臺灣博碩士論文加值系統

利用大腸桿菌 (Escherichia coli；E. coli) 大量表達pac 基因生產盤尼西林水解酵素蛋白 (penicillin acylase ； PAC) 時會產生大量內涵體 (inclusion body) ，如果能減少內涵體的累積，可以提高具有活性的 PAC 產量，得到較高的比活性 (Specific PAC Activity)。在之前的研究中發現利用具有細胞間質蛋白水解酵素 (periplasmic protease) 及保護子 (chaperone) 活性的 DegP 酵素在大腸桿菌中的共同表現 (coexpression) 可以增加可溶解並具有活性的PAC。本研究利用沒有蛋白分解酵素的DegP突變種DegP S210A進行過度表達 pac ，找出DegP在大量表達 pac 基因時提高PAC活性所表現的活性以熱衝擊蛋白為主。並以DegP 的同源間質蛋白水解酵素， DegQ 及 DegS 進行進一步的研究發現 DegP 的保護子活性在大量表達 pac 基因可能也具有改善系統生理毒素的作用，所以DegP 在大量表達pac 基因才具有異於同源蛋白的改善效果，另一方面探討類似保護子作用的FkpA蛋白進行pac 基因的大量表達，發現FkpA也具有不錯的改善效果。第二部份利用基因工程技術將PAC基因去除信號胜月太，使PAC在細胞質中成熟並探討數種保護子系統對形成PAC 成熟蛋白的影響。本實驗的保護子系統有GroEL-GroES 和 DnaK-DnaJ-GrpE及類似保護子的表現因子trigger factor。

Coexpression of DegP resulted in a shift of in vivo penicillin acylase (PAC) synthesis flux from the nonproductive pathway to the productive one when pac was overexpressed。 The number of inclusion bodies，which consist primarily of protein aggregates of PAC precursors in the periplasm，was highly reduced， and the specific PAC activity was highly increased。DegP was a heat shock protein induced in response to pac overexpression，suggesting that the protein could possibly suppress the physiological toxicity caused by pac overexpression。Coexpression of DegPS210A，a DegP mutant without protease activity but retaining chaperone activity，could not suppress the physiological toxicity，suggesting that DegP protease activity was primarily responsible for the suppression， possibly by degradation of abnormal proteins when pac was overexpressed. However, a shortage of periplasmic protease activity was not the only reason for the deterioration in culture performance upon pac overexpression because coexpression of a DegP-homologous periplasmic protease，DegQ or DegS，could not suppress the physiological toxicity。The chaperone activity of DegP is proposed to be another possible factor contributing to the suppression。
In the second part of this study， the DNA fragment encoding the pac signal peptide was removed so that proPAC was expressed in the cytoplasm。 Effect of various cytoplasmic chaperones， including DnaK/J，GroEL/ES，and trigger factor，on the expression of cytoplasmic proPAC was investigated。

中文摘要………………………………………………………………..Ⅰ
Abstract…………………………………………………………………Ⅱ
目錄…………………………………………………………………….Ⅲ
表目錄………………………………………………………………….Ⅶ
圖目錄………………………………………………………………….Ⅷ
第一章緒言
1.1前言………………………………………………………………….1
1.2盤尼西林水解酵素之用途介紹…………………………………….2
1.3 PAC酵素的來源以及種類…………………………………………3
1.4 PAC酵素的成熟途徑………………………………………………4
1.5大量表達重組蛋白的策略…………………………………………6
1.6改善內涵體在過度基因表達的策略………………………………6
1.7研究動機與方向……………………………………………………8
第二章實驗方法 ……………………………………………………..11
2.1菌種的處理………………………………………………………..11
2.1.1本研究所使用菌種種類……………………………………….11
2.1.2培養基的製備………………………………………………….11
2.1.3抗生素的配製………………………………………………….11
2.1.4誘導劑的配置………………………………………………….11
2.1.5菌種的儲存與活化……………………………………………..11
2.1.6接菌 (inoculum) 的方法與細胞濃度的測量…………………12
2.2處理DNA技術……………………………………………………14
2.2.1本研究所使用質體DNA種類…………………………………14
2.2.2基因表達質體之建構………………………………………….14
2.2.3可轉形性細胞 (competent cell) 的製備……………………...15
2.2.4化學法轉形 (transformation)………………………………….15
2.3菌種培養…………………………………………………………..17
2.3.1批次菌種醱酵培養…………………………………………….17
2.3.2生化反應器之醱酵培養……………………………………….18
2.4酵素活性之定量分析……………………………………………..18
2.4.1 PAC 酵素活性之分析原理…………………………………....18
2.4.1.1酵素樣品之製備…………………………………………….19
2.4.1.2酵素反應…………………………………………………….19
2.4.1.3呈色反應…………………………………………………….19
2.4.2 b-半乳糖苷酶 (b-galactosidase；b-gal) 分析………………..20
2.5蛋白質SDS-PAGE分析法………………………………………..20
2.5.1蛋白質電泳樣品配置………………………………………….20
2.5.1.1可溶性蛋白電泳樣品的製備………………………………21
2.5.1.2不可溶性蛋白電泳樣品的製備…………………………….21
2.5.2進行電泳分離………………………………….……………….21
2.5.3蛋白質電泳凝膠顯色………………………………………….22
2.5.4西方墨漬製法的免疫分析方法……………………………….22
2.6實驗藥品…………………………………………………………..24
2.6.1培養基及誘導劑部分………………………………………….24
2.6.2化學轉形部分………………………………………………….25
2.6.3酵素分析部分………………………………………………….25
2.6.4 b-半乳糖苷酶分析部分………………………………………..25
2.6.5蛋白質電泳部分……………………………………………….26
2.6.6免疫分析部分………………………………………………….26
2.7實驗設備…………………………………………………………..27
第三章結果與討論 …………………………………………………..29
3.1在 pac 基因過度表達下共同表達DegP酵素………………….29
3.1.1過度表達pac基因及共同表達degP的結果………………..29
3.1.2過度表達pac基因及共同表達degP的其他效應…………..30
3.1.3過度表達 pac 基因誘導DegP 酵素表達熱衝擊蛋白的活性32
3.1.4利用 DegPS210A 的共同表達探討DegP酵素的活性作用…...33
3.1.5利用DegP的同源蛋白進行探討……………………………..34
3.1.6利用FkpA共同表達在 pac 基因的過度表現………………35
3.2利用保護子系統進行過量表達細胞質中的 PAC 酵素………...36
3.2.1細胞質中的 pac 基因過度表現………………………….…..36
3.2.2利用保護子系統的共同表現在細胞質中過量表現pac基因.37
3.3 DegP對大腸桿菌過度表現pac基因之影響及保護子對PAC先驅體之穩定性探討……………………………………………..39
第四章結論……………………………………………………………45
參考文獻 ………………………………………………………………62

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