水稻蔗糖合成酶 RSuS2 之 N 端序列對其酵素活性之影響

effect of N-terminal sequence of RSuS2 on its enzyme activity

梁成芝(Cheng-Tze Liang)

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(2007年)

https://doi.org/10.6342/NTU.2007.10581

摘要

蔗糖合成酶可催化蔗糖及 UDP 轉換為果糖及 UDPG 的可逆反應。在水稻中，此酵素可能由六種 RSus 基因所表現。先前研究中，RSus2 基因之表現產物已經成功在異源系統 Pichia pastoris 中表現並進行性質檢定。然而，表現質體上的 RSus2 open reading frame (ORF) 被發現在 5’-端具有一個點突變,，此突變可能使重組 RSuS2 缺少 N 端 39 個胺基酸。為了研究重組 RSuS2 的生化性質是否受到 N 端胺基酸序列的影響，本研究建構了帶有野生形 RSus2 ORF 之表現質體並轉形入 P. pastoris X33 中表現。此野生形重組 RSuS2 經由陰離子交換管柱、膠體過濾層析管柱及鎳離子親和層析管柱純化後，用於生化性質分析。酵素動力學結果顯示，RSuS2 N 端部分可能參與 RSuS2 和蔗糖之結合。

關鍵字

水稻 ； 蔗糖合成酶蔗糖 ； 糖類代謝

並列摘要

Sucrose synthase catalyzes the reversible conversion of sucrose and UDP into fructose and UDPG. In rice, the enzyme may be encoded by six RSus genes. The expression product of RSus2 gene has been heterologously expressed and characterized in Pichia pastoris in the previous study. However, a point mutation was found in the 5’-end of RSus2 open reading frame. The mutation may cause the deletion of 39 a.a. residues of the recombinant RSuS2 protein. In order to investigate whether the biochemical properties of the recombinant RSuS2 protein are affected by its N-terminal amino acid sequence, the expression plasmid carrying the wild-type RSus2 open reading frame was constructed and transformed into P. pastoris X33 for expression. The wild-type recombinant RSuS2 protein was purified by DEAE SephacelTM ion exchange chromatography, SephacrylTM S-300 gel filtration chromatography and Ni-NTA affinity chromatography, and used for biochemical characterization. The results of enzyme kinetic analysis revealed that the N-terminal part of RSuS2 may involve in sucrose binding.

並列關鍵字

Rice ； sucrose synthase ； sucrose ； sugar metabolism

參考文獻

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