Pseudomonas tamsuii TKU015發酵烏賊軟骨生產殺蟲蛋白之研究

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Title:	Pseudomonas tamsuii TKU015發酵烏賊軟骨生產殺蟲蛋白之研究
Other Titles:	Studies on the production of insecticidal proteins by Pseudomonas tamsuii TKU015 fermentation on squid pen
Authors:	甄家宏;Chen, Chia-Hung
Contributors:	淡江大學化學學系碩士班王三郎
Keywords:	殺蟲蛋白;烏賊軟骨;靈菌紅素;Pseudomonas tamsuii;insecticidal protein;prodigiosin
Date:	2012
Issue Date:	2013-04-13 11:04:01 (UTC+8)
Abstract:	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.
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