建立海藻糖合成酶之高效率純化暨活性篩選系統

張紘綸

指導教授 ： 李冠群

國立臺灣師範大學/理學院/生命科學系/碩士(2010年)

摘要

海藻糖（trehalose）為兩個葡萄糖以α1-1鍵結形成的非還原性雙糖，存在於許多生物體當中，可以作為能量的儲存形式，在脫水等逆境時穩定蛋白質和生物膜的結構，保護細胞免於環境壓力。在食品、化妝、醫藥業上都有廣泛的應用。海藻糖合成酶（trehalose synthase、TreS）為催化麥芽糖分子內鍵結轉化成海藻糖的酵素，並產生副產物葡萄糖，為海藻糖的生合成路徑之一。因為是單一酵素反應，在應用上易於控制流程，且原物料便宜與穩定，在生產海藻糖上有很大的潛力。傳統海藻糖合成酶活性測定的方式有HPLC與海藻糖水解酶（trehalase）呈色法，但這兩種方式有費時、費用昂貴與測定標的物專一性等問題，無法快速大量分析不同活性之TreS。本實驗建立了一套新的微量盤式麥芽糖水解酶（maltase）呈色法來進行大量且快速的活性分析。本法在TreS活性測定上更有效率，準確度高且不會有專一性不足的問題，適用於大量TreS樣品的分析。未來在TreS的蛋白質工程上，如定向演化上可以對大量的突變TreS進行活性篩選，來挑選出高轉化率或是高熱穩定性的突變TreS。

關鍵字

海藻糖 ； 海藻糖合成酶

並列摘要

Trehalose (α-D-glucopyranosyl-1, 1-α-D-glucopyranoside), a non-reducing disaccharide, existing in various organisms can serve as energy storage, and as protectants of protein and lipid from various stress such as cryoprotectants. It has become as an important compound in foods, cosmetics and pharmaceuticals industrials. Trehalose synthase (TreS) is one of the biosynthesis pathways of trehalose, which reversibly catalyzes the intramolecular transglucosylation (isomerization) of maltose to produce trehalose, as well as the minor side-reaction of irreversible hydrolysis of maltose to make glucose. Because maltose is an inexpensive and readily available sugar, it has great potential to produce trehalose from maltose by utilizing the one-step isomerization reaction of TreS. A number of methods have been currently available to measure TreS activity, including HPLC and trehalase-coupled TreS assay. However, these methods are either time-consuming, expensive or with low substrate specificitied, and this makes them not suitable for characterizing TreS in a high-throughput manner. This proposal established a novel 96-well format assay, maltase-coupled TreS assay to colorimetrically determine the isomerization activity of TreS. We also establish a 96-well format protein purified system for high-throughput TreS purification. With these new colorimetric method and purification method, we can perform the biochemical characterization more efficiently, especially, in the directed enzyme evolution of TreS for screening mutant TreS with improved properties, such as high trehalose yield or high thermostability.

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