臺灣博碩士論文加值系統

九孔(Small abalone, Sulculus diversicolor aquatilis)內臟中的二種澱粉酶(Ⅱ-1,Ⅱ-2)已經被純化出來。澱粉酶之純化係利用含5 mM 氯化鈣的10 mM tris-maleate (pH 7.0) 緩衝溶液抽取後，再經硫酸銨分劃及sepharose CL-6B、CM sepharose CL-6B 和sephacryl S-100等一系列管柱層析，純化倍率前者為2,436倍，後者為1,395倍，回收率則分別為5.7 ﹪及2.5 ﹪，經SDS-PAGE膠體電泳分析，證實此兩種澱粉酶已經純化，分子量分別是55.7 KDa及65 KDa。最適溫度分別為45oC及50oC；最適pH兩者都是pH6.0附近；在pH穩定度方面，前者在pH 5.0-8.0、而後者在pH 6.0-8.0時最穩定。鋇、鎂、鈣、鉀、鈉、鎳、銀、銨等離子可將澱粉酶peakⅡ-1活性提高，而僅有鋇、鎂、錳等三種二價金屬離子可將澱粉酶Ⅱ-2之活性提高。EDTA對澱粉酶Ⅱ-1及Ⅱ-2的影響較其他抑制劑大，顯示著純化出來的兩種澱粉酶皆為鈣依存性酵素；IAA、PMSF、NEM對澱粉酶Ⅱ-1及Ⅱ-2皆有明顯的抑制作用，由此可推論在其活性部位 (active site)可能含有硫氫基 (-SH group)。這兩種澱粉酶可以水解直鏈澱粉(amylose)、洋菜膠(agarose)、pullulan、肝糖(glycogen)、支鏈澱粉(amylopectin)、龍鬚菜(Gracilaria tenuistiplitata)、cellulose、agar-agar、及alginin，顯示此兩種澱粉酶皆具有水解α-1,4及α-1,6糖苷鍵能力，但又不相似於市售之α-、β-、γ-amylase及agarase，因此，推測此兩種澱粉酶Ⅱ-1、Ⅱ-2應該為新澱粉酶(neo-amylase)。

Amylases Ⅱ-1 and Ⅱ-2 were purified from small abalone (Sulculus diversicolor aquatilis) in this study. Both of the two enzymes were extracted with 10mM Tris-maleate buffer (pH 7.0) containing 5mM CaCl2 and further fractionated by ammonium sulfate. The crude enzyme was then chromatographied with sepharose CL-6B, CM-sepharose CL-6B and sephacryl S-100 columns. By these steps, the purifications of the two enzymes increased to 2436 and 1395 fold and the recovery of the two enzymes activity were 5.7% and 2.5%, respectivily. According to SDS -polyacrylamide gel electrophoresis, the purified two amylases appeared a single protein band with molecular weight 55.7 kDa and 65 kDa, respectivily. The optimal temperature were 45℃ and 50℃, respectively. The optimal pH of peakⅡ-1 and peakⅡ-2 for digesting the soluble starch, under the optimal temperature, were found both to be 6.0. In pH stability, Ⅱ-1 and Ⅱ-2 were more stable at pH5.0-8.0 and pH6.0-8.0, respectively. AmylaseⅡ-1 was activated by Ba2+,Mn2+,Ca2+, K+,Na+,Ni+,Ag+ and NH4 ions, and Ⅱ-2 was activated by Ba2+,Mn2+,Mn2+, but completely inhibited by Hg2+, Zn2+, Cu2+ and Fe3+ ions. The inhibitors, EDTA was found to have the most efficient than others to inhibite the two enzymes. And that shows both two enzymes were Ca-dependent enzymes. However, IAA、PMSF、NEM were also found to have partially inhibited ability on both the two enzymes. Then we could suggest that the two enzymes might to have —SH group at their active site. From the results of digesting amylose、agarose、pullulan、glycogen、amylopectin、Gracilaria tenuistiplitata、cellulose、agar-agar、and alginin. . The two enzymes have hygrolizing both α-1,4 and α-1,6 glucosidic bond, so they could be neo-amylase.

目錄
頁次
中文摘要…………………………………………………………………1
英文摘要…………………………………………………………………2
壹、實驗目的………………………………………………………..3
貳、文獻整理………………………………………………………..5
一、澱粉酶…………………………………………………………..5
二、α-澱粉酶………………………………………………………...7
1.分子量……………………………………………………….8
2.各種離子對α-澱粉酶之影響……………………………….9
Ⅰ.陽離子………………………………………………….9
Ⅱ.陰離子………………………………………………..10
3.物理化學性質……………………………………………...10
4.作用方式…………………………………………………...10
5.構造………………………………………………………...11
6.新奇的α-澱粉酶…………………………………………...12
7.異構酶……………………………………………………...13
三、β-澱粉酶……………………………………………………...16
1.分子量……………………………………………………...16
2.一般性質…………………………………………………...16
3.物理化學性質……………………………………………...17
4.作用方式…………………………………………………...17
四、γ-澱粉酶……………………………………………………….19
1.分子量及組成份…………………………………………...19
2.物理化學性質……………………………………………...20
五、澱粉酶在工業上的應用………………………………………23
參、材料與方法……………………………………………………27
一、澱粉酶的純化…………………………………………………27
1.實驗材料………………………………………………………...27
2儀器……………………………………………………………...28
3.緩衝溶液………………………………………………………...28
4.藥品……………………………………………………………...28
5.澱粉酶的活性測定方法………………………………………...28
6.蛋白質定量……………………………………………………...29
7.純化方法………………………...…...…….……………………29
(1)粗酵素溶液…………………………………………………...29
(2)硫酸銨分劃…………………………………………………...29
(3)Sepharose CL-6B 膠體過濾層析法…………………………30
(4)CM-sepharose CL-6B 陽離子交換層析法………………….30
(5)Sephacryl S-100 膠體過濾層析法…………………………..30
8.電泳分析法……………………………………………………...30
9.蛋白質分子量的測定…………………………………………...31
二、生化特性………………………………………………………31
1.酸鹼度對澱粉酶的影響….…………………..………………...31
2.溫度對澱粉酶的影響…………………………………………...31
3.基質特異性………..…………………………….……………...31
4.反應生成物探討………………………………………….……..32
5.金屬離子的影響………………………………………………...32
6.抑制劑的影響…………………………………………………...32
肆、結果與討論………………………………………………………..33
一、澱粉酶的純化………………………………………………….33
1. 硫酸銨分劃…………………………………………………….33
2. Sepharose CL-6B 膠體過濾層析法……………………………33
3. CM-sepharose CL-6B 陽離子交換層析法…………………….34
4. Sephacryl S-100 膠體過濾層析法……………………………..35
5. 電泳分析鑑定及分子量估算………………………………….35
二、澱粉酶的生化特性…………………………………………….35
1.溫度對澱粉酶的影響…………………………………………36
2.酸鹼度對澱粉酶的影響………………………………………36
3.基質特異性……………………………………………………37
4.反應生成物探討………………………………………………38
5.金屬離子的影響………………………………………………39
6.抑制劑的影響………………………………………………....40
伍、結論及其應用……………………………………………………..41
陸、參考文獻…………………………………………………………..61
圖表……………………………………………………………………..46
圖一、九孔內臟澱粉酶的Sepharose CL-6B 膠體過濾層析…………46
圖二、九孔內臟澱粉酶的CM-Sepharose CL-6B陽離子交換層析….47
圖三、九孔內臟澱粉酶澱粉的Sephacryl S-100膠體過濾層析……..48
圖四、 SDS-PAGE估算之九孔內臟澱粉酶分子量………………….49
圖五、 溫度對九孔內臟澱粉酶 (Ⅱ-1)活性的影響………………….50
圖六、 溫度對九孔內臟澱粉酶 (Ⅱ-2)活性的影響….……………….51圖七、九孔內臟澱粉酶 (Ⅱ-1)的溫度安定性…..……………………..52
圖八、九孔內臟澱粉酶 (Ⅱ-2 )的溫度安定性………………………..53
圖九、 pH對九孔內臟澱粉酶(Ⅱ-1)的影響………………………….54
圖十、pH對九孔內臟澱粉酶(Ⅱ-2)的影響…………………………...55
圖十一、九孔內臟澱粉酶 (Ⅱ-1)的pH安定性………………………56
圖十二、九孔內臟澱粉酶 (Ⅱ-2)的pH安定性………………………57
圖十三、九孔內臟澱粉酶水解直鏈澱粉反應生成物之薄層分析…...58
圖十四、九孔內臟澱粉酶水解支鏈澱粉反應生成物之薄層分析…...59圖十五、九孔內臟澱粉酶水解肝糖反應生成物之薄層分析…………60圖十六、九孔內臟澱粉酶水解龍鬚菜反應生成物之薄層分析………61
圖十七、九孔內臟澱粉酶水解agar-Agar反應生成物之薄層分析….62
表一、九孔內臟澱粉酶的硫銨分劃………………………………..…63
表二、 九孔內臟澱粉酶的純化表……………………………………64
表三、九孔內臟澱粉酶對基質的特異性……………………………..65表四、 各種金屬離子對九孔內臟澱粉酶的影響……………………66
表五、各種抑制劑對九孔內臟澱粉酶的影響………………………..67

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