臺灣博碩士論文加值系統

澱粉分支酶 (starch branching enzyme; SBE, EC 2.4.1.18) 是澱粉生合成路徑中的酵素之一，在合成支鏈澱粉 (amylopectin) 分子上扮演重要角色。本實驗室先前已由成長中之台南五號綠豆 (Vigna radiata cv. Tainan no. 5) 選殖出了 SBE 異構型 VrsbeI 與 VrsbeII’ cDNA。此研究分為兩部份: 重組蛋白 rVrSBEI 的表現分析與嵌合基因設計。首先，以 pET-28a-VrsbeI 質體 DNA 轉形至 BL21 (DE3) 宿主中，嘗試尋找出可溶性重組蛋白質 rVrSBEI 之最適誘導條件。於 37℃ 震盪培養至 OD600 到 0.6 後，以 0.4 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) 誘導 15 小時後發現一條約 117 kDa 橫紋較顯著出現，可能為 rVrSBEI，但經由 SDS-PAGE 分析與質譜儀確認其蛋白質身份卻為 LacZ protein。嵌合基因是以 VrsbeI 與 VrsbeII’cDNA 序列之功能區間 (functional domain) 的特色來設計，以專一性引子經 PCR 量化並定序出 VrsbeI 的 1643 bp (VrsbeI 5’-1643) 片段及 VrsbeII 的 360 bp (VrsbeII 3’-360) cDNA 片段，未來可將兩片段接合成為嵌合型 sbe cDNA (cVrsbeI-II) 並作表現與活性分析，期待嵌合型酵素能應用於澱粉分子的修飾。

Starch branching enzyme (SBE, EC 2.4.1.18) is one of the enzymes vital for amylopectin synthesis. The sequences of the previously obtained full-length cDNA of mungbean (Vigna radiata, cv. Tainan no. 5) sbeI (VrsbeI) and sbeII (VrsbeII’) were cloned. This study contains two parts: recombinant protein rVrSBEI expression analysis and chimeric gene design. First, pET-28a-VrsbeI plasmid DNA was transformed into BL21 (DE3) host cell and to find the optimal induction condition. The conditions for rVrSBEI induction were evaluated when the cells were grown at 37℃ until OD600 to 0.6 and then induced with 0.4 mM IPTG for 15 hrs; it showed seemingly a 117 kDa protein was induced. This recombinant protein was separated in and isolated from sodium dodecyl sulfate (SDS)-
polyacrylamide gels for mass spectrometric identification. The induced protein band, however, is identified to be LacZ protein. The sequence feactures of the functional domain of VrsbeI and VrsbeII’ were used to design chimeric genes, then using specific primers that amplified two cDNA fragments: VrsbeI 1643 bp (VrsbeI 5’-1643) and VrsbeII 360 bp (VrsbeII 3’-360). In the future, we can ligate the two fragments into a chimeric sbe cDNA (cVrsbeI-II). Next, we can transform, express and analyze the chimeric enzyme activity to further use the enzyme in the modification of starch molecules.

總 目 錄
總目錄-----------------------------------------------------I
圖目錄---------------------------------------------------III
表目錄-----------------------------------------------------V
中文摘要----------------------------------------------------1
英文摘要----------------------------------------------------2
第一章 前言-----------------------------------------------3
第二章 文獻回顧--------------------------------------------9
第一節 澱粉簡介-----------------------------------------9
2.1.1 澱粉的組成與顆粒結構----------------------------9
2.1.2 參與澱粉生合成路徑之酵素------------------------10
2.1.3 澱粉生合成路徑--------------------------------12
第二節 澱粉分支酶之相關研究-----------------------------13
2.2.1 澱粉分支酶及其異構型之分類----------------------13
2.2.2 胺基酸序列和結構探討--------------------------14
2.2.3 澱粉分支酶的 N 端與 C 端功能分析----------------16
2.2.4 澱粉結構對功能性的影響-------------------------------17
2.2.5 修飾後的澱粉對於食品工業上的影響---------------------18
第三節 綠豆簡介--------------------------------------------19
第三章 材料與方法------------------------------------------27
第四章 結果-----------------------------------------------40
第五章 討論-----------------------------------------------45
第六章 結論-----------------------------------------------49
第七章 參考文獻--------------------------------------------79
附錄------------------------------------------------------85

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