臺灣博碩士論文加值系統

德基水庫因過度優養化而發生由二角多甲藻（Peridinium bipes）----後文簡稱二甲藻形成之淡水紅潮，本研究以水混合海砂對所採集之二甲藻進行研磨萃取，發現所得之水層粗萃取物具有多樣生物活性。13種測試藻株中有7種對水層粗萃取物較具敏感性，其生長速率皆受到不同程度的抑制，本研究進一步以銅綠微囊藻（Microcystis aeruginosa）進行生理活性之抑制測試。發現在二甲藻水層粗萃取物處理下，可觀測到微囊藻產氧速率隨著劑量與測試時間增加而遞減，耗氧速率則反之。光合作用之第二光系統之螢光發散量（685 nm）亦與所添加之劑量成正相關，故推論可能藉阻斷電子傳遞鏈來抑制光合作用。二甲藻水層粗萃取物亦造成微囊藻無法維持正常細胞質之酸鹼度，同時發生大量鉀離子滲漏至胞外的現象。初步純化後之水層萃取物分樣，在10分鐘內甚至可造成胞內鉀離子之完全滲漏，此現象推論係細胞膜破裂之效應，而不只是膜上特定離子運輸酵素受到抑制。另以二甲藻水層粗萃取物對11種消化酶進行敏感度測試，其中以thrombin最為敏感，不過，酵素受抑制作用之機制則未知。進一步純化水層粗萃取物後發現，活性物質之分子量均小於3 kD，再進行陽離子交換層析分離後得到三個毒性分樣，藉由其210 nm與254 nm之吸收值與蛋白質含量測定等性質，推測活性物質可能為具有環狀構造之胺基酸之胜（peptides）。

Water bloom resulted from Peridinium bipes, a freshwater dinoflagellate, has commonly occurred in Dergie dam of the mid of Taiwan. Allelopathic activity was found in the water-soluble extracts of this alga. This study is aimed to study the toxic effects and to characterize the allelochemicals. Seven of thirteen strains of phytoplankton tested were susceptible to the water-soluble extracts of P. bipes. A further study done with Microcystis aeruginosa showed that the photosynthetic O2-evolution and electron transfer between photosystems I and II, but not the respiration, were inhibited in the presence of extracts. This agreed with the phenomenon of the enhancement in the emission of PSII fluorescence when the extract was added to M. aeruginosa cells.
The treatment with P. bipes extract has caused the concentration- and time-dependent reductions in uranine accumulation in M. aeruginosa cells, associated with a significant and rapid leakage of K+ from intra- to extra-cellular medium. This was considered a phenomenon of damage in membrane integrity of the treated cells. As a result, a change in the membrane permeability along with a loss in the pH gradient across the plasma membranes might occur.
Further study was done to test the inhibition of proteases in vitro by the presence of P. bipes extracts. Among 11 proteases tested, thrombin was the most sensitive one. When the extracts were further purified by centricon and centriprep, the fractions with toxic activity were found at those <3 kD. From them, three fractions were obtained when purified with ion chromatography. These fractions showed positive reaction with protein staining, suggesting that they were peptides.
Although P. bipes seems to be cable of producing various kinds of inhibitors, present study suggests that peptide is one of such compounds accounting for allelopathic effects. The primary effects of such allelochemicals seem to act on the plasma membranes of the target algal cells and to inhibit the proteases of the predators.

中文摘要………………………………………………………1
英文摘要………………………………………………………2
一、前言………………………………………………………3
二、材料與方法………………………………………………7
2.1二甲藻萃取物對藻種生長速率之影響………………7
2.1.1材料………………………………………………………7
2.1.2二甲藻粗萃取物的製備…………………………………7
2.1.3供試藻種…………………………………………………7
2.1.4培養條件…………………………………………………8
2.1.5測試條件…………………………………………………10
2.1.6葉綠素a濃度測定法……………………………………10
2.1.7生長速率之計算…………………………………………11
2.1.8半抑制濃度（EC50）之求取……………………………11
2.2二甲藻水層萃取物對消化酶之抑制測試…………………11
2.2.1測試方法…………………………………………………11
2.2.1.1 胰蛋白酶（trypsin）活性檢測……………………11
2.2.1.2 血纖維蛋白溶酶（plasmin）活性檢測……………12
2.2.1.3 木瓜蛋白酶（papain）活性檢測…………………12
2.2.1.4 彈性蛋白酶（elastase）活性檢測………………13
2.2.1.5 凝血酶（thrombin）活性檢測……………………14
2.2.1.6 鹼性磷酸酶（alkaline phosphatase）活性檢測…14
2.2.1.7 脂肪酶（lipase）活性檢測…………………………15
2.2.1.8 a-澱粉酶（a-amylase）活性檢測…………………16
2.2.1.9 a-胰凝乳蛋白酶（a-chymotrypsin）活性之檢測…16
2.2.1.10a-葡萄糖甘酶（a-glucosidase）活性檢測………17
2.2.1.11 胃蛋白酶（pepsin）活性檢測……………………18
2.2.2.生成物濃度檢量線之求取………………………………19
2.2.3酵素活性之求取……………………………………………19
2.2.3半抑制濃度（IC50）之求取………………………………19
2.2.4可溶性蛋白質濃度測定法（bio-rad protein assay）…20
2.2.4.1 一般性分析………………………………………………20
2.2.4.2 中微量分析………………………………………………20
2.2.4.3 微量分析…………………………………………………20
2.3 二甲藻水層萃取物對天使蝦之急毒性測試…………………20
2.3.1 二甲藻水層萃取物對天使蝦之毒性測試…………………20
2.3.2半致死濃度（LC50）之求取…………………………………21
2.4二甲藻萃取物對於微囊藻光合系統的抑制測試………………21
2.4.1微囊藻培養條件………………………………………………21
2.4.2微囊藻生理測試時間之確定…………………………………22
2.4.3以氧電極測量二甲藻萃取物對於微囊藻光合系統之抑制測試……22
2.4.4呼吸作用與光合作用的速率計算……………………………22
2.4.4.1淨產氧速率的計算…………………………………………22
2.4.4.2呼吸作用速率的計算………………………………………23
2.4.4.3光合作用速率的計算………………………………………23
2.4.5二甲藻水層萃取物對微囊藻第二光系統的影響……………23
2.5二甲藻水層萃取物對於微囊藻膜系完整性的影響……………23
2.5.1二甲藻水層萃取物對於微囊藻細胞內pH值的影響…………23
2.5.1.1藻細胞的uranine染色法……………………………………23
2.5.1.2細胞內uranine螢光量的測量………………………………24
2.5.1.3以細胞流測儀測量微囊藻細胞內pH值受二甲藻萃取物干擾之測試………………………………24
2.5.1.4以螢光光度計測量微囊藻細胞內pH值受二甲藻萃取物干擾之測試………………………………25
2.5.2二甲藻水層萃取物對於微囊藻細胞鉀離子滲漏之影響……………25
2.5.2.1藻細胞內之鉀離子濃度測定…………………………………25
2.5.2.2二甲藻水層萃取物造成藻細胞鉀離子滲漏之試驗………………26
2.5.2.3鉀離子濃度的測定…………………………………………26
2.6 二甲藻水層活性物質之初步純化…………………………… 26
2.6.1二甲藻水層粗萃取物依分子量之分離………………………26
2.6.2陽離子交換層析………………………………………………27
三、結果………………………………………………………………28
3.1二甲藻萃取物對不同藻種生長速率影響之差異………………28
3.2二甲藻水層萃取物對不同消化酶抑制程度之差異………………28
3.3二甲藻水層萃取物對天使蝦（T. platyrus）之毒性測試………28
3.4微囊藻對數生長期之確定…………………………………………28
3.5二甲藻萃取物對於微囊藻光合系統與呼吸系統的影響…………29
3.5.1 對光合作用產氧及呼吸作用耗氧之影響……………………29
3.5.2二甲藻水層粗萃取物對微囊藻光系統中電子傳遞鏈的抑制…………………………………………………29
3.6二甲藻萃取物對於微囊藻膜系的影響………………………29
3.6.1二甲藻萃取物對於微囊藻胞內正常酸鹼中性的破壞…………30
3.6.2二甲藻毒性物質造成微囊藻細胞的鉀離子滲漏…………30
3.7二甲藻毒性物質之初步純化…………………………………31
3.7.1二甲藻水層粗萃取物之純化……………………………………31
3.7.2陽離子交換層析…………………………………………………31
四、討論…………………………………………………………………33
五、參考文獻……………………………………………………………39

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