臺灣博碩士論文加值系統

摘要
由Streptomyces sp. 所選殖出的cytochrome P-450 monooxygenase gene轉型至Escherichia coli或S. lividans中，發現轉型菌株將compactin轉換成pravastatin的能力有限，這些數據顯示可能需要P-450 reductase基因搭配，才能提昇其轉換效率。比對已知的P-450 reductase基因的高度保留區域，根據其胺基酸序列設計退化性引子(degenerated primer)，以Amycolata autotrophica CCRC 12444及Streptomyces griseolus CCRC 13677的染色體DNA作為模板，利用nested PCR，可以分別合成一DNA片段。上網比對確定此片段為P-450 reductase基因的一部份，其中，以A. autotrophica的染色體DNA當作模板所合成的DNA片段和已發表的Bacillus subtilis NADPH-P450 reductase基因比對，相似度為72﹪；以S. griseolus的染色體DNA當作模板所合成的DNA片段和已發表的B. subtilis NADPH-P450 reductase基因比對，相似度為70﹪。並以此片段作為探針(probe)，由A. autotrophica和S. griseolus的部分染色體基因庫(partial library)以Southern blot的方式繼續進行篩選出完整的P-450 reductase基因。為得到更長的探針，於是再設計一組退化性引子，並由所得到的250 bp DNA片段中設計一組專一性引子，以S. griseolus的染色體DNA當作模板進行PCR，得到一約800 bp DNA片段，經上網比對後發現此片段類似redoxin基因片段，並以此片段當作探針篩選得到全長的基因。Redoxin基因全長276 bp，此段ORF可以轉譯出91個胺基酸，預估分子量為10.6kDa，其G+C含量為67.8%，由胺基酸組成估算此蛋白質疏水性胺基酸含量為34.1%，極性胺基酸含量為17.6%，等電點為7.16，pH 7.0時所帶的電量為0.17。將redoxin基因選殖入E. coli表現載體pET21b中，並使其大量表現，再利用帶有金屬的親和性管柱來回收redoxin。另一方面，將已發表的B. subtilis NADPH P-450 reductase基因利用兩組specific primer從B. subtilis DB2 中選殖出來。所得到兩個NADPH P-450 reductase基因分別命名為yetO ( yfnJ )和yrhJ，彼此間的胺基酸序列相似度為61%，yetO 基因全長3,183 bp，此段ORF可以轉譯出1061個胺基酸，預估分子量為116.7 kDa；yrhJ 基因全長為3,162 bp，此段ORF可以轉譯出1054個胺基酸，預估分子量為115.9 kDa。其G+C含量依次為50.8%、46.6%。將yetO、yrhJ基因分別選殖入表現載體pQE-30中，並使其在E. coli Nova Blue中大量表現，惟yetO 基因無法大量表現。隨後利用帶有金屬的親和性管柱來回收P-450 reductase。在菌體內的分析結果指出，單獨攜帶redoxin及yrhJ 基因的E. coli轉型株無法將Na-compactin轉換成為pravastatin。再以經由 compactin 誘導 2~3 天之放線菌菌體萃取液來當作此催化反應系統，當單獨加入P450 monooxygenase時，可提升其轉換率，表示P450 monooxygenase在此反應條件下具有轉換compactin成pravastatin的能力，提高約1.0 %，此時再加入純化之 redoxin 或 P450 reductase 並搭配純化之 P450 monooxygenase 進行酵素反應，也會有 pravastatin 的生成， 但轉換率提昇的比率最高只有1.1 %。

Abstract
Cytochrome P-450 monooxygenase gene cloned from Streptomyces sp. was transformed into Escherichia coli or S. lividans. It was found that transformants have poor abilities to convert the compactin to pravastatin. These data show that cytochrome P-450 reductase may be needed to participate the reaction, then the conversion efficiency can be elevated. Four primers were designed according to the conserved region of P-450 reductase genes. A DNA fragment was amplified from Streptomyces spp. chromosomal DNA using nested polymerase chain reaction. DNA sequence analysis revealed that this DNA fragment belongs to part of P-450 reductase gene. Amino acid sequence of this fragment shows 70 % identity with NADPH-P450 reductase gene from B. subtilis. Using this P-450 reductase gene fragment as probe, the full length of the P-450 reductase gene was screened by Southern blot. Two degenerated primers and two specific primers were designed to gain longer DNA as probe, A 800 bp DNA fragment was amplified from S. griseolus chromosomal DNA. DNA sequence analysis revealed that this DNA fragment implies a redoxin gene. The ORF of redoxin gene consisted of 276 bp and predicted to encode a protein with a calculated molecular mass of 10.6 kDa. The GC content of this gene was 67.8 %, the polar amino acid content of redoxin was 17.6 %, pI was 7.16 and the charge was 0.17 while pH 7.0. Redoxin gene was cloned into pET-21b expression vector and expressed in E. coli BL21(DE3). The enzyme was purified by metal chelate column chromatography. Meanwhile, NADPH-P450 reductase genes were cloned from B. subtilis with specific primers. Two NADPH-P450 reductase gene, named yetO ( yfnJ ) and yrhJ ,showed 61% amino acid identity with each other. The ORF of yetO gene consisted of 3,183 bp and predicted to encode a protein with a calculated molecular mass of 116.7 kDa. The ORF of yrhJ gene consisted of 3,162 bp and predicted to encode 1054 amino acids with a calculated molecular mass of 115.9 kDa. The GC contents of yetO and yrhJ genes were 50.8% and 46.6%, respectively. The yrhJ gene was cloned into pQE-30 expression vector and expressed in E. coli Nova Blue. The P450 enzymes were also purified by metal chelate column chromatography. This enzyme can’t convert compactin to pravastatin. Cell crude extract of Streptomyces spp. which was induced by adding compactin as the catalytic reaction system. Adding solely P450 monooxygenase，the conversion system of compactin to pravastatin is worked, the result shows P450 monooxygenase has the ability to convert the compactin to pravastatin with the elevated conversion ratio of 1.0 %. Adding redoxin or P450 reductase with P450 monooxygenase in this enzyme reaction, pravastatin can be produced and the elevated conversion ratio is 1.1 %.

目錄
中文摘要…………………………………………………………………1
英文摘要…………………………………………………………………4
前言………………………………………………………………………7
材料與方法
一、 藥品……………………………………………………………18
二、 菌株、質體及培養基…………………………………………18
三、 E. coli 質體DNA的小量抽取……………………………………19
四、 DNA的洋菜膠體電泳分析……………………………………..20
五、 DNA片段的回收及純化………………………………………...20
六、 DNA純度之鑑定及定量分析…………………………………..22
七、 DNA黏接反應…………………………………………………...22
八、 引子的設計……………………………………………………23
九、 退化性引子的設計……………………………………………24
十、 聚合酉每連鎖反應………………………………………………25
十一、 質體轉型作用
(1) E. coli的質體轉型…………………………………………27
(2) B. subtilis DB2的轉型作用………………………………….27
十二、 P450 reductase基因的選殖
(1) S. griseolus染色體DNA的抽取……………………………..28
(2) B. subtilis染色體DNA的抽取………………………………28
(3) DNA 探針的製備…………………………………………..29
(4) 南方轉漬雜交法………………………………………….30
(5) S. griseolus染色體部分基因庫的建立…………………….31
(6) 菌落雜交法………………………………………………..32
十三、 P450 reductase基因在E. coli的表現
(1) 將來自於B. subtilis NADPH-P450 reductase基因選殖入E. coli..33
(2) P450 reductase基因的大量表現…………………………..33
(3) P450 reductase的純化……………………………………..34
十四、 Redoxin基因在E. coli的表現
(1) 將來自於S. griseolus的redoxin基因選殖入E. coli…………35
(2) Redoxin基因的大量表現…………………………………...35
(3) Redoxin的純化……………………………………………...35
十五、 P450 reductase基因在放線菌菌體內活性的分析
(1) 放線菌接種量的標準化…………………………………..36
(2) 放線菌菌體外之生物轉換………………………………..36
(3) 以高效液相層析法分析compactin及pravastatin…………37
十六、 分析compactin及pravastatin之穩定性………………………..38
十七、 蛋白質之定量
(1) 製作標準曲線…………………………………………….38
(2) P450 reductase濃度的測定………………………………..38
十八、蛋白質的電泳分析……………………………………………..39
結果
一、 能將compactin轉換成parvastatin菌株的篩選………………42
二、 P450 reductase基因的篩選
(一) Streptomyces spp.部分
1、 探針的設計………………………………………...42
2、 S. griseolus部分染色體基因庫的構築……………...44
3、 Redoxin基因的選殖…………………………………44
4、 Redoxin基因在E. coli之大量表現…………………..45
5、 Redoxin基因在放線菌菌體內的表現情形…………45
(二) Bacillus 部分
1、 NADPH-P450 reductase基因的選殖…………………..46
2、 NADPH-P450 reductase基因在E. coli之大量表現…….47
3、 NADPH-P450 reductase基因在B. subtilis之大量表現…48
4、 NADPH-P450 reductase基因在放線菌菌體內的表現...48
三、 compactin及pravastatin之pH穩定性………………………….49
討論
一、 Redoxin基因的胺基酸序列…………………………………..50
二、 Redoxin及P450 reductase基因在E. coli菌體內的表現………51
三、 Redoxin及P450 reductase基因在放線菌菌體內的表現……..53
四、 Compactin及pravastatin之pH穩定性…………………………53
圖表……………………………………………………………………...55
參考文獻……………………………………………………………….101

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