| Summary: | 博士 === 國立臺灣大學 === 植物科學研究所 === 107 === 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.
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