Summary: | 碩士 === 逢甲大學 === 化學工程研究所 === 86 === PAC酵素不僅具工業應用價值,在於學術上也是一個很好的研究對象。本
研究利用PCR技術將Providencia rettgeri ATCC31052的pac基因植株,並
將其副植株於tac啟動子的表現載體上,建構出數個重組質體以於E. coli
生物系統中進行基因之異形表現(heterologous expression)。對於PAC酵
素生成的環境調節方面,例如誘導劑與不同種類碳源的添加及菌種培養溫
度的影響,我們也做了初步的測試。結果發現,Providencia rettgeri
ATCC31052與新重組菌種均不受苯乙酸(phenylacetic acid, PAA)的誘導
。另外,對Providencia rettgeri ATCC31052的測試中發現,並沒有葡萄
糖降解物抑制的現象(catabolite repression),但對JM109(
pTrcKnPAC2601)則出現了明顯的葡萄糖降解物抑制。對於另一株新重組菌
種JM109(pUTKnPAC2601)而言,適量的葡萄糖或甘油添加下,則提高了PAC
酵素的體積活性(volumetric activity)。此外,PAC酵素的生成原本需在
低的培養溫度(28℃)下,才有較佳的表現,在37℃時具有活性的PAC酵素
則不易生成。然而,以33℃的高溫在E. coli中進行異形表現,PAC酵素的
產量依然良好,即使將溫度提升至37℃,仍有三成比例以上的PAC酵素生
成。JM109(pUTKnPAC2601)可在較高溫(如33℃)的培養條件之下,生產出
具有活性的PAC酵素,其與一般的PAC酵素之產生菌種相較之下,存有著明
顯的差別。
In this study , we PCR-cloned the pac gene from Providencia
rettgeri ATCC31052 and constructed several expression plasmids
by subcloning thepac gene into tac-expression vectors for
heterologous expression in E. coli .Environmental conditions ,
such as inducer , carbon source , andtemperature , on regulation
of PAC expression were characterized. PAC wasnot induced by
PAAin both Providencia rettgeri ATCC31052 and therecombinant
strains . In addition ,expression of PAC was not subjected to
glucose catabolite repression in Providencia rettgeri ATCC31052
, but wasrepressed by glucose in JM109(pTrcKnPAC2601) . However
, adding an appropriate amount of glucose or glycerol into the
medium increased the volumetric PAC activity in JM109(
pUTKnPAC2601) . In addition , native expression of PAC was
optimal at a low temperature of 28℃and was minimal at 37℃ ,
whereas heterologous expression in E. coli was normal at a
temperature as high as 33℃and more than thirty percent of PAC
activity still remained at 37℃ .
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