Heterologous Expression of Penicillin Acylase in E. coli by PCR- Cloning pac Gene from Providencia rettgeri ATCC31052

• Main Authors: Lin, Ming-I, 林明毅
• Other Authors: C.Perry Chou
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/46330078520033018207

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℃ .