竹纖維素合成酶在酵母菌之異源表現、純化及功能分析

Heterologous expression, purification and functional analysis of bamboo cellulose synthases from yeast

黃暄友(Hsuan-Yu Huang)

指導教授 ： 鄭貽生

國立臺灣大學/生命科學院/植物科學研究所/博士(2019年)

https://doi.org/10.6342/NTU201903344

摘要

纖維素是世界上最大量的生質物，年生成量約1兆5千億噸。植物纖維素在初級細胞壁含量約占30%，次級細胞壁則約為55%。纖維素由纖維素合成酶複合體 (Cellulose synthase complex, CSC) 合成，CSC係由數個纖維素合成酶 (Cellulose synthases, CesAs) 於細胞膜上組成，纖維素合成酶複合體的組成結構及其纖維素合成的機制仍不清楚，原因在於纖維素合成酶屬膜蛋白質，第一步就是要取得大量表達及純化的蛋白質。本研究以異源表現的方式純化六種綠竹 (Bambusa oldhamii) 纖維素合成酶 (BoCesA1, 2, 3, 4, 5, 7) ，並分析其酵素活性。分別將Maltose-binding protein (MBP) 序列以及BoCesA序列構築在pYES2/CT表現載體上，並透過酵母菌細胞 (BCY123) 在醱酵槽內大量培養及誘導蛋白質表現。分別使用固定化金屬離子親和層析 (Immobilized metal affinity chromatography) 純化MBP-BoCesAs以及粒徑排阻層析 (Size-exclusion chromatography) 分離多聚體BoCesAs，自120 g酵母菌可純化約7.2 mg的BoCesA蛋白質。在電子顯微鏡的觀察下，可以觀察到多聚體BoCesAs加入基質UDP-Glucose後所生產的纖維素產物，並使用泛甲基醣醇乙酸 (Partially methylated alditol acetate) 衍生化配合氣相層析質譜儀 (Gas chromatography-mass spectrometry) 鑑定纖維素產物的鍵結為β-1,4-glucan。本研究建立了一套大量表現以及純化纖維素合成酶的流程，並確定所純化的蛋白質具有酵素活性，此酵素將可用於纖維素生合成相關研究以及蛋白質結構與功能分析。

關鍵字

纖維素合成酶 ； 醣基轉移酶 ； 膜蛋白純化 ； 纖維素微纖維 ； 纖維素合成酶複合體

並列摘要

Cellulose is the most abundant biomass in the world with an estimated 1500 billion tons produced by plants per year. About 30% of primary cell wall and 55% of secondary cell wall consist of cellulose in plant cells. Cellulose is synthesized by the cellulose synthase complex (CSC) which contains several cellulose synthases (CesAs) in the plasma membrane. The molecular structure of CSC and the mechanism of cellulose synthesis by CesAs in plant cells remain unclear. Since CesAs are membrane proteins, the first step is to overexpress and purify a large quantity of CesAs protein. In this study, the overexpression and purification procedure for 6 genes of cellulose synthase (BoCesA1, 2, 3, 4, 5, 7) from Bambusa oldhamii were established. The Maltose-binding protein (MBP) -tagged BoCesAs were cloned into pYES2/CT and transformed into BCY123 yeast cells. A fermentor is used to incubate the BCY123 yeast cells in a large quantity. After breaking cells, the recombinant BoCesA proteins were purified by immobilized metal affinity chromatography and size-exclusion chromatography. This method resulted in 7.2mg total proteins from 120 g yeast cells. In the enzyme activity assay of BoCesAs, the long fiber-like products could be observed by a transmission electron microscope and confirmed as the product β-1,4-glucan by partially methylated alditol acetate-coupled gas chromatography-mass spectrometry analysis. Therefore, a procedure by a heterologous expression, purification strategy, and enzyme activity analysis for BoCesAs was established for further studies.

並列關鍵字

CesA protein ； Glycosyltransferase ； Membrane protein purification ； Cellulose microfibril ； Cellulose synthase complex

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