초록 ▼

파킨슨병 (Parkinson's Disease) 등의 노인성 신경장해에 대하여 탁월한 치료효과를 나타내는 의약용 아미노산인 L-DOPA (3,4-Dihydroxyphenylalanine)의 합성 및 생산기술을 개발하기 위해서는 L-DOPA 합성이 가능한 다기능효소인 β-Tyrosinase를 이용하는 것이 화학합성법보다 앞으로는 더욱 더 유리할 것이라고 판단된다. β-Tyrosinase를 이용한 L-DOPA의 합성법은 기존의 화학합성법의 제반 문제점의 해결 (D,L형의 Recemic DOPA로 부터 L-DOPA만을 제조하는 공정등이

파킨슨병 (Parkinson's Disease) 등의 노인성 신경장해에 대하여 탁월한 치료효과를 나타내는 의약용 아미노산인 L-DOPA (3,4-Dihydroxyphenylalanine)의 합성 및 생산기술을 개발하기 위해서는 L-DOPA 합성이 가능한 다기능효소인 β-Tyrosinase를 이용하는 것이 화학합성법보다 앞으로는 더욱 더 유리할 것이라고 판단된다. β-Tyrosinase를 이용한 L-DOPA의 합성법은 기존의 화학합성법의 제반 문제점의 해결 (D,L형의 Recemic DOPA로 부터 L-DOPA만을 제조하는 공정등이 필요없으므로 L-DOPA생산공정의 단순화가능)과 환경오염의 문제점을 해결할 수 있으므로, 무공해 합성 및 생산공정인 β-Tyrosinase를 이용한 L-DOPA 생산방법이 일본에서는 활발히 연구되고 있는 추세이다. 그리고, 아직 국내에서 L-DOPA를 생산하고 있지 않고 있으므로, 더욱더 국내에서는 β-Tyrosinase를 이용한 L-DOPA의 합성 및 생산법 개발이 더욱 절실히 요구된다. 따라서, 본 연구에서는 L-DOPA 합성효소인 β-Tyrosinase의 탐색, Gene Cloning, Sequence 결정 및 유전공학적 기법에 의한 재조합 β-Tyrosinase를 사용하여 기질인 Pyruvic Acid, Ammonia, Pyrocatechol로 부터 Product인 L-DOPA를 반응 8시간째 60mM까지 생산하는 L-DOPA 생산기술(Produdctivity 1.48gr/1/hr)을 개발하였다.

Abstract ▼

Studies on Synthesis and Production of L-3,4-dihydroxy phenylalanine by the Microbial Enzyme 3,4-Dihydroxyphenyl-L-alanine (L-DOPA) was first isolated by Guggenheim in 1913 from peels and buds of Vicia faba. This amino acid is contained in large amounts in brain organs, and has attracted much atte

Studies on Synthesis and Production of L-3,4-dihydroxy phenylalanine by the Microbial Enzyme 3,4-Dihydroxyphenyl-L-alanine (L-DOPA) was first isolated by Guggenheim in 1913 from peels and buds of Vicia faba. This amino acid is contained in large amounts in brain organs, and has attracted much attention as a specific drug for treatment of Parkinson''s disease. There are many reports concerning its production. L-DOPA has been obtained so far by extraction from vegetable organs or prepared by a synthetic method. However, the extraction method is not applied to large scale production and the synthetic method contains a step for optical resolution. The microbial production of this amino acid has been extensively studied. The yields in these microbial methods, however, are not high enough to be applied to industrial production. β-tyrosinase(tyrosine phenol-lyase, E.C.4.1.99.2) was found by Yamada and his group to catalyze the synthesis of L-DOPA from pyruvic acid, ammonia, and pyrocatechol in significantly high yields. This enzymatic method is simple and is one of the most economical processes to date for the preparation of L-DOPA. In this study, we described the screening of β-tyrosinase activity in mesophiles and thermophiles, gene cloning, and sequence determination of β-tyriosinase. We also established the method for the production of recombinant β-tyrosinase. Finally we studied the enzymatic synthesis and production method of L-DOPA by the recombinant β-tyrosinase. The results are as follows: 1. We have studied the occurrence of β-tyrosinase in mesophiles and ther mophiles, and found that most enteric bacteria including Citrobacter freundii (KCTC 2006, KCTC 2195, KCTC 2209, KCTC 2359, KCTC 2509), and Erwinia hebicola( KCTC 2104, KCTC 2479 ) had activity of β-tyrosinase. 2. Using direct phenol detection method, We isolated the gene encoding β-tyrosinase from Sau3AI partial digested fragments of Citrobacter freundii KCTC 2006 genomic DNA. A plasmid, pGY 1000 ( 8.7 kb) carring a 6.0 kb Sau3AI partial fragment thus obtained was subcloned into pGY1100(5.7 kb) carring a 3.0kb EcoRI-PvuⅡ fragment, which was found to contain the complete β-tyrosinase gene by restriction mapping and assay of β-tyrosinase. As the result of DNA sequence analysis of this 3.0kb EcoRI-Pvu II fragment, a single open reading frame of 1,368bp nucleotides starting with initiation codon ATG and ending with termination codon TAA at position 1,369 was found. On the basis of the predicted amino acid (456 residues), the molecular mass of the enzyme subunit is calculated to be 51,499 daltans. 3. In order to increase the expression level of β-tyrosinase, plasmids (pGY2000, pGY3000, and pGY4000) containing the coding sequence of β-tyrosinase under the control of trc promter was constructed. The expression of β-tyrosinase in E. coli/pGY2000, E. coli/pGY3000, and E.coli/pGY4000 were confirmed by the β-tyrosinase assay, and the production level of recombinant β-tyrosinase was more than 30 fold of Citrobacter freundii KCTC 2006. A band corresponding with recombinant β-tyrosinase (appximately M.W, 50,000) was observed on 12% SDS-polyacryl amide gel (PAGE), and the recombinat β-tyrosinase has been expressed more than 30% of total soluble proteins in recombinant E. coli 4. The synthesis of L-DOPA from pyruvic acid, ammonia, and pyrocatechol was studied with overproduced recombinant β-tyrosinase.The optimum pH for this reaction was around 8.0, and optimum temperature was 20 ℃. As the high concentration of substrates ( pyrocatechol and pyruvic acid) denatured the enzyme, we studied the optimum concentration of pyrocatechol and pyruvic acid. For L-DOPA synthesis, the optimum concentration of pyrocatechol and pyruvic acid was 40mM. Finally, we tried production of L-DOPA from pyruvic acid, ammonia, and pyrocatechol. The production of L-DOPA was carried out at 20℃ for 8 hrs. At 2 hrs intervals, pyrocatechol and pyruvate were added to maintain the initial substrate concentration (40 mM). The production level of L-DOPA in this reaction reached 60 mM in 8 hrs (productivity 1.48 g/l/hr ).

목차 Contents

• 제1장 서론...17
• 제2장 실험재료 및 방법...19
• 제1절 L-DOPA 합성효소 (β-tyrosinase)의 탐색...19
• 제2절 β-tyrosinase의 gene cloning 및 sequence 결정과 유전공학적 기법에 의한 대량생산...20
• 제3절 재조합 β-tyrosinase에 의한 L-DOPA의 합성 및 생산...22
• 제3장 결과 및 고찰...25
• 제1절 L-DOPA 합성효소 (β-tyrosinase)의 탐색...25
• 제2절 β-tyrosinase의 gene cloning 및 sequence 결정과 유전공학기법에 의한 대량 생산...26
• 제3절 재조합 β-tyrosinase에 의한 L-DOPA의 합성 및 생산...36
• 제4장 결론...41
• 참고문헌...43