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研究生:蕭正忠
研究生(外文):Cheng Chung Hsiao
論文名稱:水稻懸浮細胞不溶性鹼性轉化脢之純化與生化性質研究
論文名稱(外文):Purification and Characterization of Insoluble Alkaline Invertase from Rice Suspension Cells
指導教授:宋賢一
指導教授(外文):Hsien Yi Sung
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:農業化學研究所
學門:農業科學學門
學類:農業化學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:中文
論文頁數:106
中文關鍵詞:不溶性轉化脢水稻懸浮細胞
外文關鍵詞:Insoluble InvertaseRice Suspension Cell
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以台農 67 號水稻懸浮細胞為材料,經 PB-7.0 粗抽後所得的細胞碎片,以 1.0 M NaCl 溶離後,溶離液經硫酸銨分劃、Con A-Sepharose、Sepharose CL-6B及 DEAE-Sepharose 管柱層析可得鹼性轉化脢 ITa,而本實驗結果,乃首次發現植物不溶性鹼性轉化脢。經 1.0 M NaCl 溶離後的沈澱物再以 5% EDTA 進行溶離,純化步驟同上,可得到另一種鹼性轉化脢 ITb。以 Superose 6 管柱層析 (FPLC) 測定兩種蛋白質的原態分子量皆為 250 kD,以 SDS-PAGE 分析次單元分子量皆為 60 kD,判定為四元體。
在生化性質分析上,兩種轉化脢的特性極為相似,可能是同一種蛋白質。以 ITa 進行分析,最適溫度在 40-50℃ 之間,最適作用 pH 值為 7.6,pI 值為 4.7,能夠水解蔗糖與棉仔糖,但不能對麥芽糖作用,故為 β-fructosidase。以蔗糖為基質時,ITa 和 ITb 的 Km 值分別為 15.28 mM 及 14.02 mM。此酵素不被 Con A 所吸附;產物葡萄糖和果糖對其活性有抑制作用,外加 BSA 會有活化的效果,而 Tris 會對其活性有抑制,影響硫氫基的試劑 DTT 會對其有活化作用,HgCl2 與 PCMB 則會有抑制作用。
A novel insoluble alkaline invertase was isolated from rice (Oryza sativa) suspension cells. The alkaline invertase (ITa) could be released by 1.0 M NaCl and purified further by ammonium sulfate fractionation, Con A-Sepharose affinity chromatography (non-retained), gel-filtration (Sepharose CL-6B) and DEAE-Sepharose chromatography. Another bound-form invertase (ITb) can be released by 5% EDTA from cell fragments after treated 1.0 M NaCl and purified further through the same steps. The molecular masses of ITa and ITb estimated by Superose 6 (FPLC) were 250 kD, and subunit of molecular mass determined by SDS-PAGE was 60 kD. It is apparently a homotetrameric enzyme. The characteristics of these two alkaline invertases are similar. The pH optimum of ITa was 7.6. The optimum temperature was 40℃ - 50℃. Isoelectric focusing analysis indicated that the pI value of ITa was 4.7. ITa could hydrolyze sucrose and raffinose, but not maltose. The Km values of ITa and ITb for sucrose as substrate were 15.28 mM and 14.02 mM, respectively. This insoluble alkaline invertase did not have affinity toward Con A. Glucose and fructose inhibited the enzyme activity. The additional protein such as BSA could activate ITa. The enzyme activity was inhibited by Tris and metal ions (Cu2+, Ag+). Some thiol reagents (HgCl2, PCMB) inhibited ITa, while DTT could activate its activity. It suggested that sulf hydryl-group(s) either in the active site or nearly active site of ITa, which did participate the sucrose hydrolysis.
縮寫表…………………………………………………………………………III
中文摘要………………………………………………………………………V
英文摘要………………………………………………………………………VI
第一章緒論 …………………………………………………………1
第一節蔗糖與植物蔗糖的代謝………………………………………1
第二節植物蔗糖之運輸途徑…………………………………………3
第三節蔗糖轉化脢的分類與生理功能………………………………6
第四節受蔗糖轉化脢催化的其他反應………………………………8
第五節蔗糖轉化脢在植物體中的分布………………………………8
第六節蔗糖轉化脢的調節機制………………………………………9
第七節蔗糖轉化脢分子生物學的研究………………………………11
第八節實驗緣起及目的………………………………………………14
第二章材料與方法…………………………………………………15
第一節材料與培養方法………………………………………………15
第二節酵素活性分析法………………………………………………17
第三節蛋白質定量法…………………………………………………21
第四節不溶性鹼性蔗糖轉化脢的純化方法…………………………22
第五節電泳檢定………………………………………………………26
第六節不溶性鹼性蔗糖轉化脢生化性質之檢定……………………33
第三章結果……………………………………………………………39
第一節 水稻懸浮細胞不溶性轉化脢於不同培養時期的活性變化…39
第二節 水稻懸浮細胞之各種轉化脢活性分布的探討………………39
第三節不溶性鹼性轉化脢的確定……………………………………40
第四節水稻懸浮細胞不溶性鹼性轉化脢的純化……………………41
第五節不可溶性鹼性轉化脢的生化性質分析………………………44
第六節不溶性鹼性轉化脢之調控機制………………………………46
第四章討論 …………………………………………………………48
第一節水稻懸浮細胞不溶性鹼性蔗糖轉化脢的首次發現…………48
第二節水稻蔗糖轉化脢之比較………………………………………49
第三節水稻懸浮細胞不溶性鹼性蔗糖轉化脢所在位置的推測……50
第四節各種植物鹼性蔗糖轉化脢之比較……………………………51
第五章未來展望………………………………………………………52
結果之圖與表…………………………………………………………………53
參考文獻………………………………………………………………………96
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