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研究生:魏佳俐
研究生(外文):Chia-Li Wei
論文名稱:定向演化改良Streptomycesclavuligerus擴環酶對青黴素G之基質特異性
論文名稱(外文):Directed evolution of deacetoxycephalosporin C synthase from Streptomyces clavuligerus for improved conversion of penicillin G
指導教授:蔡英傑蔡英傑引用關係
指導教授(外文):Ying-Chieh Tsai
學位類別:博士
校院名稱:國立陽明大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:英文
論文頁數:140
中文關鍵詞:頭孢素7-胺基去乙醯基頭孢素酸青黴素G定向演化法擴環酵素
外文關鍵詞:cephalosporins7-aminodeacetoxycephalosporanic acidpenicillin Gdirected evolutiondeacetoxycephalosporin C synthase = expandase
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頭孢素(cephalosporins)是臨床上用來治療細菌感染的抗生素。7-胺基去乙醯基頭孢素酸(7-aminodeacetoxycephalosporanic acid,簡稱7-ADCA)為生產三種重要口服頭孢素的中間體。目前工業上生產7-ADCA主要是利用化學法將青黴素G (penicillin G)擴環為頭孢素G,接著再利用酵素法移去側鏈製得7-ADCA。由於這種化學擴環不僅製程複雜,而且會對環境造成相當程度的損害,因此一種利用酵素的生物擴環法取代目前的化學法是被高度期盼的。
因此本研究的主要目的便是利用定向演化法(directed evolution)改良Streptomyces clavuligerus的擴環酵素以增加其對青黴素G的酵素活性。首先使用化學致變劑羥胺(hydroxylamine)對擴環酵素基因做隨機突變,再利用為進行本實驗所開發的二步驟篩選法分析約5,500個突變株,三個對青黴素G具有提高酵素動力參數kcat/Km二到六倍的突變株G79E、V275I和C281Y被篩選出來。接著再利用error-prone PCR對擴環酵素基因做隨機突變,經分析約6,400個突變株後,又篩選到十個對青黴素G具有提高酵素催化效率參數kcat/Km二到六倍的突變株M73T、T91A、A106T、C155Y、Y184H、M188V、M188I、H244Q、L277Q和I305L。最後這13個突變株,外加二個由駿翰公司楊運博博士,利用定點突變法獲得的對青黴素G具有提高酵素催化效率參數kcat/Km 11到14倍的突變株,利用DNA shuffling法加以組合,在篩選過約10,600個突變株後,得到三個具有提高酵素催化效率參數kcat/Km 高達41、51和60倍的突變株。
本研究是第一個利用定向演化法改良擴環酵素,增加其對青黴素G的酵素活性。在此之前僅有以定點突變法修改擴環酵素羧基端的胺基酸區域,被發現具有提高酵素活性近二倍的研究被發表過。因此本研究不僅為工業生產之7-ADCA,提供具有更強酵素活性的擴環酵素,同時在酵素活性機制上,本研究更指出多個對此具有重要角色的胺基酸殘基。

Cephalosporins are antibiotic compounds used clinically for the treatment of bacterial infection. Three pharmaceutically important oral cephalosporins, including cephradine, cephalexin and cephadroxil, are derived from 7-aminodeacetoxycephalosporanic acid (7-ADCA) to which different side chains are added. The industrial process to synthesize 7-ADCA mainly involves a chemical ring expansion of penicillin G to phenylacetyl-7-ADCA, and followed an enzymatic side chain cleavage of phenylacetyl-7-ADCA. The chemical ring expansion is complex and expensive, and causes a significant negative environmental impact. Therefore, an enzymatic reaction is greatly desirable to replace such chemical reaction.
The primary object of this study was to engineer Streptomyces clavuligerus deacetoxycephalosporin C synthase (DAOCS) using directed evolution in an effort to improve conversion of penicillin G for industrial production of 7-ADCA. A random library of DAOCS mutants was firstly created using hydroxylamine as a chemical mutagen and subjected to a developing 2-step screening procedure. After analysis of 5,500 clones, three mutants G79E, V275I, and C281Y were selected and showed 2- to 6-fold increases in kcat/Km values compared to the wild-type enzyme. Another PCR-based random mutagenesis of the enzyme followed by screening of 6,400 clones identified additional 10 mutations, M73T, T91A, A106T, C155Y, Y184H, M188V, M188I, H244Q, L277Q, and I305L with 2- to 6-fold increased kcat/Km ratio. These mutants together with mutations N304K and I305M, which were produced by Dr. Yunn-Bor Yang (Synmax Biochemical) using site-directed mutagenesis and had 11- and 14-fold increased kcat/Km ratio, were further combined using DNA shuffling. After analysis of 10,600 clones, three mutants with 41-, 51-, and 60-fold increased kcat/Km ratio were thus obtained.
This study showed clearly that directed evolution could be used to optimize DAOCS-penicillin G reaction. Prior to this work, it is only an approximately 2-fold increase in penicillin G conversion activity were found from mutants with modifications on the C-terminal part. Therefore, our work not only provided powerful DAOCSs for industrial usage, but also revealed more residues that are significantly involved in the enzymatic catalysis.

中文摘要 1
Abstract 3
Introduction 4
A. Survey of Antibiotics 4
A.1. Introduction 4
A.2. Nomenclature 5
A.3. Classification 5
A.4. Targets for Antibacterial Agents 7
A.5. Bacterial Survival Strategies to Combat Antibiotics 8
B. Beta-Lactam 9
B.1. Introduction 9
B.2. Early History 9
B.3. Classification 9
B.4. Mode of Action 11
B.5. Mechanisms of Resistance 12
C. Penicillins 13
C.1. Introduction 13
C.2. Classification 14
D. Cephalosporins 16
D.1. Introduction 16
D.2. Classification 17
E. Genes in Penicillin/Cephalosporin Biosynthesis 18
E.1. Introduction 18
E.2. Common Early Genes 19
E.3. Late Genes in the Biosynthesis of Hydrophobic Penicillins 19
E.4. Common Genes in the Cephalosporin Pathway 20
E.5. Late Genes in Cephalosporin C Biosynthesis 21
E.6. Late Genes in Cephamycin C Biosynthesis 21
E.7. Genes Arrangement 22
F. 7-ADCA 23
F.1. Introduction 23
F.2. Properties of 7-ADCA Derivatives 24
F.3. Current Process of 7-ADCA 24
G. Recent Studies for Generation of 7-ADCA Precursor 25
G.1. Introduction 25
G.2. Fermentation of 7-ADCA Precursor 26
G.3. In Vitro Enzymatic Properties of DAOCS 27
G.4. Modifying the Reaction Conditions for Increasing Production of G-7-ADCA 29
G.5. Crystallographic Studies on DAOCS 30
G.6. Protein Engineering of DAOCS 32
Specific Aims and Approaches 35
Materials and Methods 36
A. Materials 36
A.1. Chemicals and Enzymes 36
A.2. Media 36
A.3. Strains 37
A.4. Plasmids 38
A.5. Apparatus 38
B. PCR 39
C. DNA Ligation 39
D. Electroporation (Electrotransformation) 39
E. Random Mutagenesis by a Chemical Mutagen 40
F. Random Mutagenesis by Error-Prone PCR 40
G. DNA Shuffling 41
H. Screening for Increased Activity 41
I. Mutant Generation by Site-Directed Mutagenesis 42
J. Purification of DAOCSs 43
K. DAOCS Activity Assayed by HPLC 44
Results 45
A. Development of a Screening Method for Selection of Mutant DAOCSs with Improved Penicillin G Conversion 45
B. Random Mutagenesis by a Chemical Mutagen and Screening 46
C. Random Mutagenesis by Error-Prone PCR and Screening 47
D. Purification of Mutant DAOCSs and Kinetic Analysis 49
E. Construction of Combined Mutants by DNA Shuffling and Screening 50
F. Purification of Combined Mutant DAOCSs and Kinetic Analysis 51
G. Second Round of Random Mutagenesis of Combined Mutants and Screening 52
Discussion 53
A. Experimental Strategy for Directed Evolution of DAOCS 53
B. Analysis of the Effect of the Mutations in DAOCS 54
B.1. Mutations in the C-terminal Lid of DAOCS 54
B.2. Mutations of V275I, L277Q, and C281Y 56
B.3. Mutations of H244Q, C155Y, and H184H 57
B.4. Mutations of M188V and M188I 58
B.5. Mutations of M73T, G79E, T91A, and A106T 59
B.6. Mutants with Combined Substitutions 60
C. Substrate Inhibition of DAOCS 60
D. Future Work of Engineering DAOCS for Penicillin G Conversion 61
References. 62
Tables. 71
Figures. 76
Appendixes. 100

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