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研究生:羅雪霞
研究生(外文):Hsueh-Hsia Lo
論文名稱:利用新的酵素法生產L-Homophenylalanine
論文名稱(外文):Novel Enzymatic Methods for the Production of L-Homophenylalanine
指導教授:許文輝許文輝引用關係
指導教授(外文):Wen-Hwei Hsu
學位類別:博士
校院名稱:國立中興大學
系所名稱:分子生物學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:105
中文關鍵詞:L-homophenylalaninehydantoinaseL-N-carbamoylasehomophenylalanylhydantoin高效能液相層析法aminotransferaseN6-protecting-2-oxo-6-aminohexanoic acid定點突變
外文關鍵詞:L-homophenylalaninehydantoinaseL-N-carbamoylasehomophenylalanylhydantoinHPLCaminotransferaseN6-protecting-2-oxo-6-aminohexanoic acidsite-directed mutagenesis
相關次數:
  • 被引用被引用:10
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  • 下載下載:1
  • 收藏至我的研究室書目清單書目收藏:0
本研究運用二種生物轉換法,hydantoinase法及aminotransferase法,進行血管收縮素轉化酶抑制劑前驅物L-homophenylalanine (L-HPA) 的合成。在hydantoinase法的部份,以hydantoinase搭配L-N-carbamoylase,可以將racemic homophenylalanylhydantoin (rac-HPAH) 轉換產生L-HPA。為分析此反應,建立高效能液相層析法 (high performance liquid chromatography) 搭配Chirobiotic T管柱,於移動相EtOH/H2O = 10/90 (pH 4.2-4.5),發現能同時解析rac-HPAH、rac-N-carbamoyl-homophenylalanine (rac-NCaHPA)及rac-HPA。純化的重組Bacillus caldolyticus CCRC 11954及Brevibacillus agri NCHU 1002 hydantoinase,以及選殖自Arthrobacter aurescens DSM 9771及Methanococcus jannaschii DSM 2661的hydantoinase基因在Escherichia coli表現後,取其細胞萃取液,分別進行活性分析。結果顯示,重組之A. aurescens、B. agri及B. caldolyticus hydantoinases對HPAH的活性分別為0.8、7.9及7.8 U/mg,且均為非立體異構物選擇性。而M. jannaschii hydantoinase則對HPAH無活性。利用含有HPAH當作唯一氮源的基底培養基來篩選產L-hydantoinase之土壤菌,結果發現具有D-hydantoinase活性的菌株有23株,另有7株菌具有DL-hydantoinase活性。以B. agri hydantoinase搭配對NCaHPA為絕對L-選擇性之Bacillus kaustophilus L-N-carbamoylase,能將rac-HPAH轉換產生L-HPA,但會有D-NCaHPA的積聚。
在aminotransferase法的部份,以定點突變方式,改造E. coli aspartate aminotransferase (AAT) 的受質特異性,俾運用於L-HPA以及N6-protecting-2-oxo-6-aminohexanoic acid (N6-protecting-OAHA) 之生合成。以2-oxo-4-phenylbutyric acid (OPBA) 為胺基接受者,lysine為胺基供應者,野生型AAT的活性為0.13 U/mg。針對Arg292,Ile17及Leu18進行定點突變,結果發現R292E/L18H變異酵素的活性最高,達1.81 U/mg。利用大量表現R292E/L18H變異酵素之E. coli進行生物轉換,L-HPA轉換率可達100% (>99% e.e.),證明lysine是一種具潛力的胺基供應者,以HPLC/MS/MS分析,發現在此生物轉換過程中,lysine去胺後的產物,2-oxo-6-aminohexanoic acid (OAHA),會自然環化成∆1-piperideine 2-carboxylic acid。L-HPA的低溶解度與OAHA的環化可能與此反應能完全朝向產物的方向進行有關。為了同步合成血管收縮素轉化酶抑制劑前驅物L-HPA及N6-protecting-OAHA,分別以2-amino-6-benzyloxycarbonylamino-hexanoic acid (BOC-lysine) 及2-amino-6-(2,2,2-trifluoro-acetylamino)-hexanoic acid (TFA-lysine) 為胺基供應者,野生型AAT的活性分別為0.11及0.33 U/mg。以BOC-lysine為胺基供應者時,R292E/L18H變異酵素的活性最高,達0.70 U/mg。若以TFA-lysine為胺基供應者,則以R292E/L18T變異酵素活性最高,達0.67 U/mg。分別以BOC-lysine或TFA-lysine為胺基供應者,利用大量表現變異酵素之E. coli進行生物轉換,分別可達到42及35%轉換率,證明aminotransferase法亦可用於同步合成L-HPA及N6-protecting OAHA。
Hydantoinase and aminotransferase methods were used for the biosynthesis of L-homophenylalanine (L-HPA), an angiotensin-converting enzyme inhibitor (ACEI) precursor. In the combination of hydantoinase and L-N-carbamoylase, rac-homophenylalanylhydantoin (rac-HPAH) was converted to L-homophenylalanine (L-HPA). High performance liquid chromatography (HPLC) with Chirobiotic T column could enantioseparate rac-HPAH, rac-N-carbamoyl-homophenylalanine (rac-NCaHPA), and rac-HPA using EtOH/H2O = 10/90 (pH range from 4.2 to 4.5) as mobile phase at a flow-rate of 0.6 ml/min. Hydantoinase activities in purified recombinant Brevibacillus agri NCHU 1002 and Bacillus caldolyticus CCRC 11954 hydantoinase, and crude extracts of Escherichia coli expressing Arthrobacter aurescens DSM 9771 and Methanococcus jannaschii DSM 2661 were respectively determined. The hydantoinases of A. aurescens, B. agri and B. caldolyticus showed non-stereoselective toward HPAH, with specific activities of 0.8, 7.9, and 7.8 U/mg, respectively. However, M. jannaschii DSM 2661 hydantoinase showed no enzymatic activity toward HPAH. Minimal medium containing HPAH as sole nitrogen source was used to screen L-hydantoinase-producing soil bacteria. A total of 23 bacterial strains with D-hydantoinase activity and 7 strains with DL-hydantoinase were found. In the combination of B. agri hydantoinase and strictly L-selective Bacillus kaustophilus L-N-carbamoylase, rac-HPAH could be converted to L-HPA with the accumulation of D-NCaHPA.
Site-directed mutagenesis was performed to alter the substrate specificity of E. coli aspartate aminotransferase (AAT). AAT mutants were then used to synthesize L-HPA. Using 2-oxo-4-phenylbutyric acid (OPBA) as amino acceptor and lysine as amino donor, the specific activity of wild-type AAT was 0.13 U/mg. AAT mutants targeted at Arg292, Ile17, and Leu18 were subjected to activity assay. The specific activity of R292E/L18H variant toward lysine increased to 1.81 U/mg. The E. coli cells expressing R292E/L18H variant were used as biocatalyst for the transamination of lysine to OPBA and the yield of L-HPA (>99% e.e.) reached 100%. The formation of ∆1-piperideine 2-carboxylic acid, the spontaneous cyclization product of 2-oxo-6-aminohexanoic acid (OAHA), was demonstrated by HPLC/MS/MS method. The low solubility of L-HPA and the spontaneous cyclization of OAHA possibly cooperated to drive the reaction to completion. Thus, lysine was potential amino donor for the synthesis of L-HPA by aminotransferase process. For the simultaneous synthesis of L-HPA and N6-protecting-OAHA, both are ACEI precursors, 2-amino-6-benzyloxycarbonylamino-hexanoic acid (BOC-lysine) or 2-amino-6-(2,2,2-trifluoro-acetylamino)-hexanoic acid (TFA-lysine) was used as amino donor. The specific activity of wild-type AAT toward BOC-lysine or TFA-lysine was 0.11 and 0.33 U/mg, respectively. The specific activity of R292E/L18H or R292E/L18T variant toward BOC-lysine or TFA-lysine was 0.70 and 0.67 U/mg, respectively, which was the highest activity among the examined mutants. The E. coli cells expressing AAT variant were used as biocatalysts and the conversion yields using BOC-lysine or TFA-lysine as amino donor were 42 and 35%, respectively. Our data indicated that aminotransferase could also be used for the simultaneous synthesis of L-HPA and N6-protecting-OAHA.
表次 VII
圖次 VIII
縮寫表 X
中文摘要 1
英文摘要 3
第一章:以Hydantoinase法進行L-Homophenylalanine之生物轉換
一、緒論
1.1.血管收縮素轉換酶抑制劑 (ACEI)的發展 6
1.2. L-Homophenylalanine的合成 8
1.3. Hydantoinase產程在工業上的應用 9
1.4. Hydantoinase的分類 9
1.4.1. D-Hydantoinase 10
1.4.2. L-Hydantoinase 10
1.4.3.非立體異構物選擇性hydantoinase 11
1.4.4. Hydantoinases之間的類緣關係 11
1.5. N-Carbamoylase 12
1.5.1. D-N-Carbamoylase 12
1.5.2. L-N-Carbamoylase 12
1.6. 研究動機 12
二、材料與方法
2.1. 化學藥品 14
2.2. 質體、菌株、酵素與培養基 14
2.3. Hydantoinases基因的選殖、表現與純化 14
2.3.1. A. aurescens DSM 9771 14
2.3.2. Methanococcus jannaschii DSM 2661 15
2.3.3. Brevibacillus agri NCHU 1002 16
2.3.4. Bacillus caldolyticus CCRC 11954 16
2.4. N-Carbamoylase基因的選殖、表現與純化 16
2.5. 酵素活性分析 17
2.6. 化學分析方法 17
2.6.1. 呈色法分析hydantoinase之活性 17
2.6.2. 薄層液相層析法分析N-carbamoylase之活性 18
2.6.3. 高效能液相層析法分析rac-HPAH、NCaHPA及HPA 18
2.7. 有機溶劑的作用 18
2.8. 以基底培養基(minimal medium)篩選耐熱L-hydantoinase生產菌 19
2.8.1. 篩選方法 19
2.8.2. Hydantoinase的部份純化 19
三、結果
3.1. Hydantoinases基因的選殖與表現 21
3.1.1. A. aurescens DSM 9771 21
3.1.2. M. jannaschii DSM 2661 21
3.2. Hydantoinase活性之分析 22
3.2.1. 利用HPLC法分析rac-HPAH, NCaHPA及HPA的建立 22
3.2.2. 呈色法測hydantoinase活性及TLC法測HPA的產生 23
3.2.3. Hydantoinase的立體異構物選擇性 24
3.3. N-Carbamoylase的立體異構物選擇性 24
3.4. Hydantoinase搭配N-carbamoylase生產L-HPA 25
3.5. 耐熱L-Hydantoinase生產菌之篩選 25
3.5.1. 菌種篩選 25
3.5.2. Hydantoinase的硫氨純化及活性分析 25
四、討論
4.1. rac-HPAH、NCaHPA及HPA的解析 27
4.2. Hydantoinase對立體異構物的選擇性 28
4.3. N-Carbamoylase活性分析 29
4.4. Hydantoinase搭配N-carbamoylase進行生物轉換 30
4.5. 結語 30
參考文獻 32
圖表 40
第二章:利用基因重組之Escherichia coli Aspartate Aminotransferase進行L-Homophenylalanine之不對稱合成
一、緒論
1.1. 利用aminotransferase產程製備L-HPA 62
1.2. 胺基供應者 63
1.3. L-Lysine-2-aminotransferase 63
1.4. Aminotransferase之分類 64
1.5. E. coli AAT 65
1.6. 2-Oxo-6-amino-hexanoic acid衍生物的製備 66
1.7. 研究動機 66
二、材料與方法
2.1. 化學藥品、酵素、培養基及菌株 68
2.2. 重組AAT之構築、表現與純化 68
2.2.1. E.快速製備coli之染色體 68
2.2.2. 重組AAT之構築 68
2.2.3. 重組AAT之表現與純化 69
2.3. 酵素活性分析 69
2.4. AAT之定點突變 69
2.5. 受質抑制作用之分析 70
2.6. 使用表現變異AAT基因之菌體生產L-HPA及N6-protecting-OAHA 70
2.7. 受質及產物之分析 70
三、結果
3.1. 野生型及變異AAT之活性分析 72
3.2. 受質抑制現象 72
3.3. 利用變異AAT合成L-HPA 73
3.4. P2C及OAHA的生成 73
3.5. 以N6-protecting-lysine當胺基供應者進行生物轉換 74
3.6. 利用大量表現變異AAT基因的菌體同步合成L-HPA及N6-protecting-OAHA 75
3.7. BOC-lysine、BOC-OAHA、TFA-lysine及TFA-OAHA的分析 75
四、討 論
4.1. 變異AAT對lysine的轉胺能力 76
4.2. AAT變異酵素對的N6-protecting-L-lysine活性 77
4.3. V39L變異酵素的活性 78
4.4. OPBA濃度對活性的影響 79
4.5. 利用基因重組E. coli 菌株當生物轉換劑 79
4.6. OAHA的環化 80
4.7. 結 論 80
五、參考文獻 81
圖表 85
總結 104
附錄
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