• 한국미생물·생명공학회지
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• 제20권2호
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• pp.143-149
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• 1992

호화전분이 포함된 김치로부터 유산을 생성하는 6균주를 분리하였으며, 분리된 균주는 soluble starch가 포함된 APT 액체배지에서 분리균주의 생육과 $\alpha$-glucosidase 활력이 우수한 No.2 균주를 선별하였다. 이 분리균은 Pediococcus halophilus 또는 그 유연균으로 동정되었다. 효소의 정제는 protamine sulfate에 의한 핵산의제거, ammonium sulfate 분획, gel filtration 및 ion exchange 등의 4단계 정제과정을 거친 결과 20.17배 정제되어 단일 band 효소로 분리되었다.

• Animal cells and systems
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• 제1권4호
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• pp.583-587
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• 1997

Incubation of an NADPH-dependent succinic semialdehyde reductase from bovine brain with N-bromosuccinimide (NBS) resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo-first-order kinetics with the second-order rate constant of $6.8\times{10}^3$ $M^-1$ $min^{-1}$. The inactivation was prevented by preincubation of the enzyme with substrate succinic semialdehyde, but not with coenzyme NADPH. There was a linear relation-ship between oxindole formation and the loss of enzyme activity. Spectro-photometric studies indicated that about one oxindole group per molecule of the enzyme was formed following complete loss of enzymatic activity. It is suggested that the catalytic function of succinic semialdehyde reductase is modulated by binding of NBS to a specific tryptophan residue at or near the substrate binding site of the enzyme.

• Journal of Microbiology and Biotechnology
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• 제7권1호
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• pp.37-42
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• 1997

A bacterial strain L1 producing a thermostable esterase was isolated from soil taken near a hot spring and identified as Bacillus stearothermophilus by its microbiological properties. The isolated thermostable esterase was purified by ammonium sulfate fractionation, ion .exchange and hydrophobic interaction chromatographies. The molecular weight of the purified enzyme was estimated to be 50,000 by SDS-PAGE. Its optimum temperature and pH for hydrolytic activity against PNP caprylate were $85^{\circ}C$ and 9.0, respectively. The purified enzyme was stable up to $70^{\circ}C$ and at a broad pH range of 4.0-11.5 in the presence of bovine serum albumin. The enzyme was inhibited by phenylmethylsulfonyl fluoride and diethyl p-nitrophenyl phosphate, indicating the enzyme is a serine esterase. The enzyme obeyed Michaelis-Menten kinetics in the hydrolysis of PNPEs and had maximum activity for PNP caproate ($C_6$) among PNPEs ($C_2-C_12$) tested.

• 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.

• 한국미생물·생명공학회지
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• 제9권3호
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• pp.165-171
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• 1981

E. coil ATCC 9637의 균체를 젤라틴과 DEAE-cellulose의 혼합 성형 후 글루트알데히드 가교법으로 제조론 고정화 penicillin amidase의 differential column reactor에서의 반응속도를 논의하였다. 이러한 반응조의 최적 조작조건은 효소충진량 1g, 기질농도30mM(0.1M 인산완충액, pH8.0), 유출속도 4 $m\ell$/min, 온도 4$0^{\circ}C$이었다. 이 최적조건에서 고정화효소의 일반적인 성질을 조사하였다. Km 상수는 4.8mM 이었고 specific activity 308 units/g 고정화 효소이 었다. 또한 고정화효소에서는 기질에 의한 효소반응 저해효과가 보이지 않았다. 이러한 differential column reactor에서는 column내에서의 pH 감소효과 및 외부 화산효과가 없어지기 때문에 이러한 외부적 영향을 받지 않는 고정화효소의 반응 속도론적 연구에 적합함을 알았다.

• 산업식품공학
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• 제15권3호
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• pp.214-220
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• 2011

중고압 하에서 $\beta$-glucosidase효소반응을 물리화학적 관점에서 연구하였다. 모델 기질 (p-nitrophenyl-${\beta}$-D-glucopyranoside)에 대한 $\beta$-glucosidase 효소의 작용에 대한 압력 효과를 실험 하였다. 즉, 압력 조건(25MPa, 50 MPa, 75 MPa, 100 MPa)과 시간 (10분, 60분, 1시간, 6시간, 24시간, 40시간)의 처리 조건에서 효소 활성도를 분광학적인 표준방법에 따라 측정하였다. 효소-기질 반응의 단계를 크게 kinetic 구간과 평형 구간으로 구분하여 물리화학적 모델을 적용하여, 정 역반응속도 상수, 평형상수, 압력에 의한 부피 감소 등을 산출하였다. 대기압에서 100MPa까지 압력이 증가할수록 효소-기질 반응의 생성물이 더 많이 형성되었으며 전형적인 kinetic 구간과 평형 구간이 나타났다. 압력, 시간, 생성물농도 등의 데이터로부터 kinetic 구간과 평형에서의 생성물 예측 모델을 완성하였다. 결론적으로 중고압 처리에 의하여 효소-기질 반응이 촉진됨을 알 수 있었고, 임의의 압력 및 시간 조건에 따른 생성물의 농도를 예측할 수 있게 되었다.

• BMB Reports
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• 제28권1호
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• pp.72-78
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• 1995

Time-dependent inactivation of E. coli GTP cyclohydrolase I with a 2',3'-dialdehyde derivative of GTP (oGTP) was directed to the active site of the enzyme, and was dependent on the concentration of oGTP. The kinetics of inactivation were biphasic with a rapid reaction occurring immediately upon exposure of the enzyme to oGTP followed by a slow rate of inactivation. The $K_i$ value of oGTP for the enzyme was 0.25 mM. Inactivation was prevented by preincubation of the enzyme with GTP, the substrate of the enzyme. At 100% inactivation, 2.3 mol of [8.5'-$^3H$]oGTP were bound per each enzyme subunit, which consists of two identical polypeptides. The active site residue which reacted with the affinity label was lysine. oGTP interacted selectively with the ${\varepsilon}$-amino group of lysine in the GTP-binding site to form a morpholine-like structure which was stable without sodium borohydride treatment. However, triphosphate group was eliminated during the hydrolysis step. To identify the active site of the enzyme, [8.5'-$^3H$]oGTP-labeled enzyme was cleaved by endoproteinase Lys-C, and the $^3H$-labeled peptide was purified by HPLC. The amino acid sequence of the active site peptide was Pro-Ser-Leu-Ser-Lys, which corresponds to the aminoterminal sequence of the enzyme.

• Food Science and Biotechnology
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• 제17권4호
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• pp.766-771
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• 2008

The proteolytic enzyme from Saccharomyces sp. B101 was purified to homogeneity by ammonium sulfate fractionation, ultrafiltration, diethyl aminoethyl (DEAE)-Sephadex A-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography from the culture supernatant of Saccharomyces sp. B101. The specific activity and the purification fold of the purified enzyme were 4,688.9 unit/mg and 18, respectively. The molecular weight of the purified enzyme was estimated to be 33 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature for the enzyme activity were pH 8.5 and $30^{\circ}C$, respectively. The enzyme activity was relatively stable in the pH range of 6.5-8.5 at below $35^{\circ}C$. The salt-tolerance and stability for the enzyme activity were relatively stable even at NaCl concentrations of 10 and 15%. The activity of enzyme was inhibited by $Ag^{2+}$ and $Fe^{2+}$, and activated by $Mn^{2+}$. In addition, the enzyme activity was potently inhibited by ethylenediaminetetraacetic acid (EDTA) and phenylmethyl sulfonylfluoride (PMSF). Based on these findings we concluded that the purified enzyme was a serine protease. Km and Vmax values for hammastein milk casein were 1.02 mg/mL and 278.38 unit/mL, respectively.

• 분석과학
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• 제10권1호
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• pp.66-74
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• 1997

지름 2.54cm, 길이 10cm인 유리관에 tyrosinase(EC. 1.14.18.1)를 입자의 크기 $550{\mu}m$인 탄소에 고정시켜 충진하고, 페놀과 산소를 기질로 사용하여 tyrosinase의 반응 특성을 조사하기 위해 axial dispersion 모델을 제안하였다. 본 논문에서 페놀의 농도는 55.5mM로 고정시키고 산소(2.7ppm, 5.4ppm, 그리고 9.5ppm)와 유속 (1~3mL/s)을 변화시키면서 탄소에 고정된 tyrosinase의 반응을 관찰하였다. 또한, Damkolher수를 계산하고 분산 특성과 식으로부터 효소반응 속도 및 분산의 영향을 예측하기 위해 수치적 해석을 하였다. 연구 결과 물질저항은 주로 외부 전달과 내부확산이었으며, 제안된 모델에서 Biot수는 64.25였다. 페놀은 1.0mL/s 정도의 느린 속도에서 산소의 농도가 높을수록 높은 전환율을 나타내었다. 한편, axial dispersion 모델과 plug flow 모델의 비교에서는 모두 같은 전환율을 나타내어 axial dispersion 모델이 반응속도와 무관함을 알 수 있었다.

• 한국포장학회지
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• 제13권3_4호
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• pp.113-120
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• 2007

The method to characterize the fresh produce respiration was presented with possible application of modified atmosphere package design. Particularly the respiration model based on enzyme kinetics was introduced as function of oxygen and carbon dioxide concentrations. The method to estimate the equilibrated package atmosphere for any package conditions was presented by incorporation of $O_2$ and $CO_2$ permeabilities of the packaging film. Temperature dependences for fresh produce respiration and gas permeation were given by Arrhenius equation and then used to analyze the effect of temperature on the package atmosphere. An example analysis was presented for better understanding of the concept.