Pseudomonas tamsuii TKU015這株新種細菌，係為以烏賊軟骨為唯一碳/氮源，篩選自淡水土壤之納豆激酶生產菌。液相層析串聯式質譜(LC/MS)分析此菌所生產一種蛋白質與Pseudomonas entomphila L48之殺蟲蛋白具有高相似性的胺基酸序列，轉殖此蛋白質基因於大腸菌之結果顯示其具殺果蠅活性。因此本研究主要探討此菌生產殺蟲蛋白之較佳發酵條件，將所得發酵菌液分成全菌液、上清液、菌體、破菌菌體，分別探討此四種樣品之殺果蠅活性。
結果顯示較適培養條件為，將菌株P. tamsuii TKU015接種於100 mL含有1 %烏賊軟骨粉、0.1 % K2HPO4及0.05 % MgSO4．7H2O (pH 7) 之液體培養基（裝填於250 mL錐形瓶）於30℃經搖瓶(150 rpm)培養5天，所得破菌菌體的致死率可達81%。此外亦探討Serratia marcescens TKU011所生產之靈菌紅素及Paenibacillus macerans TKU029所生產之生物界面活性劑對果蠅之半致死濃度(LC50)，所得到的結果靈菌紅素LC50為1350 ppm，生物界面活性劑LC50則為75 ppm。

Pseudomonas tamsuii TKU015, a new species strain, is a nattokinase- producing bacterium isolated from Tamsui soil using squid pen as the sole carbon/nitrogen source. LC/MS analyzed results showed that there is a Pseudomonas tamsuii TKU015 protein with high similarity to the insecticidal protein of Pseudomonas entomphila L48 in amino acid sequences. Gene cloning and expression of the protein has been studied and showed with insecticidal activity against fruit fly. The purpose of this study was to investigate better culture conditions for the production of insecticidal proteins. Samples of culture broth、culture supernatant、cell pellet and broken cells are tested for their insecticidal activities. 
Results showed that when cultured in the media containing 1% squid pen powder as the sole carbon and nitrogen source ,0.1% K2HPO4,and 0.05% MgSO4．7H2O(pH=7), Pseudomonas tamsuii TKU015 produced insecticidal proteins. The culture was incubated in 100 mL of medium in an Erlenmeyer flask (250 mL) containing 1% SPP at 30℃for 5 days . Broken cells showed higher larval mortality 81%.
Insecticidal activities of yellow pigment NO.4 and red pigment NO.40  were also performed and compared that of Serratia marcescens TKU011 produced prodigiosin. Commercial surfactants and Paenibacillus macerans TKU029 bio-surfactant were also studied. The LC50 of fruit fly for prodigiosin and bio-surfactant were 1350 ppm and 75 ppm respectively.

目錄
	頁次

簽名頁
授權書
中文摘要	I
英文摘要 III
誌謝	V
目錄	VI
圖目錄	IX
表目錄	XI

第一章 緒論	1
第二章 文獻回顧	2
     2.1 菌種之簡介	2
     2.2 蝦蟹殼及烏賊軟骨之應用	2
2.3殺蟲劑分類簡介	3
     2.4微生物殺蟲劑	4
2.4.1 Pseudomonas spp.源之殺蟲物質	5
2.4.2靈菌紅素	6
        2.4.3生物界面活性劑	6

第三章 材料與方法	9
     3.1 實驗菌株	9
3.1.1 Pseudomonas tamsuii TKU015	9
3.1.2 Serratia marcescensTKU011	9
3.1.3 Paenibacillus macerans TKU029	9
     3.2 實驗材料	9
     3.3 實驗儀器	11
     3.4 殺蟲最適培養條件探討	12
3.4.1 碳/氮源之選擇	12
3.4.2 碳/氮源濃度之影響	12
3.4.3 培養溫度	12
3.4.4 培養體積 (通氣量)	12
3.4.5 培養時間	13
     3.5果蠅培養基	13
     3.6果蠅殺蟲活性測試	13
     3.7破菌方法	13
     3.8 蛋白質電泳分析	14
3.9 Paenibacillus macerans TKU029界面活性劑之分離純化	14
3.10 靈菌紅素之萃取	14
第四章 結果與討論	19
     4.1殺蟲蛋白較適生產條件探討	19
4.1.1 碳/氮源之選擇	19
4.1.2 培養溫度探討	20
4.1.3 碳/氮源濃度探討	20
4.1.4 培養體積 (通氣量)探討	20
4.1.5綜合培養條件	20

     4.2靈菌紅素對果蠅之半致死濃度(LC50)測量	32
4.2.1靈菌紅素與兩種食用色素對果蠅幼蟲致死率之比較	32
     4.3 生物界面活性劑對果蠅半致死濃度測定	40
4.3.1生物界面活性劑與非離子型界面活性劑對果蠅幼蟲致死率比較	40
4.3.2生物界面活性劑與離子型界面活性劑對果蠅幼蟲致死率比較	41

第五章 結論	47
參考文獻	48
圖目錄
圖3.1發酵菌液處理流程圖	16
圖3.2 果蠅生長流程	17
圖3.3 Prodigiosin 定量方法	18
圖4.1在不同的培養基下P. tamsuii TKU015之生長曲線	22
圖4.2P. tamsuii TKU015在於不同碳氮源培養基培養所得四種樣品之幼蟲致死率	23
圖4.3 P. tamsuii TKU015殺蟲蛋白隨培養溫度及時間之變化	24
圖4.4培養溫度對P. tamsuii TKU015生產殺蟲蛋白之影響	25
圖4.5 SPP濃度對P. tamsuii TKU015生產殺蟲蛋白之影響	26
圖4.6培養體積對P. tamsuii TKU015生產殺蟲蛋白之影響	27
圖4.7 P. tamsuii TKU015發酵SPP培養基一至五天所得破菌體蛋白質之SDS-PAGE圖	28
圖4.7 P. tamsuii TKU015於不同培養基培養五天所得破菌體蛋白質之SDS-PAGE圖	29
圖4.9靈菌紅素在四種樣品中含量對果蠅幼蟲致死率之影響	34
圖4.10 DMSO濃度對果蠅幼蟲致死率之影響	35
圖4.11靈菌紅素濃度對果蠅幼蟲致死率之影響	36
圖4.12食用色素紅色四十號濃度對果蠅幼蟲致死率之影響	37
圖4.13  R40,Y4,PG對果蠅幼蟲致死率之比較	38
圖4.14 P. macerans TKU029所生產之生物界面活性劑濃度對果蠅幼蟲致死率之影響	42
圖4.15 Tween20, Tween40濃度對果蠅幼蟲致死率之影響	43
圖4.16 CTAB濃度對果蠅幼蟲致死率之影響	44
圖4.17 SDS濃度對果蠅幼蟲致死率之影響	45

表目錄
表2.1 Pseudomonas spp. 殺蟲活性整理表	8
表4.1培養條件探討前後所得果蠅幼蟲殺蟲活性比較	30
表4.2各種菌種對果蠅幼蟲致死率之比較	        33
表4.3各種色素對果蠅幼蟲的半致死濃度(LC50)	39
表4.4各種界面活性劑對果蠅幼蟲的半致死濃度(LC50)	46

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