2,6 bisphosphate.
On the other hand, membrane-bound RSuS was observed in the crude extract of recombinant RSuS2. The cleavage activity of purified RSuS2 was activated by Triton X-100, while the synthetic activity was activated by phospholipids.

並列關鍵字

sucrose synthase ； SuS ； Pichia pastoris

參考文獻

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