綠竹 (Bambusa oldhamii) 蔗糖轉化酶活化因子之純化與檢定

Purification and characterization of invertase activator from Bambusa oldhamii

范峻維(Jiun-Wei Fan)

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(2009年)

https://doi.org/10.6342/NTU.2009.10345

摘要

蔗糖轉化酶 (EC 3.2.1.26, β-D-fructofuranosidase)，為高等植物中參與蔗糖代謝其中一個重要酵素，可水解蔗糖產生葡萄糖及果糖。蔗糖轉化酶對於植物生長及發育扮演重要的角色。綠竹筍 (Bambusa oldhamii) 中至少擁有三種不同型態的蔗糖轉化酶，過去研究也指出綠竹筍中可能有蔗糖轉化酶活性的調控因子存在。本研究利用酵母菌 Picahia pastoris 表現重組蛋白質 rBoIT2 和 rBoIT3 作為分析綠竹筍中蔗糖轉化酶活化因子活性測定所使用。

綠竹筍的可溶性蛋白質粗抽液經硫酸銨分劃後發現，酸性蔗糖轉化酶在飽和濃度 50 ~ 80% 的硫酸銨分劃中具有最高的比活性；而在飽和濃度 50 ~ 100% 的分劃中含有可提升 rBoIT2 活性的活化因子。硫酸銨分劃經熱處理後內生性蔗糖轉化酶活性會被除去，但活化因子具有很高的熱穩定性。活化因子利用 Sepharose CL-6B 膠體過濾層析再經 Mono Q 陰離子交換層析純化，或是先進行DEAE-Sephacel 陰離子交換層析再經 Superdex G200 膠體過濾層析純化。這兩種純化方法的結果以 SDS-PAGE 檢定呈現出相似的蛋白質色帶。在分子量大小 60 kD, 45 kD, 38 kD, 25 kD 可以觀察到蛋白質色帶。純化結果顯示綠竹筍中極有可能存在蔗糖轉化酶的活化因子

關鍵字

綠竹 ； 蔗糖轉化酶活化因子

並列摘要

Invertase (EC 3.2.1.26, β-D-fructofuranosidase) is one of the major enzymes involved in sucrose metabolism of higher plants. The enzyme catalyzes the hydrolysis of sucrose to glucose and fructose, and is crucial for plant growth and development. There are at least three invertase isozymes in green bamboo (Bambusa oldhamii), and inhibitor and activator of bamboo invertase have been found in previous studies. To purify and characterize the activator of invertase from bamboo, the recombinant proteins, rBoIT2 and rBoIT3 were purified from Picahia pastoris for analysis of the activities of activator.

Soluble proteins from green bamboo shoots were fractionated by ammonium sulfate. The highest specific activity of invertase was found in the fraction of 50 ~ 80% saturation of ammonium sulfate. Activators were present in the fraction of 50 ~ 100% saturation of ammonium sulfate. After heat treatment, the activities of invertase were removed, while activators showed high thermo stability. Activators were purified by Sepharose CL-6B gel-filtration chromatography and then by Mono Q ion-exchange chromatography or first by DEAE-Sephacel ion-exchange chromatography and then by Superdex G200 gel-filtration chromatography. SDS-PAGE analysis of proteins purified by these two methods showed a similar protein pattern, which contained four major proteins with molecular masses of 60 kD, 45 kD, 38 kD, 25 kD, respectively. The results of this study indicated that the activator of invertase did exist in green bamboo.

並列關鍵字

Bambusa oldhamii ； invertase ； activator

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