• 대한화학회지
• /
• 제53권5호
• /
• pp.499-504
• /
• 2009

금속은 생명현상에 필수적인 효소반응에서 핵심적인 역할을 수행한다. 따라서 금속효소 안에서 일어나는 반응에 대한 구체적인 메커니즘은 많은 과학자의 오랜 관심사였다. Methane MonoOxygenase (MMO)는 메탄을 메탄올로 산화시키는 반응을 일으키는 효소이며 최근 Tolman등은 MMO의 모델로서 Bis(μ-oxo)dicopper Complexes의 Hydroxide Transfer 반응에 대한 몇 가지 가능한 메커니즘을 제시하였다. 이후 Hydrogen Transfer와 Hydroxide Rebound의 2단계로 이루어지는 메커니즘이 이론적으로 제시 되었다. 본 연구에서는 Bis($\mu$-oxo)dicopper Complexes 의 반응단계 중 첫 번째 단계인 Hydrogen Transfer 반응과정의 반응물과 전이상태, 생성물의 구조를 최근에 개발된 M06계열의 M06, M06L, M06-2X를 비롯한 여러 가지 DFT방법을 이용하여 계산하였다. M06/6-31G(d,p)/LANL2DZ방법을 사용하여 계산된 반응물의 구조가 실험에서 얻은 XRD구조와 가장 잘 일치했으며 기저함수의 크기에 따라 전이상태의 구조, 활성화 에너지 및 반응에너지에 큰 차이를 보였다.

• 한국미생물·생명공학회지
• /
• 제24권2호
• /
• pp.222-226
• /
• 1996

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% by solubilizing and recrystallyzing the precipitates.

• 한국어병학회지
• /
• 제21권3호
• /
• pp.181-188
• /
• 2008

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was used to detect and identify four fish viruses, fish iridovirus, viral hemorrhagic septicaemia virus (VHSV), viral nervous necrosis virus (VNNV), hirame rhabdovirus (HRV). Four viruses were detected by PCR with each specific primers. Identification of iridovirus was achieved by digesting the PCR amplified fragment with a restriction enzyme ApaⅠ. It was possible to distinguish positive from false positive PCR amplicons of VHSV by RFLP of PstⅠ or HindⅢ restriction enzymes. VNNV was identified using RFLP of BamHⅠrestriction enzyme and HRV was identified by XbaⅠ restriction enzyme. This approach can be used for more rapid, simple and specific diagnosis of fish viral diseases.

• 한국환경보건학회지
• /
• 제30권2호
• /
• pp.79-83
• /
• 2004

The oxidative degradation of BPA with laccase from Trametes versiclor was conducted in a closed, temperature controlled system containing acetate buffer for pH control. The effects of medium pH, buffer concentration, temperature and mediator on degradation of BPA were investigated. The inactivation of the enzyme by temperature and reaction product was also studied. The optimal pH for BPA degradation showed about 5. Buffer concentration did not affect BPA degradation. On the other hand, the enzyme stability was higher at low concentration buffer(25 mM). Temperature rise increased the degradation rate of BPA up to 45$^{\circ}C$. The valuable mediator of laccase for BPA was ABTS. Elevated temperature and reaction product irreversibly inactivated the enzyme.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제8권4호
• /
• pp.257-262
• /
• 2003

A straightforward and effective method is presented for immobilizing enzymes on a microchip platform without chemically modifying a micro-channel or technically microfabricating a column reactor and fluid channel network. The proposed method consists of three steps: the reconstitution of a nitrocellulose (NC) membrane on a plane substrate without a channel network, enzyme immobilization on the NC membrane, and the assembly of another substrate with a fabricated channel network. As a result, enzymes can be stably and efficiently immobilized on a microchip. To evaluate the proposed method, two kinds of enzymatic reaction are applied: a sequential two-step reaction by one enzyme, alkaline phosphatase, and a coupled reaction by two enzymes, glucose oxidase and peroxidase, for a glucose assay.

• Journal of Microbiology and Biotechnology
• /
• 제15권3호
• /
• pp.573-578
• /
• 2005

Pyrimidine nucleotide N-ribosidase (pyrimidine 5'-nucleotide phosphoribo(deoxyribo)hydrolase/pyrimidine 5'-nucleotide nucleosidase, EC 3.2.2.10) catalyzes the breakdown of pyrimidine 5'-nucleotide into pyrimidine base and ribose(deoxyribo)-5-phosphate. However, detailed characteristics of the enzyme have not yet been reported. The enzyme was purified to homogeneity 327.9-fold with an overall yield of $6.1\%$ from Pseudomonas oleovorans ATCC 8062. The enzyme catalyzed cytidine monophosphate (CMP) and uridine monophosphate (UMP), but not adenosine monophosphate (AMP) and guanosine monophosphate (GMP). The enzyme optimally metabolized CMP at pH 6.0 and UMP at around 8.5, and the optimum temperature for the overall enzyme reaction was found to be $37^{\circ}C$. The $K_m$ values of the enzyme for CMP (at pH 6.0) and UMP (at pH 8.5) were 1.6 mM and 1.1 mM, respectively. AMP, deoxyCMP, and deoxyUMP were very effective inhibitors of the reaction. Double-reciprocal plots obtained in the absence and in the presence of AMP revealed that this inhibitory effect was of the mixed competitive type with respect to the breakdown of CMP and of the noncompetitive type with respect to the breakdown of UMP. In the presence of AMP, the enzyme followed sigmoid kinetics with respect to each substrate.

• Applied Biological Chemistry
• /
• 제11권
• /
• pp.55-61
• /
• 1969

본(本) 연구(硏究)에서 사용(使用)한 K-17 균주(菌株)의 효소생성(酵素生成)및 그의 금속염(金屬鹽) 등(等)에 대(對)한 영향(影響)등(等)을 요약(要約)하면 대략(大略) 다음과 같다. 1. 본(本) 균주(菌株)는 Glucose Isomerizing Enzyme 생성(生成)에 있어서 Xylose를 절대적(絶對的)으로 요구(要求)하지 않는다. 2. 본(本) 효소(酵素)는 Free Incubation 상태(狀態)에서는 $75^{\circ}C$, 30시간(時間)의 가열(加熱)로서 대부분(大部分)의 효소(酵素)가 실활(失活)된다. 3. 금속(金屬) Ion 중(中) $Mg^{++}$는 효소작용(酵素作用)에 있어 일종(一種) 의 Activator로 작용(作用)하며 $Co^{++}$는 Activator로 서의 작용(作用)은 없고 효소(酵素)의 열(熱) 실활(失活)을 보호(保護)하는 작용(作用)을 나타낸다. 4. 본(本) 효소작용(酵素作用)에 있어서 Glucose의 용매(溶媒)로서는 Phosphate Buffer가 가장 우수(優秀)한 결과(結果)를 나타내었다. 5. 본(本) 균주(菌株)의 효소(酵素) 생성(生成) 배지(培地)로서는 탄소원(炭素源)으로서 주어지는 당류(糖類)의 혼용(混用)이 단용(單用)에 비(比)해 균체(菌體)의 수량(收量)이나 효소능(酵素能)이 다같이 높아짐을 알게 되었다.

• Journal of Biosystems Engineering
• /
• 제32권6호
• /
• pp.455-461
• /
• 2007

The electric current of a flow injection type enzyme-sensor was measured to confirm the stable operating conditions of the sensor. The current of the sensor was decreased as the buffer solution velocity increased. Under the limitation of the cycle time to be below 10 minutes, the effective ranges of the buffer solution velocity were suggested $0.10{\sim}0.26$, $0.12{\sim}0.24$, $0.1{\sim}0.25$ and $0.05{\sim}0.10\;cm/s$ of 1.0, 1.4, 2.4 and 3.4 mm of the electrode diameters, respectively. As the reaction time of the enzyme and the substrate was increased, the current was decreased because of the dilution between the sample and buffer solution. Therefore, it could be recommended that the reaction time was able to be selected as shortly as possible in consideration of the total cycle time. As the result of the experiments using a different volume ratio of the enzyme to substrate, it was concluded that the substrate had to be mixed with the same amount of the enzyme. The current have increased remarkably in proportion to the electrode diameter under 0.1 cm/s of the buffer solution velocity but there was no difference over 0.1 cm/s of the buffer solution velocity. The cross type arrangement of the electrode was highly suggested for application and machining of the sensor.

• 한국식품영양과학회지
• /
• 제24권6호
• /
• pp.943-947
• /
• 1995

고구마의 효율적 활용을 위하여 고구마가 지니 여러가지 가능성을 이용한 새로운 가공식품인 음료를 물 첨가량과 효소처리 유무에 따라 제조하고 그 음료의 품질특성을 조사하였다. 환원당은 효소처리를 하므로서 증가하였으며 pH 조절시에도 다소 증가하였으나 큰 차는 나타나지 않았다. 점도는 각 처리구 마다 효소처리되었을 때 낮아졌으며 효소처리(pH조절)에서는 더 낮아졌다. 또한 색도는 각 처리구에서 효소 무처리구 보다 효소처리구에서 색의 밝기와 황색도는 낮아졌으나 적색도는 고구마 1 : 물 1에서는 낮아졌고 고구마 1 : 물 2, 3에서는 증가하였다. 관능검사 결과는 색, 맛, 향기, 조직감 모두 고구마 1 : 물 1로 섞은 시료에 pH를 4.5로 조절하고 효소 처리하여 제조한 음료가 가장 좋은 것으로 나타났으며 고구마 1 : 물 3으로 하여 제조한 음료에서는 모두 가장 나쁜 것으로 나타났다. 이상의 결과는 고구마 1 : 물 1의 비율액을 pH 4.5로 조절하고 $60^{\circ}C$에서 복합효소로 처리하여 만든 음료가 품질면에서 우수한 것으로 나타났다.

• Journal of Microbiology and Biotechnology
• /
• 제12권2호
• /
• pp.189-195
• /
• 2002

Using Streptomyces sp. YU100 isolated from Korean soil, the fermentative production of phospholipase D was attempted along with its purification and characterization studies. When different carbon and nitrogen sources were supplemented in the culture medium, glucose and yeast extract were found to be the best. By varying the concentration of nutrients and calcium carbonate, the optimal culture medium was determined as 2.0% glucose, 1.5% yeast extract, 0.5% tryptone 0.3% calcium carbonate. During cultivation, the strain secreted most of the phospholipase D in the early stage of growth within 24 h. The phospholipase D produced in the culture broth exhibited hydrolytic activity as well as transphosphatidylation activity on lecithin (phosphatidylcholine). In particular, the culture broth showed 8.7 units/ml of hydrolytic activity when cultivated at $28^{\circ}C$ for 1.5 days. The phospholipase D was purified using 80% ammonium sulfate precipitation and DEAE-Sepharose CL-6B column chromatography, which produced a major band of 57 kDa on a 10% SDS-polyacrylamide gel with purity higher than 80%. The enzyme showed an optimal pH of 7 in hydrolytic reaction, and at pH 4 in a transphosphatidylation reaction. The enzyme activity increased until the reaction temperature was elevated to $60^{\circ}C$. The enzyme was relatively stable at high temperatures and neutral pH, but significantly unstable in the alkaline range. Among the detergents tested as emulsifiers of phospholipids, the highest enzyme activity was observed when 1.5% Triton X-100 was employed. However, no inhibitory effect by metal ions was detected. Under optimized reaction conditions, the purified enzyme not only completely decomposed PC to phosphatidic acid within 1 h, but also exhibited higher than 80% conversion rate of PC to PS by transphosphatidylation within 4 h.