• Title/Summary/Keyword: 단일 효소 반응

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Real-time Fluorescence Assay of DNA Polymerase Using a Graphene Oxide Platform (산화 그래핀 플랫폼을 이용한 DNA 중합효소의 실시간 형광에세이)

  • Gang, Jongback
    • Microbiology and Biotechnology Letters
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    • v.41 no.4
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    • pp.456-461
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    • 2013
  • Using the different adsorption properties of ssDNA and dsDNA to GO, this study used a real time and efficient fluorescence assay to detect the enzymatic activity of the Klenow fragment with the adsorbed DNA to GO. Results showed that adsorption of fluorescein-tagged ssDNA to GO resulted in fluorescence quenching and DNA was released from GO by adding complementary DNA. In addition, fluorescence restoration was increased through a polymerization reaction by the Klenow fragment in the presence of a fluorescein-attached template, GO, and primer. Gel electrophoresis was conducted to confirm the hybridization and DNA polymerization reactions on GO.

Purification and some Properties of Keratinolytic Protease Produced by Pseudomonas sp. KP-364. (Pseudomonas sp. KP-364가 생산하는 Keratinolytic Pretense의 정제 및 성질)

  • 전동호;강상모;권태종
    • Microbiology and Biotechnology Letters
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    • v.31 no.3
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    • pp.224-229
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    • 2003
  • A keratinolytic protease was purified from the culture medium of Pseudomonas sp. KP-364 by use of an assay of the hydrolysis of feather keratin. Membrane ultrafiltration and DEAE-cellulose ion-exchange resin and Sephadex G-150 gel chromatographies were used to purify the enzyme. The specific activity of the purified keratinolytic protease relative to that in the original medium was approximately 72-fold high. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Sephadex G-150 chromatography indicated that the purified keratinolytic protease is monomeric and has a molecular weight of 36 kDa. The optimal pH and temperature of the keratinolytic protease activity were 6.6 and 37 C, respectively, and the keratinolytic protease was relatively stable at pH value from 3.0 to 10.0 at 37 C for 1hour. The keratinolytic protease was inhibited by EDTA and EGTA, indicating that the keratinolytic protease was a kind of metalloprotease that require Li+ for cofactor.

Purification and Properties of Alkaline Pretense from Xanthomonas sp. YL-37 (Xanthomonas sp. YL-37 균주가 생산하는 Alkali성 단백질분해효소의 정제 및 성질)

  • 장형수;권태종
    • Microbiology and Biotechnology Letters
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    • v.26 no.5
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    • pp.427-434
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    • 1998
  • An alkaline protease was 4-fold purified, yielding 2.3% of recovery by ammonium sulfate precipitation, CM-cellulose column chromatography and Sephadex G-100 column chromatography. The purified enzyme was estimated to be monomeric with molecular weight of about 62,000 from polyacrylamide gel eletrophoresis (PAGE) and sodiumdodecylsulfate polyacrylamide gel electrophoresis (SDS-FAGE). The optimal pH and temperature of the alkaline pretense activity were 11.0 and 50$^{\circ}C$, respectively, exhibiting high stability at pH value from 6.0 to 11.0 at 50$^{\circ}C$ for 30 minute. The alkaline pretense was activated by MnSO$_4$, CaCl$_2$, and was inhibited by CuSO$_4$, ZnSO$_4$, HgCl$_2$, EDTA and EGTA. Also, the enzyme was found to be a metaloenzyme requiring Mn$\^$2+/ as cofactor. The NH$_2$-terminal amino acid of alkaline protease was alanine. The Km and Vmax values of this enzyme for casein was 4.0 mg/$m\ell$ and 5,500 unit/$m\ell$, respectively.

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Characterization and Evolutionary Relationship of Lactate Dehydrogenase in Liver of Lampetra japonica and Liver-specific C4 Isozyme in Gadus macrocephdus. (칠성장어(Lampetra japnica) 간조직 젖산탈수소효소와 대구(Gadus macrocephalus) liver-Specific C4동위효소의 특성 및 진화적 관계)

  • 박선영;조성규;염정주
    • Journal of Life Science
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    • v.14 no.4
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    • pp.708-715
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    • 2004
  • The lactate dehydrogenase (EC 1.1.1.27, LDH) in liver of Lempetra japonica was purified in buffer of affinity chromatography. The liver-specific $C_4$ isozyme of Gadus macrocephalus was purified by heat treatment, affinity chromatography, and DEAE-Sephacel chromatography. The liver-specific $C_4$ isozyme was eluted in a buffer containing NAD+ and was coeluted with $B_4$isozyme in plain buffer of affinity chromagraphy. Liver-specific $C_4$ isozyme in G. macrocephalus was the most thermostable, and$B_4$isozyme was more stable than $A_4$. The LDH in the fraction of pH 7.45 purified from the liver of L. iaponica by chromatofocusing was more inhibited by pyruvate than purified LDH. The optimum pH of the LDH isozyme in the liver of L. japonica was 7.5 and that of liver-specific$C_4$ isozyme was 8.5. The LDH in liver of L. japonica made complexes more with antibody against Coreoperca herzi$A_4$ and liver-specific $C_4$ than with that against eye-specific $C_4$. Therefore, the structure of the LDH in liver of L. japonica might be similarly evolved to that of subunit A and liver-specific $C_4$ isozyme in liver tissue of G. macrocephalus. The evolution rate of subunit C is faster than that of subunit A. LDH in liver of L. japonica has not one isozyme but isozymes and it was also found out to have not only subunit A and B but also subunit C.

Kinetic Measurement of the Step Size of DNA Unwinding by Bacteriophage T7 DNA Helicase gp4 (T7 박테리오파지 gp4 DNA helicase에 의한 DNA unwinding에서 step size의 반응속도론적 측정)

  • Kim, Dong-Eun
    • Journal of Life Science
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    • v.14 no.1
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    • pp.131-140
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    • 2004
  • T7 bacteriophage gp4 is the replicative DNA helicase that unwinds double-stranded DNA by utilizing dTTP hydrolysis energy. The quaternary structure of the active form of T7 helicase is a hexameric ring with a central channel. Single-stranded DNA passes through the central channel of the hexameric ring as the helicase translocates $5'\rightarrow3'$ along the single-stranded DNA. The DNA unwinding was measured by rapid kinetic methods and showed a lag before the single-stranded DNA started to accumulate exponentially. This behavior was analyzed by a kinetic stepping model for the unwinding process. The observed lag phase increased as predicted by the model with increasing double-stranded DNA length. Trap DNA added in the reaction had no effect on the amplitudes of double-stranded DNA unwound, indicating that the $\tau7$ helicase is a highly processive helicase. Global fitting of the kinetic data to the stepping model provided a kinetic step size of 10-11 bp/step with a rate of $3.7 s^{-1}$ per step. Both the mechanism of DNA unwinding and dTTP hydrolysis and the coupling between the two are unaffected by temperature from $4∼37^{\circ}C$. Thus, the kinetic stepping for dsDNA unwinding is an inherent property of tile replicative DNA helicase.

Measurement of the Affinity Constant of Monoclonal Antibody to Human Apolipoprotein A-I by ELISA (효소면역 분석법에 의한 아포지단백질 A-I 단일클론항체의 친화상수의 측정)

  • Mic Hung Yoon;Hyun Hee Lee
    • Biomedical Science Letters
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    • v.1 no.1
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    • pp.1-8
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    • 1995
  • The present study was undertaken to determine the dissociation constant (Kd)of monoclonal antibody to human apolipoprotein A-I (apo A-I) using enzyme-linked immunosorbent assay (ELISA). First the monoclonal antibody was incubated in solution with the antigen until the equilibrium was reached; then the free antibody which remains unsaturated at equilibrium was captured by binding to antigen on the microtiter plate and be measured by a classical indirect ELISA. The value of Kd determined from Scatchard plot was 0.625$\times$10^{-9}$ for purified antibody and 0.720$\times$10$^{-9}$ for unpurified antibody. This method was valuable for the measurement of true dissociation constant and found to be simple, reproducible, and accurate.

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A Study on the Detection Characteristics in Glucose and Fabrication of Bi-Enzyme Electrode using Electrochemical Method (전기화학적 방법을 이용한 다중 효소 전극 제작 및 글루코스 검출 특성에 관한 연구)

  • Han, Kyoung Ho;Shin, In Seong;Yoon, Do-Young
    • Journal of the Korean Electrochemical Society
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    • v.23 no.3
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    • pp.66-72
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    • 2020
  • In this study, the development of biosensors capable of bi-enzyme reactions by including Horseradish peroxidase and glucose oxidase was carried out for detection of glucose. The sensors were manufactured using electro deposition method to reduce production time, and screen printed electrodes (SPE) were used to produce economical sensors. To check the bienzyme effect, the sensor was compared and analyzed with single enzyme biosensor. The characteristics of the sensor were evaluated using scanning electron microscopy(SEM), cyclic voltammetry(CV), electrochemical impedance spectroscopy(EIS), chronoamperometry(CA), and flow injection analysis(FIA). Analysis results from SEM, CV and EIS confirmed that the enzymes are well fixed to the electrode surface. In addition, it was confirmed that bi-enzyme biosensors manufactured from the CA method improved signal performance by 200% compared to single enzyme biosensors. From this results, we were able to explain that HRP and GOD react catalyzed to each other. And the results of FIA showed that the intensity of each current signal was constant when the same concentration of glucose was injected four times. In addition, by analyzing the intensity of current signals for glucose concentrations, the biosensors manufactured in this study showed excellent trends in signal sensitivity, reproducibility and stability.

Production of Lactulose by Biological Methods and Its Application (생물학적 방법을 통한 기능성 이당 lactulose의 생산과 응용 연구)

  • Kim, Yeong-Su;Kim, Do-Yeon;Park, Chang-Su
    • Journal of Life Science
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    • v.26 no.12
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    • pp.1477-1486
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    • 2016
  • Lactulose (4-O-${\beta}$-D-galactopyranosyl-D-fructose) is a non-digestible synthetic ketose disaccharide which can used in food and pharmaceutical fields due to its useful functions for encephalopathy, chronic constipation, hyperammonemia, etc. Therefore, the lactulose is regarded as one of the most important disaccharides and have been concentrated much interesting as an attractive functional material in the current industry. From this reason, the research related on the production of lactulose has been carried out various academic and industrial research groups. To produce lactulose, two main methods, chemical production and enzymatic production have been used. Commercially lactulose produced by alkaline isomerization of lactose as chemical production method but it has many disadvantages such as rapid lactulose degradation, purification, and waste management. From these reasons, lactulose produced by enzymatic method which solves these problems has been suggested as a proper method for lactulose production. Two different enzymatic methods have been reported as methods for lactulose production. Lactulose can be obtained through hydrolysis and transfer reaction catalyzed by a ${\beta}$-galactosidase which requires fructose as co-substrate and exhibits a low conversion. Alternatively, lactulose can be produced by direct isomerization of lactose to lactulose catalyzed by cellobiose 2-epimerase which requires lactose as a single substrate and achieves a high lactulose yield. This review summarizes the current state of lactulose production by chemical and biological methods.

Sludge reduction by Enzyme Pretreatment (효소 전처리를 통한 슬러지 저감)

  • 김정래;심상준;최수형;염익태
    • KSBB Journal
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    • v.19 no.2
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    • pp.93-97
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    • 2004
  • We investigate the effect of enzyme pretreatment using protease, carbohydrase, and lipase on improvement of sludge treatment efficiency by measuring SCOD and TCOD. The enzyme-pretreatment increases SCOD of excess sludge. In addition, the amount of sludge reduction during digestion, in terms of SCOD and TCOD, are enhanced by enzyme-pretreatment. Among pretense, carbohydrase, and lipase, pretense showed the best enhancement of the sludge treatment efficiency. Sludge digestion followed by ozone and enzyme treatments showed more effective sludge treatment when compared with ozone treatment alone. Therefore, we expect that enzyme pretreatment can be used as a useful tool for enhancing the sludge treatment efficiency.

Electrochemical characterization of 3-mercaptopropionic acid self-assembled monolayer for urea sensor (요소센서를 위한 3-mercaptopropionic acid 자기조립 단일층의 전기화학적 특성 분석)

  • Yun, Dong-Hwa;Song, Min-Jung;Kim, Jong-Hoon;Kang, Moon-Sik;Min, Nam-Ki;Hong, Suk-In
    • Proceedings of the KIEE Conference
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    • 2004.07c
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    • pp.1579-1581
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    • 2004
  • 바이오센서는 효소(enzyme), 생분자(biomolecule), 항체(antibody), 세포(cell) 등의 biological agent를 인지 물질(recognition material)로 하여 측정하고자하는 분석 대상(analyte)과 높은 선택성으로 반응을 일으키게 하여 그 결과를 기존의 물리, 화학센서로 감지 해내는 방식이므로 기존의 의료용 화학센서를 대체하는 추세이다. 바이오센서가 기존의 센서와 구별되는 점은 생물질의 선택적인 반응 및 결합을 이용하는 것이므로 바이오센서의 실용화에 있어서 가장 중요한 것은 생체 반응 물질의 고정화 기술과 고정화막의 선택이라 할 수 있다. 일정전압법을 이용한 요소센서는 많이 연구되어 오고 있으나 낮은 농도에서의 감도저하에 따른 단점으로 상용화에 이르지 못하고 있다. 본 논문은 요소센서의 이용하기 위한 고정화막으로 3-mercaptopropionic acid 자기조립 단인층의 전기화학적 특성을 고창하였다. 자기조립 단일층은 직접적인 전자전달로 인하여 낮은 요소 농도에서 뛰어난 강도와 빠른 반응 시간을 보였으며, 특히 다공질 실라콘을 기질로 사용한 경우 평면 전극 보다 약 3배의 감도 증가 효과를 가져왔다. 자기조립 단일층의 표면 분석은 X-ray photoelectron spectroscopy(XPS)를 이용하였다.

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