• KSBB Journal
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• 제6권2호
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• pp.167-174
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• 1991

섬유소 물질을 유용한 자원으로 전환시키기 위하여 섬유소 분해요소를 상요하여 가수분해 반응을 시킬 때 섬유소의 구조적 특성, 섬유소-효소계의 상관성, 가수분해 저해효과, 섬유소 분해효소의 불활성화 등에 의하여 섬유소 가수분해 반응 속도에 상당한 영향을 주는 것으로 알려져 있다. 가수분해 반응이 진행함에 따라 높은 결정성을 가진 섬유소인 Avicel PH 101의 결정화도는 서서히 증가하였으며 무정형이 많은 Solka Floc BW 40은 반응초기에 급격한 증가를 보여주었고 실험적으로 결정성 부분과 무결정성 부분이 동시에 분해됨을 알 수 있었으며, 반응 초기에 급격한 효소의 흡착이 일어나며 반응이 진행함에 따라 서서히 탈착됨을 볼 수 있었다. 반응 생성물인 글루코오스와 셀로바이오스는 초기 가수분해 반응속도에 상당한 저해효과를 주며 또한 생성물의 농도가 증가할수록 효소 흡착량이 감소함을 알 수 있었다. 섬유소 분해요소의 불활성도는 반응시간에 따라 지수 함수적으로 변화함을 실험적으로 알 수 있었다. 셀로바이오스의 초기 가수분해 반응속도는 상대적으로 높은 ${\beta}-glucosidase$의 활성도에 의해 비교적 높은 셀로바이오스의 농도(5-10mg/ ml)에서도 기질의 저해효과가 적음을 볼 수 있었다.

• Toxicological Research
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• 제12권2호
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• pp.155-161
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• 1996

The enzymatic properties for NADPH-P450 reductase domain of a fusion protein between human cytochrome P450 1A1 and rat NADPH-P450 reductase expressed in Escherichia coli were investigated. The fusion plasmid pCW/1A1OR-expressed E. coli membrane showed high NADPH-cytochrome c reductase activity ($830.1\pm 85.8 nmol\cdot min^{-1}\cdot mg protein^{-1}$), while pCW control vector and P 450 1A1 expression vector pCW/1A1 showed relatively quite low activity ($4.35\pm 0.49, 3.27\pm 0.50 nmol\cdot min^{-1}\cdot mg protein^{-1}$, respectively). The kinetic curves for NADPH-cytochrome c reductase followed typical Michaelis-Menten kinetics. The $K_{max}$ and $V_{max}$ for NADPH-dependent reductase activity were $8.24\pm 2.61\mu $and $817.9\pm 60.8 nmol\cdot min^{-1}\cdot mg protein^{-1}$, respectively, whereas those for cytochrome c-dependent reductase activity were $19.97\pm 2.86\mu M$ and $1303.5\pm 67.1 nmol\cdot min^{-1}\cdot mg protein^{-1}$. The reductase activities were also compared with those of rat, porcine and human liver microsomes. The activity of pCW/ 1A1OR-expressed E. coli membrane was 15.2-fold higher than that of rat liver microsome. Treatment with benzo(a)pyrene, 7-ethoxyresorufin and $\alpha$-naphthofiavone which are known as specific substrates or inhibitor for human P450 1A1 increased NADPH-cytochrome c reductase activity of fusion protein in E. coli membrane dose-dependently. These results demonstrate that the membrane topology of fused enzyme may be important for activity of its NADPH-P450 reductase domain.

• Journal of Microbiology and Biotechnology
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• 제30권12호
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• pp.1905-1911
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• 2020

Homoserine dehydrogenase (HSD) catalyzes the reversible conversion of ʟ-aspartate-4-semialdehyde to ʟ-homoserine in the aspartate pathway for the biosynthesis of lysine, methionine, threonine, and isoleucine. HSD has attracted great attention for medical and industrial purposes due to its recognized application in the development of pesticides and is being utilized in the large scale production of ʟ-lysine. In this study, HSD from Bacillus subtilis (BsHSD) was overexpressed in Escherichia coli and purified to homogeneity for biochemical characterization. We examined the enzymatic activity of BsHSD for ʟ-homoserine oxidation and found that BsHSD exclusively prefers NADP+ to NAD+ and that its activity was maximal at pH 9.0 and in the presence of 0.4 M NaCl. By kinetic analysis, Km values for ʟ-homoserine and NADP+ were found to be 35.08 ± 2.91 mM and 0.39 ± 0.05 mM, respectively, and the Vmax values were 2.72 ± 0.06 μmol/min-1 mg-1 and 2.79 ± 0.11 μmol/min-1 mg-1, respectively. The apparent molecular mass determined with size-exclusion chromatography indicated that BsHSD forms a tetramer, in contrast to the previously reported dimeric HSDs from other organisms. This novel oligomeric assembly can be attributed to the additional C-terminal ACT domain of BsHSD. Thermal denaturation monitoring by circular dichroism spectroscopy was used to determine its melting temperature, which was 54.8℃. The molecular and biochemical features of BsHSD revealed in this study may lay the foundation for future studies on amino acid metabolism and its application for industrial and medical purposes.

• Journal of Microbiology and Biotechnology
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• 제11권4호
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• pp.649-655
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• 2001

The kinetics of the thermolysin-catalyzed synthesis of N-(benzyloxycarbonyl)-L-phenylalanyl-L-leucine ethyl ester (Z-Phe-LeuOEt) from N-(benzyloxycarbonyl)-L-phyenylalanine (Z-Phe) and L-leucine ethyl ester (LeuOEt) in an ethyl acetate saturated aqueous system in a batch operation were studied. The kinetics for the synthesis of Z-Phe-LeuOEt were expressed using a rate equation for the rapid equilibrium random bireactant mechanism. The four kinetic constants involved in the rate equation were determined numerically by the quasi-Newton method so as to fit the calculated results with the experimental data. Within the pH and temperature range examined, the $K_{cat}$ value for the synthesis of Z-Phe-LeuOEt reached a maximum at pH 7.0 and $45^{\circ}C$, whereas the affinity between Z-Phe and thermolysin reached a maximum at pH 6.0 adn $40^{\circ}C$. The inhibitory effect of Z-Phe on the condensation reaction decreased as the pH and temperature decreased. In contrast, they affinity between LeuOEt and thermolysin remained unchanged within the pH and temperature range examined. Therefore, it was concluded that the protonation state of the carboxyl groups. of Z-Phe was more imprtant than that of the amono groups of LeuOEt for the synthesis of Z-Phe-LeuOEt in the present solvent system. The equilibrium yield at pH 6.0 and $30^{\circ}C$ was 8% higher than that at pH 7.0 and $40^{\circ}C$, although the rate was much slower. This result suggested that the affinity between the enzyme and the substrate rather than the overall rate was a more important factor affecting the equilibrium yield, when the peptide synthesis was carried out in a product-precipitation system.

• 한국근적외분광분석학회:학술대회논문집
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• 한국근적외분광분석학회 2001년도 NIR-2001
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• pp.1158-1158
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• 2001

Fast and dynamic biochemical, enzymatic and morphological changes occur during the so-called generative development and during the vegetative processes in seeds. The most characteristic biochemical and compositional changes of this period are the formation and decline of storage components or their precursors, the change of their degree in polymerization and an extensive change in water content. The aim of the present study was to detect the maturation processes in seed nondestructively and to verify the applicability of near infrared spectroscopic methods in the measurement of physiological, chemical and biochemical changes in wheat seed. The amount and variation of different water “species” has been changed intensively during maturation. Characteristic changes of three water absorption bands (1920, 1420 and 1150 nm) during maturation were analysed. It was concluded that the free/bound transition of water molecules could be followed sensitively in different region of NIR spectra. Kinetic changes of carbohydrate reserves were characteristic during maturation. An intensive formation and decline of carbohydrate reserves were observed during early stage of maturation (0 -13 days, high energy demand). An accelerated formation of storage carbohydrates (starch) was detected in the second phase of maturation. Five characteristic absorption bands were analysed which were sensitive indicators the changes of carbohydrates occurred during maturation. Precursors of protein synthesis and the synthesis of reserve proteins and their kinetic changes during maturation were followed from NIR spectra qualitative and qualitatively. Dynamic formation of amino acids and the changes of N forms were detected by spectroscopic, chromatographic and by capillary electrophoresis methods. Calibration equations were developed and validated in order to measure the optimal maturation time protein and moisture content of developing wheat seeds. The spectroscopic methods are offering chance and measurement potential in order to detect fine details of physiological processes. The spectra have many hidden details, which can help to understand the biochemical background of processes.

• Journal of Microbiology and Biotechnology
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• 제17권11호
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• pp.1789-1796
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• 2007

A ${\beta}$-glucosidase with the molecular mass of 160,000 Da was purified to homogeneity from cell extract of a cellulolytic bacterium, Cellulomonas uda CS1-1. The kinetic parameters ($K_m$ and $V_{max}$) of the enzyme were determined with pNP-cellooligosccharides (DP 1-5) and cellobiose. The molecular orbital theoretical studies on the cellulolytic reactivity between the pNP-cellooligosaccharides as substrate (S) molecules and the purified ${\beta}$-glucosidase (E) were conducted by applying the frontier molecular orbital (FMO) interaction theory. The results of the FMO interaction between E and S molecules verified that the first stage of the reaction was induced by exocyclic cleavage, which occurred in an electrophilic reaction based on a strong charge-controlled reaction between the highest occupied molecular orbital (HOMO) energy of the S molecule and the lowest occupied molecular orbital (LUMO) energy of the hydronium ion ($H_3O^+$), more than endocyclic cleavage, whereas a nucleophilic substitution reaction was induced by an orbital-controlled reaction between the LUMO energy of the oxonium ion ($SH^+$) protonated to the S molecule and the HOMO energy of the $H_2O_2$ molecule. A hypothetic reaction route was proposed with the experimental results in which the enzymatic acid-catalyst hydrolysis reaction of E and S molecules would be progressed via $SN_1$ and $SN_2$ reactions. In addition, the quantitative structure-activity relationships (QSARs) between these kinetic parameters showed that $K_m$ has a significant correlation with hydrophobicity (logP), and specific activity has with dipole moment, respectively.

• Biomolecules & Therapeutics
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• 제31권1호
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• pp.82-88
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• 2023

Genomic analysis indicated that the genome of Drosophila melanogaster contains more than 80 cytochrome P450 genes. To date, the enzymatic activity of these P450s has not been extensively studied. Here, the biochemical properties of CYP6A8 were characterized. CYP6A8 was cloned into the pCW vector, and its recombinant enzyme was expressed in Escherichia coli and purified using Ni²⁺-nitrilotriacetate affinity chromatography. Its expression level was approximately 130 nmol per liter of culture. Purified CYP6A8 exhibited a low-spin state in the absolute spectra of the ferric forms. Binding titration analysis indicated that lauric acid and capric acid produced type I spectral changes, with K_d values 28 ± 4 and 144 ± 20 µM, respectively. Ultra-performance liquid chromatography-mass spectrometry analysis showed that the oxidation reaction of lauric acid produced (ω-1)-hydroxylated lauric acid as a major product and ω-hydroxy-lauric acid as a minor product. Steady-state kinetic analysis of lauric acid hydroxylation yielded a k_cat value of 0.038 ± 0.002 min^-1 and a K_m value of 10 ± 2 µM. In addition, capric acid hydroxylation of CYP6A8 yielded kinetic parameters with a k_cat value of 0.135 ± 0.007 min^-1 and a K_m value of 21 ± 4 µM. Because of the importance of various lipids as carbon sources, the metabolic analysis of fatty acids using CYP6A8 in this study can provide an understanding of the biochemical roles of P450 enzymes in many insects, including Drosophila melanogaster.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2022년도 추계학술대회
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• pp.157-157
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• 2022

Plants being sessile are prone to various abiotic challenges, including salinity. Plants generally cope with salt stress by regulating their endogenous NO levels. NO exogenously applied in various forms also successfully impedes the salt stress, but its small size, short half life, and high volatility rate hamper its application in agriculture. NO application via CS as a nanocarrier is an alternate option to ensure the optimal kinetic release of NO for a long period compared to the free NO form. Herein, we synthesized and characterized GSNO-CS NP by ionic gelation of TPP with CS and then reacting with GSH, followed by reaction with NaNO₂ suspension. The synthesized NPs were characterized using non-destructive analytical techniques such as DLS, FTIR, and SEM to ensure their synthesis and surface morphology. NO-release profile confirmed optimal kinetic NO release for 24 h from NO-CS NP as compared to free NO form. The efficiency of NO-CS NP was checked on Arabidopsis plants under salinity stress by gauging the morphological, physiological, and enzymatic antioxidant system and SOS pathway gene expression levels. Overall, the results revealed that NO-CS NP successfully mitigates salinity stress compared to free GSNO. Concluding, the findings provide sufficient experimental evidence for the application of nanotechnology to enhance NO delivery, thus inducing more benefits for the plants under stress conditions by mitigating the deleterious impacts of salt stress on the morphological and physiological status of the plants, and regulating the ions exchange by overexpression of SOS pathway candidate genes.

• Journal of Microbiology and Biotechnology
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• 제27권4호
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• pp.768-774
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• 2017

Horseradish peroxidase (HRP) catalyzes the oxidation of aromatic compounds by hydrogen peroxide via insoluble polymer formation, which can be precipitated from the wastewater. For HRP immobilization, poly(lactic-co-glycolic acid) (PLGA) fine carrier supports were produced by using the Nano Spray Dryer B-90. Immobilized HRP was used to remove the persistent 2,4-dichlorophenol from model wastewater. Both extracted (9-16 U/g) and purified HRP (11-25 U/g) retained their activity to a high extent after crosslinking to the PLGA particles. The immobilized enzyme activity was substantially higher in both the acidic and the alkaline pH regions compared with the free enzyme. Optimally, 98% of the 2,4-dichlorophenol could be eliminated using immobilized HRP due to catalytic removal and partly to adsorption on the carrier supports. Immobilized enzyme kinetics for 2,4-dichlorophenol elimination was studied for the first time, and it could be concluded that competitive product inhibition took place.

• Bulletin of the Korean Chemical Society
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• 제24권8호
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• pp.1188-1192
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• 2003

In order to study the role of residue in the active site of glutathione S-transferase (GST), Tyr 108 residue in human GST P1-1 was replaced with alanine, phenylalanine and tryptophan by site-directed mutagenesis to obtain mutants Y108A, Y108F and Y108W. These three mutant enzymes were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The substitutions of Tyr108 significantly affected $K_m^{CDNB}$ and $K_m^{ETA}$, whereas scarcely affected $K_m^{GSH}$. The substitutions of Tyr108 also significantly affected $I_{50}$ of ETA, an electrophilic substrate-like compound. The effect of these substitutions on kinetic parameters and the response to inhibition suggests that tyrosine 108 in hGST P1-1 contributes to the binding of the electrophilic substrate and a major determinant in the binding of CDNB is the aromatic ring of Tyr108, not its hydroxyl group.