綠竹筍蔗糖轉化酶在酵母菌 Pichia pastoris 中的表現與檢定

Expression and Characterization of Green Bamboo Shoot Invertase in Pichia pastoris

謝常文(Chang-Wen Hsieh)

指導教授 ： 宋賢一

共同指導教授 ： 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(2004年)

https://doi.org/10.6342/NTU.2004.01719

摘要

蔗糖是植物光合作用的主要產物，也是醣類運輸的主要形式。藉由蔗糖轉化酶 (β-fructofuranosidase, EC 3.2.1.26) 的作用，蔗糖可被水解成葡萄糖與果糖，提供植物細胞利用。大多數植物都具有多種的蔗糖轉化酶異構酶，不同異構酶的生理功能則尚未完全釐清。為了探討蔗糖轉化酶在綠竹快速生長過程中扮演之角色，本研究以含有綠竹筍蔗糖轉化酶 cDNA (Boβfruct2 及 Boβfruct3) 的表現質體轉形至酵母菌 Pichia pastoris X-33 中進行重組蛋白質的表現。在以 0.5% 甲醇誘導第 24 和 12 小時後，分別可以在培養基中測得酸性蔗糖轉化酶的活性，而在誘導第 84 和24 小時後分別可達到最佳表現活性；另外在細胞內的蔗糖轉化酶的活性都相當低，顯示兩者的重組蔗糖轉化酶可以被分泌到胞外。以硫酸銨分劃及金屬螯合層析法等步驟進行純化，可得部分純化之重組蔗糖轉化酶 (命名為 rIT2 和 rIT3)。以膠體過濾層析法得知 rIT2 之原態分子量約為 56 kD，rIT3 約為 65 kD。rIT2 之最適反應溫度在 50-60℃，最適反應 pH 值為 3，在 0-30℃、pH 3-7 的環境下較為穩定；而 rIT3 之最適反應溫度則為 40-50℃，最適反應 pH 值為 4，在 0-20℃、pH 3-6 的環境下較安定。rIT2 的 Km 為 0.42 mM，Vmax 為 0.10 μmole/min•μg；rIT3 的 Km 為 22.9 mM，Vmax 為 0.13 μmole/min•μg。它們除了可以水解蔗糖外，也都可以水解具有 β-果糖苷基的棉仔糖，但是相對活性較低。本研究亦已經製備了 rIT2 的專一性抗體，未來應該可以應用於基因表現的分析。

關鍵字

酵母菌 ； 綠竹筍 ； 蔗糖轉化酶

並列摘要

In plants, sucrose is the mainly product of photosynthesis and the major transport form of carbohydrate. Invertase (β-fructofuranosidase, EC 3.2.1.26) catalyzes the conversion of sucrose to glucose and fructose for plant cell utilization. Invertae isozymes exit in most of plants. However, the physiological functions of different isozymes have yet to be elucidated. For the purpose of gaining an insight into the role of invertase in green bamboo growth, this research is set to transform the expression vectors containing green bamboo invertase cDNA (Boβfruct2 and Boβfruct3) into yeast Pichia pastoris X-33 for expression of recombinant proteins. After induction with 0.5% methanol for 12 and 24 hours, acid invertase activities could be detected in the media, respectively, and reached the max at 84 hours and 24 hours, respectively. In addition, the invertase activity was very low in the cell extracts, indicating the recombinant proteins were secreted exogenously. The recombinant invertses (rIT2 and rIT3) were partially purified by ammonium sulfate fractionation and affinity chromatography. Native molecular mass of rIT2 and rIT3 are 56 kD and 65 kD, respectively. rIT2 has a pH optimum of 3.0, temperature optimum of 50-60℃, Km of 0.42 mM, and Vmax of 0.10μmole/min･μg. rIT3 has a pH optimum of 4.0, temperature optimum of 40-50℃, Km of 22.9 mM, and Vmax of 0.13μmole/min･μg. rIT2 is stable at 0-30℃ and pH 3-7, and rIT3 is stable at 0-20℃ and pH 3-6. Both enzymes hydrolyzed sucrose and raffinose, but the relative activity for raffinose were lower. The specific antibody of rIT2 has also prepared and it might to be applied to the analysis of expression.

並列關鍵字

Pichia pastoris ； green bamboo ； invertase

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