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) 是澱粉生合成路徑的酵素之ㄧ，在支鏈澱粉 (amylopectin) 的合成上扮演重要的角色。本論文目的是將台南五號綠豆的 (Vigna radiata cv. Tainan no.5) VrsbeII 次選殖到表現載體，並於大腸桿菌系統表現出具有活性的 rVrSBEⅡ 重組蛋白質。首先將 VrsbeII 構築於 pET-30 EK/LIC 表現載體中，並轉形到 NovaBlue 宿主細胞，希望獲得只含 ORF 之序列，但發現全長只接入約 350 bp 片段，經過慎察斷裂位置，發現存在相同 6 bp (CCAGTT) direct repeat的跳動子突變位置，因此避開此序列重新設計具有 Bam HI 和 Not I 切位的引子，PCR 量化出在C端短少24個核苷酸的目標片段，載入兩端經限制酶處理的pET21b 載體中，並轉型至大腸桿菌 BL21 表現系統，成功的表現出以可溶性形式存在於細胞質的重組蛋白。 rVrSBEⅡ在宿主的最佳誘導條件，為 37℃震盪培養至 OD600 到 0.6 後，以 0.2 mM IPTG 誘導 5 小時可獲得最佳 rVrSBEⅡ表現量，粗萃物比活性為 0.252 U/mg。利用具親和性之 Ni-NTA (Ni-Nitrilotriacetic acid) 管柱純化，以 SDS-PAGE 評估 rVrSBEⅡ 分子量為 108-kDa。並進一步利用西方點墨法來確認，結果顯示粗萃物與部份純化後的rVrSBEⅡ，皆可被專一性 Anti-6xHis tag mouse monoclonal 所雜合，可確定所誘導出的重組蛋白質。經由具親和性之 Ni-NTA resin 管柱純化，所得之純化重組蛋白質比活性提高至 6.402 U/mg，其純化倍數已達 25.4 倍。本探討已將VrsbeⅡ 基因放入大腸桿菌系統表現出具有生物活性之重組蛋白質，預期未來可進一步以基因工程改善其酵素性質利用於食品科學應用。

Starch branching enzyme (SBE, EC 2.4.1.18) is a vital enzyme for amylopectin synthesis in the starch biosynthetic pathway. The aim of this thesis was to subclone VrsbeII of mungbean (Vigna radiata L. cv Tainan no. 5) into expression vector and produce active enzymes in the E. coli system. First, VrsbeII was constructed into pET-30 EK/LIC vector and transformed into E. coli NovaBlue host cells, hopefully full length sequence of the open reading frame could be obtained. However, there was only approx. 350 bp fragment could be stably maintained in pET-30 EK/LIC system and the unexpected outcome reoccurred. The sequence in the 350 bp was carefully examined and found astonishingly that two transposon-like 6-bp (CCAGTT) direct repeat sequences were in VrsbeII. Therefore, primers designed with Bam HІ and Not І sites which skipped one direct repeat sequence were used to prepare a 24 nucleotide shortened VrsbeII at the 3’-end by PCR. The redesigned insert fragment was ligated into the parallel sites on pET21b vector, followed by transforming into E. coli BL21 (DE3) cells, and expressed successfully as a soluble protein in the cytosol. The optimal expression condition for rVrSBEⅡwas evaluated that the cells were grown at 37℃ until OD600 to 0.6, then induced with 0.2 mM IPTG for 5 hrs, and the maximal crude enzyme activity of 0.25 U/mg was obtained. The crude enzyme was purified by HisTrapTM affinity column and the molecular size of rVrSBEⅡwas 108-kDa whose activity has enriched 25.5-fold (6.402 U/mg). The 108-kDa rVrSBEⅡwas also detected by anti-6x His-tag mouse monoclonal antibodies in Western blot. In summary, VrsbeⅡ has been expressed into a biologically functional protein in E. coli system and expected to further improve its enzymatic properties by genetic engineering for application in food use.

圖 目 錄 IV
表 目 錄 IV
中文摘要 VII
英文摘要 VIII
第一章 前 言 1
第二章 文獻回顧 4
一、澱粉之簡介 4
1.1 澱粉的組成及顆粒結構 4
1.2 澱粉之生合成酵素 4
二、澱粉分支酶之研究 6
2.1 作用模式與特性 6
2.2 SBE活性分析之方法 7
2.3 基因結構之探討 8
2.4 異構酶作用之差異對澱粉結構的影響 11
2.5 澱粉結構對功能性的影響 12
第三章 材料與方法 24
3.1 實驗材料與藥品 24
3.2 綠豆之sbe II基因的製備與純化 27
3.3 建構綠豆之sbeII基因之表現載體 30
3.4 選殖載體轉形 (transformation) 進入勝任細胞 33
3.5 菌落 PCR 法 33
3.6小量質體DNA抽取 34
3.7 表現質體以限制酶切割確認 35
3.8定序與分析 35
3.9 菌株保存與活化 36
3.10 蛋白質之表現 36
3.11 蛋白質之抽取 37
3.12 重組蛋白質之純化 37
3.13 蛋白質濃度測定 39
3.14 酵素分子量之測定 39
3.15 西方點墨法 (Western blot method) 42
3.16 直鏈澱粉分支試驗 (Amylose-branching assay) 44
3.17 序列分析軟體與檢索之資料庫 45
第四章 結果與討論 51
4.1 綠豆中sbe ІІ基因表現載體之建構 51
4.2 重組rVrSBE ІІ 蛋白質之表現 52
4.3 重組rVrSBE ІІ 蛋白質之純化 54
4.4 重組rVrSBE ІІ之酵素活性分析 55
4.5 VrSBEII蛋白質之結構模擬 57
第五章 結論 76
第六章 參考文獻 77

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