• 공업화학
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• 제25권1호
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• pp.107-112
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• 2014

흑연가루의 결합재로 클로로술폰화 폴리에틸렌 고무용액을, 매개체로 ferrocene을 사용하여 장미조직을 고정한 과산화수소 정량 바이오센서를 제작하였다. 실험 전극전위 영역에서 보여준 Hanes-Woolf 도시의 선형성은 기질의 환원이 장미 과산화효소의 촉매력에 의한 것임을 보여 주었다. 또 얻어진 10개 이상의 전기화학 파라미터들은 전극이 정량적으로 성능을 발휘하고 있음을 입증하였다. 이런 사실들은 효소전극의 실용화를 위하여 장미조직이 상업용 과산화효소를 대치하여 사용될 수 있음을 확신시켜 주는 것이었다.

• Bulletin of the Korean Chemical Society
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• 제24권1호
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• pp.65-69
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• 2003

The kinetic and chemical mechanisms of AChE-catalyzed hydrolysis of short-chain thiocholine esters are relatively well documented. Up to propanoylthiocholine (PrTCh) the chemical mechanism is general acid-base catalysis by the active site catalytic triad. The chemical mechanism for the enzyme-catalyzed butyrylthiocholine(BuTCh) hydrolysis shifts to a parallel mechanism in which general base catalysis by E199 of direct water attack to the carbonyl carbon of the substrate. [Selwood, T., et al. J. Am. Chem. Soc. 1993, 115, 10477- 10482] The long chain thiocholine esters such as hexanoylthiocholine (HexTCh), heptanoylthiocholine (HepTCh), and octanoylthiocholine (OcTCh) are hydrolyzed by electric eel acetylcholinesterase (AChE). The kinetic parameters are determined to show that these compounds have a lower Michaelis constant than BuTCh and the pH-rate profile showed that the mechanism is similar to that of BuTCh hydrolysis. The solvent isotope effect and proton inventory of AChE-catalyzed hydrolysis of HexTCh showed that one proton transfer is involved in the transition state of the acylation stage. The relationship between the dipole moment and the Michaelis constant of the long chain thiocholine esters showed that the dipole moment is the most important factor for the binding of a substrate to the enzyme active site.

• 한국전기전자재료학회:학술대회논문집
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• 한국전기전자재료학회 2001년도 춘계학술대회 논문집 센서 박막재료
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• pp.6-10
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• 2001

We synthesized polypyrrole (PPy) nanotubules by oxidative polymerization of the pyrrole monomer on the pore of a polycarbonate membrane. The electrochemical behavior was investigated using cyclic voltammetry and AC impedance. The redox potential was about -0.5 V vs. Ag/AgCl reference electrode, while the potential was about 0 V for electro-synthesized PPy film. It is considered as the backbone grows according to the pore wall. Therefore, it is possible to be arranged regularly. That leads to improvement in the electron hopping. The AC impedance plot gave a hint of betterment of mass transport. PPy nanotubules have improved in mass transport, or diffusion. That is because the diffusion occurs through a thin pore wall of PPy nanotubules. The kinetic parameter of PPy nanotubules enzyme electrode with glucose solution was evaluated. The formal Michaelis constant and maximum current calculated by computer were about 23.8 mmol $dm^{-3}$ and $440\;{\mu}A$ respectively. Obviously, an affinity for the substrate and current response of the PPy nanotubules enzyme electrode are rather good, comparing with that of PPy film. What is more, the enzyme electrode is sensitive to blood sugar of a diabetic serum despite an obstruction of ascorbic acid, oxygen, some protein and/or hormone.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.445-449
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• 2003

As for the purpose, we first introduce an random mutation into wild-type gene to expand a mutation space, and then further recombine the mutant genes by staggered extension process PCR. As a result, we obtained the best clones 6-52 that showed a high activity and stability, from a round of error prone and staggered extension process PCR. The purified enzyme showed a similar pH stability to the wild-type enzyme and reveal a slightly high optimum pH at 12. In the optimum temperature, an identical dependency was also showed and a quite high stability in the thermal stability was obtained. Along with this, the enzyme was also stable at a reaction that supplement with a 15 % of ethanol as an additive. The addition of other solvents and surfactants did not improve the reaction and thus resulted in a similar profile to those of wild-type enzyme. The specific activity on the target compound rac-ketoprofen ethyl ester was calculated to be about 85, 000 unit, and the kinetic constants Km and Vmax were determined to be 0.2 mM and 90 mM/mg-protein/min respectively. The deduced amino acid alignment with the wild type enzyme revealed five mutations at L120P, I208V, T249A, D287H and T357A. Based on these observations, the site directed mutagenesis to delineate the mutagenic effect is under progress.

• BMB Reports
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• 제28권3호
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• pp.197-203
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• 1995

Famesyl protein transferase involved in the first step of post-translational modification of $p21^{ras}$ proteins transfers the famesyl moiety from famesyl pyrophosphate to a cysteine residue in $p21^{ras}$ proteins. The enzyme was first purified 30,000-fold from bovine testis by use of 30~50% ammonium sulfate fractionation, DEAE-Sephacel ion exchange chromatography, Sephacryl S-300 gel filtration chromatography, Sephacryl S-200 gel filtration chromatography, and hexapeptide (Lys-Lys-Cys-Val-Ile-Met) affinity chromatography. The molecular weight of the purified enzyme was estimated to be ~100 kDa by gel filtration and SDS-polyacrylamide gels showed two closely spaced bands of ~50 kDa protein. These indicate that the enzyme consists of two nonidentical subunits, a and 13, which have slightly different molecular weights. The enzyme was inhibited by hexapeptide (Lys-Lys-Cys-Val-Ile-Met), which acted as an alternative substrate that competed for famesylation. Kinetic analysis by measuring initial velocities showed that famesyl protein transferase is a very slow enzyme. EDTA-treated famesyl protein transferase showed little activity with $Mg^{2+}$ or $Zn^{2+}$ alone, but required both $Mg^{2+}$ and $Zn^{2+}$ for the catalytic activity.

• 한국미생물·생명공학회지
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• 제20권1호
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• pp.40-45
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• 1992

Aspergillus niger로부터 acidic nucleotidase를 Sepharose CL-6B gel 여과와 DEAE-Sephacel 이온교환수지를 이용하여 부분정제하였다. 5'-AMP 와 3'-AMP를 기질로 사용했을 경우에 효소의 최적 pH는 4.5, 그리고 최적온도는 $55^{\circ}C$였다. 그러나, p-nitrophenyl phosphate를 기질로 사용했을 경우에는 최적 pH는 변화가 없었으나, 최적 온도는 $70^{\circ}C$였다. 효소의 활성화에너지는 3'-AMP, 5'-AMP 그리고 p-nitrophenyl phosphate를 기질로 사용했을 경우에 각각 4.76kcal/mole, 6.95kcal/mole 그리고 11.82kcal/mole 였다.

• Journal of Microbiology and Biotechnology
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• 제9권3호
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• pp.249-254
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• 1999

A catechol 2,3-dioxygenase (C23O) was purified to apparent homogeneity from Pseudomonas putida SU10 through several purification steps consisting of ammonium sulfate precipitation and chromatographies on DEAE 5PW, Superdex S-200, and Resource-Q. Gel filtration indicated a molecular mass under nondenaturing conditions of about 130 kDa. The enzyme has a subunit of 34 kDa as was determined by SDS-PAGE. These results suggest that the native enzyme is composed of four identical subunits. The N-terminal amino acid sequence (30 residues) of the enzyme has been determined and exhibits high identity with other extradiol dioxygenases. The reactivity of this enzyme towards catechol and methyl-substituted catechols is somewhat different from that seen for other catechol 2,3-dioxygenases, with 4-methylcatechol cleaved at a higher rate than catechol or 3-methylcatechol. $K_m$ values of the enzyme for these substrates are between 3.5 and 5.7 M.

• Bulletin of the Korean Chemical Society
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• 제6권5호
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• pp.312-317
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• 1985

${\beta}$-Glucuronidase (EC 3.2.1.31) which hydrolizes D-glucuronate from ${\beta}$-D-glucuronide was purified from rat liver, using ammonium sulfate fractionation, DEAE-cellulose chromatography, Concanavalin-A Sepharose 4B chromatography and gel filtration on Sephadex G-200. This enzyme has the molecular weight of 280,000 daltons by gel filtration and 75,000 daltons by SDS-polyacrylamide gel electrophoresis. As its funtion is reverse of detoxification in the liver, the inhibition of the enzyme was tested with extracts of several food products and medicinal herbs, some are known as anti-cancer agents. Among them, Panax ginseng and Cortnellus shiiake inhibited the enzyme competitively and the $K_1$ values were $9.22 {\times}\;10^{-2}$ and 0.102 mg/ml, respectively. These inhibitors strongly bound to DEAE-cellulose. The negatively charged amino acids, L-aspartate and L-glutamate, inhibited the enzyme, and $K_1$ value of L-aspartate was 0.80 mM. The interaction between ${\beta}$-glucuronidase and p-nitrophenyl-${\beta}$-D-glucuronide was found to involve ionic forces by the effect of ionic strength on the kinetic constant, Vmax/Km. It was inferred from these findings that cationic group at the active center of the enzyme is probably involved in attacking the substrate.

• Journal of Microbiology and Biotechnology
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• 제24권12호
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• pp.1636-1643
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• 2014

3-Hydroxybutyryl-CoA dehydrogenase is an enzyme that catalyzes the second step in the biosynthesis of n-butanol from acetyl-CoA, in which acetoacetyl-CoA is reduced to 3-hydroxybutyryl-CoA. To understand the molecular mechanisms of n-butanol biosynthesis, we determined the crystal structure of 3-hydroxybutyryl-CoA dehydrogenase from Clostridium butyricum (CbHBD). The monomer structure of CbHBD exhibits a two-domain topology, with N- and C-terminal domains, and the dimerization of the enzyme was mostly constituted at the C-terminal domain. The mode of cofactor binding to CbHBD was elucidated by determining the crystal structure of the enzyme in complex with $NAD^+$. We also determined the enzyme's structure in complex with its acetoacetyl-CoA substrate, revealing that the adenosine diphosphate moiety was not highly stabilized compared with the remainder of the acetoacetyl-CoA molecule. Using this structural information, we performed a series of site-directed mutagenesis experiments on the enzyme, such as changing residues located near the substrate-binding site, and finally developed a highly efficient CbHBD K50A/K54A/L232Y triple mutant enzyme that exhibited approximately 5-fold higher enzyme activity than did the wild type. The increased enzyme activity of the mutant was confirmed by enzyme kinetic measurements. The highly efficient mutant enzyme should be useful for increasing the production rate of n-butanol.

• 한국식품과학회지
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• 제16권3호
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• pp.267-272
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• 1984

미생물 세포를 효소 고정화 담체로 사용하여 ${\beta}-fructofuranosidase$를 고정화 시켰다. Penicillium spp. "K-8"로 명명된 곰팡이를 배양한 뒤 균체의 세포벽에 존재하는 다당체를 periodate와 반응시켜 활성화된 알데히드기를 얻을 수 있었다. 이 때 건조 세포 g당 periodate, 1.2g이 최적 농도이었고, 이 농도하에서 온도가 알데히드 형성에 미치는 영향은 거의 없었다. 활성화된 균체에 ${\beta}-fructofuranosidase$를 공유결합에 의하여 고정화시켰다. 더 높은 효소의 고정화를 위하여 이를 glutaraldehyde를 처리한 바 0.5%의 농도와 1시간의 반응조건에서 최대 효소 고정화율 26%를 나타내었다. 이 조건에 의해 제조한 고정화 효소는 kinetic parameters로서 최적온도가 $55^{\circ}C$, 최적 pH가 5, Km값이 55mM. Ea가 19kJ $mol^{-1}$이었다. 회분식 반응조내에서는 6번의 반복된 반응기간동안 고정화 효소 활성에 약간의 감소가 있었으나 비교적 좋은 역가의 안정성을 보여주었다.