• Biomolecules & Therapeutics
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• v.21 no.6
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• pp.487-492
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• 2013

Cytochrome P450 4A11 (CYP4A11) is a fatty acid hydroxylase enzyme expressed in human liver. It catalyzes not only the hydroxylation of saturated and unsaturated fatty acids, but the conversion of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE), a regulator of blood pressure. In this study, we performed a directed evolution analysis of CYP4A11 using the luminogenic assay system. A random mutant library of CYP4A11, in which mutations were made throughout the entire coding region, was screened with luciferase activity to detect the demethylation of luciferin-4A (2-[6-methoxyquinolin-2-yl]-4,5-dihydrothiazole-4-carboxylic acid) of CYP4A11 mutants in Escherichia coli. Consecutive rounds of random mutagenesis and screening yielded three improved CYP4A11 mutants, CP2600 (A24T/T263A), CP2601 (T263A), and CP2616 (A24T/T263A/V430E) with ~3-fold increase in whole cells and >10-fold increase in purified proteins on the luminescence assay. However, the steady state kinetic analysis for lauric acid hydroxylation showed the significant reductions in enzymatic activities in all three mutants. A mutant, CP2600, showed a 51% decrease in catalytic efficiency ($k_{cat}/K_m$) for lauric acid hydroxylation mainly due to an increase in $K_m$. CP2601 and CP2616 showed much greater reductions (>75%) in the catalytic efficiency due to both a decrease in $k_{cat}$ and an increase in Km. These decreased catalytic activities of CP2601 and CP2616 can be partially attributed to the changes in substrate affinities. These results suggest that the enzymatic activities of CYP4A11 mutants selected from directed evolution using a luminogenic P450 substrate may not demonstrate a direct correlation with the hydroxylation activities of lauric acid.

• KSBB Journal
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• v.18 no.5
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• pp.352-355
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• 2003

${\beta}$-(1,6)-Branched ${\beta}$-(1,3)-D-glucans are known to enhance the immune system in human body, and in most cases have higher molecular weights over 1 MDa. In order to enhance the efficacy of glucans by decreasing their molecular weights, sonication, acid treatment, and enzymatic hydrolysis were tested and compared in this work. Treatment of sonication was effective to decrease the molecular weight to the extent of several dozens of kilo-daltons, but have a risk to disorder the triple helical structure of the glucans. Acid treatment was also an effective method to degrade polysaccharides, but ${\beta}$-(1,6)-branched of the glucan molecules was found to be also hydrolyzed. Treatment of ${\beta}$-(1,3)-glucanase was an effective method to decrease the molecular weight in mild conditions, but could not hydrolyse the highly ${\beta}$-(1,6)-branched ${\beta}$-(1,3)-glucans efficiently.

• Journal of Life Science
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• v.23 no.4
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• pp.537-541
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• 2013

We analyzed glycerol using thin-layer chromatography (TLC) and compared the separation resolution of some mobile phases. When acetonitrile:distilled water (85:15 v/v) was used as a mobile phase, the band of glycerol on the TLC was more distinctly and rapidly separated. Using TLC analysis, we prepared a calibration curve for the glycerol concentration vs. the area of the glycerol band in which the glycerol concentration of the x-axis was converted into a log-scale ranging from 3.0 to 0.0625 (%, w/v). Based on this calibration curve, the residual glycerol concentration (0.2 [%, w/v]) in biodiesel was determined successfully using TLC analysis. When the results of the TLC analysis were compared with those of a chemical and enzymatic assay, the results were fairly similar. We conclude that TLC without additional analytical instruments can be used as an alternative method for the quantitative analysis of the concentration of glycerol in biodiesel.

• Journal of Korea Technical Association of The Pulp and Paper Industry
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• v.37 no.1 s.109
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• pp.25-37
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• 2005

This study was carried out to examine the optimal cultural condition in enzyme activities of CMCase, FPase and xylanase in selected strains which secret extracellular enzymes for using deinking agent to old newsprint. The results of this study were as follow: The production of enzyme by Bacillus pumilus I was maximal as grown on the medium, containing of rice bran+xylan $2.0\%$, peptone $0.8\%,\;K_2HPO_4\;0.1\%\;and\;CaCl_2\;0.06\%$ at pH 8.0 and $28^{\circ}C$ for 72 hours. Optimal cultural condition of B. subtilis I was avicel+xylan $3.5\%,\;urea\;0.4\%,\;K_3PO_4\;0.1\%\;and\;CaCl_2\;0.015\%$ at pH 9.0 and $28^{\circ}C$ for 36 hours. The maximal enzyme production was observed in the medium, containing of avicel+xylan $3.5\%,\;urea\;1.6\%\;and\; K_2HPO_4\;0.125\%$ with pH 9.0 when B. pumilus II was cultured at $28^{\circ}C$ for 60 hours. The production of enzyme by B. subtilis IT was maximal as grown on the medium, containing of xylan $2.0\%,\;yeast\; extract\;0.6\%,\;K_2HPO_4\;0.1\%\;and\;ZnSO_4\;0.04\%$ at pH 8.0 and $34^{\circ}C$ for 36 hours. The activities of FPase and xylanase in tested 4 strains were not much different with Thermomonospora fusca.

• Korean Journal of Food Science and Technology
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• v.29 no.2
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• pp.194-197
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• 1997

Dopamine ${\beta}-hydroxylase\;(DBH)$ catalyses the enzymatic reaction of dopamine to norepinephrine. For the purpose of screening DBH inhibitory activity from edible mushrooms, Ganoderma lucidum, Agaricus bisporus and Lentinus edodes were examined by tracing inhibitory activities against bovine adrenal DBH, utilizing tyramine as a substrate. Among the three edible mushrooms tested, Ganoderma lucidum showed potent enzyme inhibitory activilies above 100% against DBH in chloroform fraction. Lentinus edodes and Agaricus bisporus showed inhibitory activities in ethylacetate fraction on 79.7% and 64.7%, respectively. Each solvent fraction of these mushrooms were assessed in the aspects of their inhibitory activities against DBH, and their $IC_{50}$ values were calculated. $IC_{50}$ value of chloroform fraction of Ganoderma lucidum was $1.60{\times}10^{-4}\;g$, and those of ethylacetate fractions of Agaricus bisporus and Lentinus edodes were $5.50{\times}10^{-4}\;g\;and\;2.35{\times}10^{-4}\;g$, respectively.

• The Journal of Korean Medicine
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• v.22 no.2
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• pp.3-9
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• 2001

Objective : Oriental medicinal plants reported to be used as anti-hypertensive drugs have been in vitro screened for inhibitory effects on angiotensin-converting enzyme (ACE). Methods : The bioassay is based on inhibition of plasma angiotensin-converting enzyme, as measured from the enzymatic cleavage of the Hip-His-Leu substrate into His-Leu. The plant material is extracted with hexane, ethylacetate, n-buthanol and water separately. Results : In total, 51 species (202 extracts) have been investigated and $400{\;}\mu\textrm{g}/ml$ of the solvent extracts from 26 extracts inhibit the enzyme activities by more than 50%. Among them, four samples of two plant species (buthanol and ethylacetate extracts of Salvia miltiorrhiza and buthanol and water extracts of Jeffersonia dubia) were found to posses a high ACE inhibition ability more than 90%. Conclusion : These results suggested that many Oriental medicinal plants have a antihypertensive effects by inhibition of ACE.

• Biomolecules & Therapeutics
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• v.27 no.2
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• pp.193-200
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• 2019

Ceramide metabolism is known to be an essential etiology for various diseases, such as atopic dermatitis and Gaucher disease. Glucosylceramide synthase (GCS) is a key enzyme for the synthesis of glucosylceramide (GlcCer), which is a main ceramide metabolism pathway in mammalian cells. In this article, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine GCS activity using synthetic non-natural sphingolipid C8-ceramide as a substrate. The reaction products, C8-GlcCer for GCS, could be separated on a C18 column by reverse-phase high-performance liquid chromatography (HPLC). Quantification was conducted using the multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z $588.6{\rightarrow}264.4$ for C8-GlcCer at positive ionization mode. The calibration curve was established over the range of 0.625-160 ng/mL, and the correlation coefficient was larger than 0.999. This method was successfully applied to detect GCS in the human hepatocellular carcinoma cell line (HepG2 cells) and mouse peripheral blood mononuclear cells. We also evaluated the inhibition degree of a known GCS inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) on GCS enzymatic activity and proved that this method could be successfully applied to GCS inhibitor screening of preventive and therapeutic drugs for ceramide metabolism diseases, such as atopic dermatitis and Gaucher disease.

• Journal of Microbiology and Biotechnology
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• v.16 no.11
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• pp.1784-1790
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• 2006

Bacillus anthracis is a causative agent of anthrax. Anthrax toxins are composed of a protective antigen (PA), lethal factor (LF), and edema factor (EF), in which the PA is a central mediator for the delivery of the two enzymatic moieties LF and EF. Therefore, the PA has been an attractive target in the prevention and vaccinization for anthrax toxin. Recently, it has been reported that the molecule consisting of multiple copies of PA-binding peptide, covalently linked to a flexible polymer backbone, blocked intoxification of anthrax toxin in an animal model. In the present study, we have screened novel diverse peptides that bind to PA with a high affinity (picomolar range) from an M13 peptide display library and characterized the binding regions of the peptides. Our works provide a basis to develop novel potent inhibitors or diagnostic probes with a diverse polyvalence.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.1
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• pp.57-60
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• 2000

$(R)-\beta-acetylmercaptoisobutyric$ acid (RAM), a chiral compound, is an important intermediate for the chemical synthesis of various antihypertensive and congestive heart failure drugs. Microorganisms capable of converting $(R,S)-\beta-acetylmercaptoisobutyric$ acid ((R,S)-ester) to RAM were screened from soil microorganisms. A strain of Pseudomonas sp. 1001 screened from a soil sample was selected to be the best. Cells showed an activity of 540 U/mL from culture broth and the enzyme was thermostable up to $70^{\circ}C$. This strain could produce RAM asymmetrically from (R,S)-ester.

• Journal of Microbiology and Biotechnology
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• v.9 no.5
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• pp.646-649
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• 1999

A thermophilic bacterium producing D-stereospecific dipeptidase was isolated from Korean soil samples. The enzyme hydrolyzed the peptide bond between D-alanyl-D-alanine (D-Ala-D-Ala). The isolated bacterial strain was rod shaped, gram-positive, motile, and formed an endospore. Morphological and physiological characteristics suggested this microorganism a thermophilic Bacillus species, and was named as Bacillus sp. BCS-l. The production of D-stereospecific dipeptidase was growth-associated and optimal at $55^{\circ}C$. The enzyme was applied for the synthesis of D-amino acid-containing peptide, N-benzyloxycarbonyl-L-aspartyl-D-alanine benzyl ester (Z-L-Asp-D-AlaOBzl), as a model reaction. A thermodynamically controlled synthesis of Z-L-Asp-D-AlaOBzl was achieved in an organic solvent.