臺灣博碩士論文加值系統

中文摘要

Pseudomonas vesicularis MA103 為一株具有分解洋菜能力的海洋細菌。將此菌於 MMB-MAEF108 發酵槽中26oC、150 rpm、48 hr、pH 6.2、通氣量 4.0 L/min培養，收集離心去除菌體後之培養液，進行洋菜酶的分離與純化。
以 DE-52 離子交換樹脂層析，可分離出三個具分解洋菜能力的蛋白質波峰劃分區域，個別收集後命名為 PV-1、PV-2、及 PV-3，其比活性分別為 5008.73 U/mg、5801.76 U/mg、及 6604 U/mg，純化倍數分別為 18.89 倍、20.69 倍、及 23.85 倍。PV-1 及 PV-2 再經 Bio-Gel P-100 膠體過濾層析其純化後，經 12% SDS-PAGE 電泳後得知酵素分子質量分別為 62.9 kDa 及 86.4 kDa
Agarase PV-1 的最適作用 pH 值為 6.0，最適作用溫度為 40oC，但於 50oC 下反應 15 min 相對活性降至 20.00%。Agarase PV-2 的最適作用 pH 值為 7.0，最適作用溫度為 40oC 或 45oC，於50oC 下反應 15 min 相對活性降至 58.86%。
Agarases PV-1 與 PV-2 以 HIMEDIATM agar 為基質時有最佳之基質特異性活性，相對 agarose 為基質分解活性分別為 1.43 及 1.24 倍。Agarase PV-2 無法分解馬尾藻 (褐藻) 多醣。
Agarases PV-1 或 PV-2 個別水解中性洋菜 (agarose) 之水解產物經 HPLC 分析，所得產物各為一介於新洋菜二糖與新洋菜六糖間之寡醣類。利用 agarases PV-1 或 PV-2 個別與新洋菜六糖 (neoagarohexaose) 混合反應，經 HPLC 分析，所得產物各為一大於六糖之寡醣類，故推測 agarases PV-1 與 PV-2 為兼具水解及合成反應活性之酵素。

Abstract

Pseudomonas vesicularis MA103 is a marine bacterium, which has the ability to degrade agar. Strain MA103 was grown at 26oC for 48 hr in MMB-MA103 broth with 150 rpm, pH 6.2, air flow rate 4.0 L/min in fermenter. After removing the cells, collected crude enzymes proceed in the separation and purification of the agarases.
The chromatograph of the wide enzymes with DE-52 gel achieved the separation of three agarases, they are named PV-1, PV-2, and PV-3 by their elution order in NaCl gradient. The following properties of these PV-1, PV-2, and PV-3 agarases were: specific activity, 5008.73, 5801.76, and 6604 U/mg; purification fold, 18.89, 20.96, and 23.85 fold; respectively. Agarases PV-1 and PV-2 were purified further by Bio-Gel P-100 chromatography. The estimated molecular mass of agarases PV-1 and PV-2 were 62.9 kDa and 86.4 kDa, respectively.
The optimum pH for agarase PV-1 was found to be 6.0. The optimum temperature for PV-1 was 40oC, but 80% of the enzyme activity was inactivated in 15 minutes under 50oC. The optimum pH for agarase PV-2 was found to be 7.0. The optimum temperature for PV-2 was 40oC or 45oC, but 58.86% of the enzyme activity was inactivated in 15 minutes under 50oC.
The best substrate specific activities of agarases PV-1 and PV-2 toward HIMEDIATM agar were 1.42 and 1.24 folds digestion activity compare to agarose, respectively. Agarase PV-2 did not have the capability to degrade Sargassum sp. polysaccharide.
Hydrolytic products of agarose digested by either agarases PV-1 or PV-2 were analyzed by HPLC, and the results showed that they could be oligosaccharides, which have molecular size between neoagarohexaose and neoagarobiose. Products of neoagarohexaose reacted with agarases PV-1 and PV-2 were analyzed by HPLC. The products examined could be oligosaccharides, whose molecular size was larger than neoagarohexaose. Based on these results, agarases PV-1 and PV-2 are enzymes that could have both hydrolytic and polymeric activitires.

目　　錄

目錄 i
圖目錄 v
表目錄 viii
附錄目錄 ix
中文摘要 x
英文摘要 (Abstract) xi
壹、前言 1
貳、文獻整理 3
一、洋菜的構造與特性 3
二、洋菜酶生產菌株及其分離來源 3
三、洋菜酶對洋菜水解的作用機制 5
四、洋菜酶之生化特性 6
4-1 溫度和 pH 值 6
4-2 分子量 7
4-3 金屬離子 8
五、海藻寡醣組成成分鑑定 9
5-1 薄層層析法 (Thin-layer chromatography, TLC) 9
5-2 高效能液相層析法 (High-performance liquid chromatography, HPLC) 10
5-3 磁核共振光譜法 (Nuclear magnetic resonance, NMR) 10
六、洋菜酶的應用 11
6-1 細胞的分離 11
6-2 DNA的分離 12
6-3 藻類原生質體的製作 12
6-4 海藻多醣的生產與黏度降低 13
6-5 藻類組成的分析 13
6-6 融合瘤細胞的分離 14
七、藻類多醣之洋菜酶水解物 14
7-1 生理活性 14
7-2 應用 15
參、實驗設計 18
肆、實驗材料與方法 19
一、實驗材料 19
1-1 實驗菌株 19
1-2 試驗藥品 19
　　　　　1-2-1 藥品 19
　　　　　1-2-2 電泳標準品 21
　　　　　1-2-3 培養基組成 22
　　　　　1-2-4 洋菜酶反應基質 22
　　　　　1-2-5 DNS 溶液 23
　　　　　1-2-6 膠過濾緩衝液 23
　　　　　1-2-7 SDS-PAGE 23
　　　　　1-2-8 電泳溶液 24
　　　　1-3 儀器設備 24
二、實驗方法 26
　　　　2-1 洋菜酶之生產及濃縮 26
　　　　　2-1-1 菌株保存 26
　　　　　2-1-2 菌株活化 26
　　　　　2-1-3 洋菜酶之生產 26
　　　　　2-1-4 洋菜酶之濃縮 27
　　　　2-2 洋菜酶的活性測定 27
　　　　2-3 蛋白質的定量 28
　　　　2-4 洋菜酶的純化分離 28
　　　　　2-4-1 DE-52 離子交換樹脂層析 28
　　　　　2-4-2 Bio-Gel P-100 膠體過濾層析 29
　　　　　2-4-3 純化酵素之命名 29
　　　　2-5 洋菜酶之生化性質測試 29
　　　　　2-5-1 分子量的鑑定 29
　　　　　2-5-2 基質特異性 30
　　　　　2-5-3 最適作用 pH 值 30
　　　　　2-5-4 pH 值的安定性 30
　　　　　2-5-5 最適作用溫度 30
　　　　　2-5-6 熱安定性 31
　　　　　2-5-7 金屬離子的影響 31
　　　　　2-5-8 儲藏安定性 31
　　　　2-6 洋菜酶之分解產物的探討 31
　　　　　2-6-1 HPLC 鑑定 31
　　　　2-7 洋菜酶定序 32
　　　　　2-7-1 內部可能胺基酸序列質譜儀分析 32
　　　　2-8 統計與分析 32
伍、結果與討論 33
　　一、洋菜酶的純化分離 33
　　二、洋菜酶的生化性質 35
　　　　2-1 最適作用溫度與溫度安定性 35
　　　　2-2 最適作用 pH 值與 pH 值安定性 38
　　　　2-3 金屬離子之影響 40
　　　　2-4 儲藏安定性 41
　　　　2-5 基質特異性 43
　　　　2-6 水解產物分析 45
　　　　　2-6-1 MA103 agarases 粗酵素液之水解產物分析 45
　　　　　2-6-2 Agarase PV-1 之水解產物分析 46
　　　　　2-6-3 Agarase PV-2 之水解產物分析 47
陸、結論 50
柒、參考文獻 52
捌、附錄 105


圖目錄

圖一、 MA103 garases 粗酵素DE-52層析圖 64
圖二、 P. vesicularis MA103 所產洋菜酶 DE-52 部份純化 之12% SDS-PAGE 圖 65
圖三、 Agarase PV-1 之 Bio-Gel P-100 膠體過濾層析圖 66
圖四、 P. vesicularis MA103所產agarase PV-1 12% SDS- PAGE 圖 67
圖五、 Agarase PV-2 之 Bio-Gel P-100 膠體過濾層析圖 68
圖六、 P. vesicularis MA103所產agarase PV-2 12% SDS- PAGE圖 69
圖七、 MA103 agarases 粗酵素液的最適反應溫度 70
圖八、 Agarase PV-1 的最適反應溫度 71
圖九、 Agarase PV-2 的最適反應溫度 72
圖十、 MA103 agarases 粗酵素液之溫度安定性 73
圖十一、 Agarase PV-1之溫度安定性 74
圖十二、 Agarase PV-2之溫度安定性 75
圖十三、 MA103 agarases 粗酵素液於不同緩衝液中的最適 作用 pH 值 76
圖十四、 Agarase PV-1 於不同緩衝液中的最適作用 pH 值 77
圖十五、 Agrase PV-2 於不同緩衝液中的最適作用 pH 值 78
圖十六、 pH 值對 MA103 agrases 粗酵素液之安定性 79
圖十七、 pH 值對 agarase PV-1 之安定性 80
圖十八、 pH 值對 agarase PV-2 之安定性 81
圖十九、 金屬離子對 MA103 agarases 粗酵素液活性之影響 82
圖二十、 金屬離子對 agarase PV-1 活性之影響 83
圖二十一、金屬離子對 agarase PV-2 活性之影響 84
圖二十二、 MA103 agarases粗酵素液對十二種藻類多醣之 基質特異性 85
圖二十三、 Agarase PV-1 對十二種藻類多醣之基質特異性 86
圖二十四、 Agarase PV-2 對十二種藻類多醣之基質特異性 87
圖二十五、 半乳糖、新洋菜二糖、及新洋菜六糖標準品之 高效能液相層析圖 88
圖二十六、 MA103 agarases 粗酵素液與 0.1% agarose 反應 1-4 hr 之高效能液相層析圖 89
圖二十七、 MA103 agarases粗酵素液與0.1% neoagarohexaose
反應 1-4 hr 之高效能液相層析圖 90
圖二十八、 MA103 agarases 粗酵素液與 0.1% neoagarobiose
反應 1-4 hr 之高效能液相層析圖 91
圖二十九、 MA103 agarases 粗酵素液與 0.1% galactose 反應 1-4 hr 之高效能液相層析圖 92
圖三十、 Agarase PV-1 與 0.1% agarose 反應 1-4 hr之高 效能液 相層析圖 93
圖三十一、 Agarase PV-1 與 0.1% neoagarohexaose 反應 1-4 hr之高效能液相層析圖 94
圖三十二、 Agarase PV-1 與 0.1% neoagarobiose 反應 1-4 hr 之高效能液相層析圖 95
圖三十三、 Agarase PV-1 與 0.1% galactose 反應 1-4 hr 之高 效能 液相層析圖 96
圖三十四、 Agarase PV-2 與 0.1% agarose 反應 1-4 hr 之高 效能液相層析圖 97
圖三十五、 Agarase PV-2 與 0.1% neoagarohexaose 反應 1-4 hr 之高效能液相層析圖 98
圖三十六、 Agarase PV-2 與 0.1% neoagarobiose 反應 1-4 hr 之高效能液相層析圖 99
圖三十七、 Agarase PV-2 與 0.1% galactose 反應 1-4 hr 之 高效能液相層析圖 100


表目錄

表一、 Pseudomonas vesicularis MA103 洋菜酶純化表 101
表二、 MA103 agarases 粗酵素液儲藏安定性 102
表三、 Agarase PV-1 儲藏安定性 103
表四、 Agarase PV-2 儲藏安定性 104

附錄目錄

附錄一、 Agarose 的基本構造及洋菜酶作用位置 105
附錄二、 Agaropectin可能組成基本單位構造 105
附錄三、 Agarases 的生產微生物、種類及分離來源 106
附錄四、 微生物所產洋菜酶分解洋菜產物 108
附錄五、 洋菜酶的生化特性 110
附錄六、 Agarase PV-2 胺基酸序列質譜儀比對報告一 112
附錄七、 Agarase PV-2 胺基酸序列質譜儀比對報告二 113
附錄八、 Agarase PV-2 胺基酸序列質譜儀比對報告三 114

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