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3,4-Dihydroxyphenyl-L-alanine의 효소적 생산에 대한 반응첨가물의 영향
이승구,노현수,홍승표,성문희 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.2
재조합 대장균에서 과발현된 Citrobacter freundii KCTC 2006 유래의 tyrosine phenol-lyase(E.C.4.1.99.2) 이용 3,4-dihydroxyphenyl-L-alanine (L-DOPA)의 생산에 대한 반응첨가물의 영향을 조사하였다. 반응액 중 효소 및 조효소 농도의 영향을 조사한 결과, 효소농도는 약 2 units/ml이 적합하였으며, 조효소인 pyridoxal -5-phosphate 는 0.1 mM 이상이 필요하였다. Pyrocatechol과 결합하여 안정한 복합체를 형성하는 sodium borate는 pyrocatechol에 의한 효소의 불활성화를 감소시키는 효과가 있었으나, 효소의 반응성을 현저히 저하시켜 L-DOPA 생산의 관점에서는 불리한 단점도 있었다. 한편, 알콜류 등의 유기용매가 L-DOPA 합성반응에 미치는 영향을 조사한 결과 methanol을 5% 농도로 반응액에 첨가하였을 때, 효소의 반응과 안정성이 크게 증가하여 L-DOPA 합성반응이 지속적으로 수행됨으로써 고농도의 L-DOPA를 효율적으로 생산할 수 있게 되었다. 생산된 L-DOPA의 약 77%가 불용성 상태로 침전되어 쉽게 회수할 수 있었으며, 침전된 L-DOPA를 1N HCI에 용해한 후 재결정화 함으로써 최종적으로 99.96%의 고순도 L-DOPA를 생산할 수 있었다. The enzymatic synthesis of 3,4-dihydroxyphenyl-L-alanine (L-DOPA) was examined for the effects of the reaction additives such as sodium borate, alcohol, and organic solvents. The enzyme used was tyrosine phenol-lyase of Citrobacter freundii KCTC 2006 produced in Escherichia coli. The amounts of tyrosine phenol-lyase and pyridoxal -5-phosphate were optimized to 2.0 units/ml and 0.1 mM, respectively, for the synthetic reaction. Sodium borate, a substance that forms a complex with pyrocatechol, reduced the enzyme deactivation by pyrocatechol although it seriously inhibited the enzyme activity. Among the organic solvents tested, dimethylsulfoxide, dimethylformamide, and alcohol increased the productivity of the L-DOPA synthesis. In a reaction system with 5% methanol, L-DOPA concentration increased up to 210 mM after 24 hours, and 77.1% of which was separated as precipitates. The L-DOPA was purified to 99.96%
제조합 대장균에서 과발현된 Citrobacter freundii KCTC2006 유래의 β-Tyrosinase를 이용한 3,4-Dihydroxyphenyl-L-alanine의 생산
이승구,노현수,홍승표,이규종,왕지원,태동년,엄기남,방상구,김영준,성문희 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.1
재조합 대장균에서 대량발현 시킨 Citrobacter freundii KCTC 2006 유래이 효소 β-tyrosinase를 이용하여 pyrocatechol, sodium pyruvate, ammonium acetate로부터 3,4-dihydroxy phenyl-L-alanine을 생산하기 위한 연구를 수행하였다. 이 효소반응에 적합한 온도 및 pH 조건은 각각 18℃와 8.5로 결정되었고, 반응액 중의 ammonium acetate와 sodium pyruvate의 농도는 각각 300 mM, 50 mM 이상으로 조절하는 것이 적합하였다. Pyrocatechol의 경우는 20 mM에서 가장 높은 반응성을 나타냈으나, 기질을 반복적으로 첨가하며 장시간 동안 효소반응을 수행하는 경우에는 pyrocatechol의 고갈을 피하기 위하여, 20 mM에서 50 mM 사이로 조절하였다. 한편, 반응액 중에 ethanol을 10% 첨가한 경우에는 반응속도가약 20% 증가하였다. 이상과 같은 효소반응특성에 기초하여 조제한 기질용액에 β-tyrosinase를 1 unit/㎖ 농도로 가하고, pyrocatechol과 pyruvate가 고갈되지 않도록 간헐적으로 첨가하면서 효소반응을 수행한 결과, 24시간 만에 85.2%의 수율로 31.6g/ℓ의 3,4-dihydroxyphenyl-L-alanine를 생산할 수 있었다. By using the β-tyrosinase of Citrobacter freundii KCTC2006, which was cloned and overexpressed in Escherichia coli, 3,4-dihydroxy phenyl-L-alanine (L-DOPA) was synthesized efficiently from pyrocatechol, sodium pyruvate, and ammonium acetate. Optimal temperature and pH for the reaction were determined to be about 18℃ and 8.5, respectively. The effects of substrate concentrations were also examined at different concentrations of ammonium acetate, sodium pyruvate, and pyrocatechol. Ammonium acetate and sodium pyruvate increased the reaction rate until the concentrations reached to 300 mM and 50 mM, respectively. Although pyrocatechol showed the optimal concentration at 20 mM, it was controlled between 20 mM and 50 mM to avoid the depletion of substrate during the enzymatic synthesis. Based on above results, a reaction medium for the production of L-DOPA was prepared and incubated with 1 unit/㎖ of β-tyrosinase. Pyrocatechol and sodium pyruvate was added to the reaction solution intermittently to avoid the substrate depletion during the enzymatic reaction. After 24 hour of reaction, 31.6 g/ℓ L-DOPA was accumulated in the reaction solution as soluble and precipitated ones and the conversion yield was about 85.2%.
분뇨처리장의 공정개선에 의한 처리효율 향상에 관한 연구
이찬원,김승현,김창수,문성원,전홍표,윤종섭 한국환경과학회 2004 한국환경과학회지 Vol.13 No.6
There is a need to improve the efficiency of the existing sanitary treatment facilities, because the effluent standard becomes more stricter and septic sludge increased. Thus, operating processes of sanitary treatment system in M city changed with installation of additional facilities. Process modifications were as follows: Dilution water was added to the next process after primary aeration tank. Some secondary sedimentation sludge was recycled to primary aerator so that most of the organics were stabilized in primary aeration tank under automatic control of dissolved oxygen. The line of effluent from dewatering process flowing to the activated sludge tank was changed to the primary aerator. The primary sedimentation sludge line was linked to a thickener. Polymer was added to the activated sludge tank. The effluent of primary aerator and aerobic digester was recycled from the 5th to the 1st sector. As consequencies of above process modifications, the improvement of removal efficiency was achieved as BOD 54%, COD 42%, SS 61%, T-N 39%, and T-P 12%, respectively.
정용훈,이일한,홍승표,성문희,홍순광 명지대학교 자연과학연구소 2004 자연과학논문집 Vol.23 No.-
방선균의 유전자는 그 구조의 특이성으로 외래 숙주에서는 발현이 거의 되지 않아 단백질 발현에 많은 어려움이 있다. 지금까지 다양한 벡터들이 방선균에서의 발현을 목적으로 개발되어 사용하여 왔지만, 이들은 유전자를 발현시키고자 할 때마다 발현 유전자의 특성에 맞게 벡터 및 유전자를 제조합시켜야 하는 불편함이 있었다. 따라서 본 연구에서는 방선균에서 쉽게 사용할 수 있는 두 가지의 초고속 발현 백터를 제작하였다. 제작된 pFSE와 pFSE-extra 벡터는 방선균에서 replication이 가능한 pIJ101 replication origin과 selection marker인 tsr(thiostrepton) 유전자를 가지고 있으며, 대장균에서도 replication이 가능한 shuttle vector 이다. 두 pFSE 벡터는 방선균에서 강력히 발현되는 강력 promoter ermEp를 가지고 있으며, pFSE-extra 벡터는 Streptomyces griseus에서 분비되는 단백질을 코드하는 유전자 sghC의 promoter 및 분비시그널을 포함하는 영역으로 구성되어 있어, 각각 하부에 연결된 유전자의 발현을 세포내에서 또는 세포 밖으로 분비하는 특성을 가지고 있다. 두 벡터의 발현시스템에는 AspEI으로 절단되는 제한 부위를 가지고 있으며, 효소로 절단 시 간단히 T-vector로 이요할 수 있는 초고속 발현 시스템이다. The genes of streptomycetes are hardly expressed in other host systems because of their unique structure. Many expression systems for the streptomycetes genes have been developed, yet modification of vector and target genes is the essential prerequisite, which make a limit in their use. To make expression easier in efficient and fast way, two kinds of expression systems, named pFSE and pFSE-Extra, were constructed, Both plasmids have the repication origins from pIJ101 and pBlescript and can be used as Streptomyces-E. coli shuttle vectors. The plasmid pFSE has the strong promoter derived from ermE and designed for the intracellular expression of streptomycetes genes in Streptomyces host system. pFSE-extra has the promoter region including the secretion signal originated from the sghC gene that was identified to encode massively secreted protein from Streptomyces griseus and can be used for the secretion of the expressed protein in Streptomyces also. Both plasmids can be used as T-vector cloning system after digestion with AspEI restriction enzyme, which make their application broader as the fast expression system in Streptomyces.
한국 주요정신장애의 유병률 및 관련요인 : 2006 전국정신질환역학조사
조맹제,장성만,함봉진,정인원,배안,이영문,안준호,원승희,손정우,홍진표,배재남,이동우,조성진,박종익,이준영,김진영,전홍진,이해우 大韓神經精神醫學會 2009 신경정신의학 Vol.48 No.3
Objectives The aims of this study are to estimate the prevalence of the DSM-IV psychiatric disorders in the Korean population using the Korean version of Composite International Diagnostic Interview (K-CIDI), and to compare those with previous studies. Methods The Korean Epidemiologic Catchment Area study Replication (KECA-R) was Conducted between August 2006 and April 2007. The sampling of the subjects was carried out across 12 catchment areas. A multistage, cluster sampling design was adopted. The target Population included all eligible residents aged 181o 64 years. Face-to-face interviews were conducted with the Korean version of Composite International Diagnostic Interview (K-CIDI) based on the DSM-IV (N=6,510, response rate=81.7%). Results A total of 6,510 participants completed the interview. The lifetime and 12-month prevalence rates for all types of DSM-IV disorders were 30.0% and 17.3%, respectively. Those of Specific disorders were as follows : 1) alcohol use disorder, 16.2% and 5.6% ; 2) nicotine use disorder, 9.0% and 6.0% ; 3) specific phobia, 3.8% and 3.4% ; 4) major depressive disorder, 5.6% and 2.5% ; and 5) generalized anxiety disorder, 1.6% and 0.8%. Data relating to nicotine and alcohol use disorder revealed a very high male/female ratio. Mood disorder and anxiety disorder were more prevalent among females than males. Conclusion The prevalence of psychiatric disorders was high. In comparison with other studies, remarkable differences in the distribution of psychiatric disorders across the country and times were observed.