• Applied Biological Chemistry
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• v.47 no.1
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• pp.41-48
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• 2004

Cyclodextrin glucanotransferase (CGTase) of Bacillus sp. isolated from soil was purified and its enzymological characteristics were investigated. It was found that the production of CGTase reached to the maximum when the strain was cultured in the broth containing 0.1 % albumin, 2% $NH_4Cl$, 2% soluble starch and 0.2% $NH_2PO_4$ for 72 hrs at $37^{\circ}C$. The purity of CGTase was increased by 9.7 folds through purification procedures by the following column chromatography DEAE-cellulose ion exchange chromatography and Sephadex G-100, G-150 gel filtration and its specific activity was 528.0 unit/mg. The optimum pH and temperature for the CGTase activity were 8.0 and $80^{\circ}C$, respectively. The enzyme was stable in pH $8.0{\sim}11.0$ at $60{\sim}80^{\circ}C$. The activity of purified enzyme was inhibited by $Pb^{2+},\;Hg^{2+}$ and $Zn^{2+}$. When CGTase was treated with each 20.5 unit, 41 unit, 205 unit and 410 unit to investigate the transglucosylation to stevioside by purified cyclodextrin glucanotransferase, transglucosylation rate to stevioside was 74.9%, 75.7%, 68.7% and 57.9%. Brown effect was observed above the concentration amounting to 205 unit of our CGTase.

• Journal of Food Hygiene and Safety
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• v.31 no.5
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• pp.319-326
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• 2016

Colistin is a last resort antimicrobial agent against multi-drug resistant Gram-negative bacteria. This study was conducted to develop an analytical method to determine colistin in fish and shrimp. The analytes were confirmed and quantified via liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the positive ion mode using multiple reaction monitoring (MRM). The sample was extracted with acidified 5% methanol (containing 0.5% formic acid). Then, solid phase extraction (SPE) was used for cleanup. Matrix-matched calibration curves were linear over the calibration ranges (0.05-1.2 mg/kg) for all the analytes into blank sample with $r^2$ > 0.99. All the values fulfilled the criteria requested by the Codex guidelines. Average recoveries ranged from 85.9% to 107.9%. The repeatability of measurements, expressed as the coefficient of variation (CV, %), was less than 15%. The limit of detection (LOD) was 0.02 mg/kg, and the limit of quantitation (LOQ) was 0.05 mg/kg. This improved method showed higher accuracy and acceptable sensitivity to meet the CAC guideline requirements and is applicable for the analysis of residual colistin (A+B) in fish and shrimp.

• Journal of the Korean Society of Food Science and Nutrition
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• v.32 no.5
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• pp.684-688
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• 2003

The objective of this study was to search for the best strain as a source of L- $\alpha$-glycerophosphate oxidase (GPO) production and to establish the process technology for the purification of GPO on an industrial scale. The GPO was produced by culturing Streptococcus faecium, and purified by ammonium sulfate, DEAE-cellulose and hydroxyapatite chromatography. The relative activity was 60 units/L for 5. faecim ATCC 12755, 65 units/L for 5. faecium ATCC 19634, and 67 units/L for 5. faecium $M_{74}$.LC, respectively. The optimum condition for fermentation was $37^{\circ}C$ for temperature, 300 rpm for stir rate, 0.5 L/min for aeration rate and 17 hours. The main culture medium prepared by the modified AC medium. AC medium consists of 0.1% glucose, 0.2% glycerol, 1.0% tryptone and 1.0% yeast extract, 0.5% $K_2HP0_4$, pH 7.0. The GPO was purified by ammonium sulfate fractionation and ion exchange column chromatography, The yield and purity were 17.2% and 5.3 fold, respectively.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.28 no.2
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• pp.227-236
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• 1995

The objective of this research was to characterize fucoidans isolated from Laminaria religiosa and Undaria pinnatifida in Korea to obtain basic data for Production of soluble dietary fiber materials with biological functionality. Fucoidans were successively extracted 3 times at $65\%$ for 1hr with arid solution of pH 2.0, and cetylpyridinium chloride was used for partial purification. The yields of partially purified fucoidans were $2.71\%$ for L. religiosa, $6.65\%$ for sporophylls of U. pinnatifida and $0.40\%$ for blade of U. pinnatifida. The yield from sporophylls of U. pinnatifida was highest among the sample tested, whereas the yield from blade of U. pinnatifida was lowest. It appeared that the fuconidans content in different parts of U. pinnatifida varied. Partially purified fucoidans were separated into 3 fractions by DEAE-Sephadex A-25 ion exchange column and the maior fractions were refractionated with tractional precipitation with ethanol. $60-70\%$ ethanol precipitated fractions of 1. religiosa and sporophylls of U. pinnatifida turned out to be homogeneous by cellulose acetate electrophoresis and gel filteration chromatography. The molar ratios of fucose, galactose, and sulfate in the purified fucoidans(ethanol precipitated fractions) were 1 : 0.31 : 2.43 for L. religiosa and 1 : 0.97 : 1.99 for sporophylls of U. pinnatifida. The averaged molecular weights of the purified fucoidans from L. religiosa and sporophylls of U. pinnatifida were 31,000 and 38,000, respectively.

• BMB Reports
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• v.33 no.4
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• pp.326-331
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• 2000

Acidic chitinases from the gizzards of a broiler were purified to homogeneity, using precipitation with $(NH_{4})_{2}SO_{4}$, ion exchanger chromatography, gel filtration, chromatofocusing and hydrophobic interaction chromatography. The enzymes, GAC1 and GAC2, were purified 180- and 194- folds with a recovery of 4.9% and 2.7%, respectively. The molecular mass of GAC1 and GAC2 were 48.2 kDa and 57.8 kDa, respectively. Chromatofocusing resulted in a pI of 3.1 for both enzymes. The purified enzymes were endochitinases that were devoid of ${\beta}-N-acetylglucosaminidase$ and lysozyme activity. Kinetic studies using $[^3H]chitin$ indicate that GAC1 has a $K_m$ and $V_{max}$ of 1.97 mg/ml and 185 mg/mg protein/h, respectively. The GAC2 has a $K_m$ and $V_{max}$ of 0.42 mg/ml and 92.3 mg/mg protein/h, respectively at optimal pH and temperature (pH 5.0 and $60^{\circ}C$). When the pentamer and hexamer of N-acetylglucosamine (GlcNAc) were used as a substrate, the major product by GAC1 was the dimer of GlcNAc with a differential accumulation of the monomer and trimer, depending upon the substrate. However, the GAC2 produced the dimer and trimer in an equal quantity, regardless of the substrate used. The first 9 $NH_2-terminal$ amino acid residues of the purified gizzard chitinase GAC1 and GAC2 shared a 100% homology. The first 25 $NH_2-terminal$ amino acid residues of GAC1 also shared 55-60% homology with animal chitinases and some animal proteins, such as whey protein and oviduct-specific proteins. However, little homology was found with either microbial and plant chitinases, or egg white lysozyme.

• Applied Biological Chemistry
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• v.41 no.1
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• pp.31-41
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• 1998

A carotenoprotein from the muscle of Ascidian (Halocynthia roretzi) was purified by ion exchange chromatography and gel filtration chromatography, and analyzed molecular weight, stability of pH and heat, effect of denaturing agents, amino acids and fatty acids composition. The purified carotenoprotein had absorption maxima of 463 nm and 439 nm. The carotenoprotein had an approxmimate molecular weight of 64.4 kDa in polyacrylamide gel electrophoresis. The amino acid compositions of carotenoprotein were mainly Gly (15.39%), Asn (11.31%), Gln (10.62%) and Ser (13.35%). The major fatty acids composition of carotenoprotein were $C_{16:1(n-7)}\;(15.4%)$, $C_{22:1(n-9)}\;(14.5%)$ and $C_{20:1(n-11)}\;(11.4%)$. The monounsaturated fatty acids (45.2%) contained abundant content compared to other saturated (38.1%) and polyunsaturated (11.7%) fatty acids.

• Applied Biological Chemistry
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• v.42 no.4
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• pp.304-309
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• 1999

Two Lectins, designated FVL-1 and FVL-2, were isolated and purified from the fruiting bodies of edible mushroom Flammulina veluripes using ammonium sulfate fractionation, ethanol treatment, DEAE-TOYPEARL ion-exchange column chromatography, and TSK-Gel HW-55F column chromatography. Specific activity increased 18 folds for FVL-1 and 7.9 folds for FVL-2 from ethanol treated sample. SDS-PAGE of FVL-1 and FVL-2 gave apparent molecular mass of 10.6 kDa and 37 kDa, respectively. FVL-2 agglutinated all type of human erythrocytes (A, B, AB, and O). However, FVL-1 agglutinated more human erythrocyte type O than type A, B, and AB. The hemagglutination activities of the FVL-1 were effectively inhibited by bovine submaxillary and porcine stomach mucins(BSM and PSM), fetuin, asialofetuin and cations, such as $Cu^{2+}$, $Mg^{2+}$, $Ca^{2+}$, $Mn^{2+}$ and $Fe^{2+}$. However, FVL-2 was not inhibited by any cations. The hemagglutination activities of the two lectins were not inhibited by the sugar, such as lactose, galactose and sugar derivatives. FVL-1 and FVL-2 were stable at pH $5{\sim}11$ and pH $4{\sim}7$, respectively. FVL-1 was stable below $55^{\circ}C$ and FVL-2 was below $45^{\circ}C$.

• The Korean Journal of Pharmacology
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• v.27 no.2
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• pp.145-153
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• 1991

Human neutrophil cathepsin-G, which has been known as one of the active enzymes causing inflammatory diseases, was purified by two steps procedure involving one size exclusion (Ultorogel AcA54) and one ion exchange (CM-Sephadex) chromatography. Purified HNCGs were cross-reacted with Anti-HNCathepsin-G antibodies which were radised in rabbits and purified by cathepsin-G labeled Sepharose 4B affinity chromatography. HNCGs were effectively inhibited by NSAIDs including phenylbutazone, sulindac, oxyphenbutazone, salicylic acid and salicyluric acid. $IC_{50}_s$ of these drugs for inhibition of Cathepsin G were 0.3-0.8 mM. Other NSAIDs including aspirin showed little or no inhibition effect on the activity of Cathepsin G. These results strongly indicated that NSAIDs which showed inhibition effect on the activity of HNCGs possibly be at least a part of mechanism of action which might be related to direct inhibition of cathepsin G at the tissue destruction sites beside of their known mechanism of action as an anticyclo-oxygenase in treatment of inflammatory diseases. Lipid soluble component of Korean Red Ginseng which was known as an anti-inflammatory agent inhibited HNCGs strongly, but no other fractions did inhibited HNCGs. Antibiotics including novobiosin and rifamycin showed some inhibition effect on HNCGs, i. e.., $IC_{50}$ of these drugs were 2.6 mM and 1.5 mM respectively, and other antibiotics including penicillin G showed no or negligible inhibition effect on the activity of HNCGs. However. tetracyclines inhibited HNCGs very effectively at the concentration of therapeutic range. The inhibition effect of the activity of HNCGs by tetracycline are not related to the N-dimethyl radical on the 4 position of the tetracycline molecule. Furthermore, N-dedimethylated tetracyclines may have beneficial effect for long term treatment of chronic inflammatory diseases without developing any drug resistance to microorganisms.

• The Korean Journal of Mycology
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• v.25 no.4 s.83
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• pp.330-339
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• 1997

Botrytis cinerea T91-1 has shown to produce at least four different polygalacturonases in a liquid medium containing citrus pectin as a carbon source. One of the enzymes, its molecular weight was estimated as 37 kDa by denatured polyacrylamide gel electrophoresis, was purified by a series of procedures including acetone precipitation, ion exchange, heparin affinity, and reverse phase column chromatographies. By viscometric analysis, the enzyme was revealed as an endo-polygalacturonase. The enzyme activity was inhibited by divalent cations such as $Ca^{2+}$, $Co^{2+}$, and $Cu^{2+}$. Km and Vmax for polygalacturonic acid hydrolysis were 0.33 mg/ml and 28.6 nM/min, respectively. The optimum temperature for enzymatic activity was $55^{\circ}C$ and the enzyme showed optimal pH values between 4.0 and 4.5. The enzyme was stable up to 12 hours in the range of pH 4 to 7 and at the temperature below $30^{\circ}C$. Amino acid sequence from N-terminal up to 6 amino acids determined by Edman degradation showed little homology with polygalacturonases from fungi and plants.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.28 no.5
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• pp.659-666
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• 1995

Fucoidans were isolated from Hizikia fusiformis and Sargassum fulvellum and characterized on their chemical properties. Crude fucoidans were extracted at $65^{\circ}C$ for 1hr with acid solution of pH 2.0, and cetylpyridinum chloride was used for partial purification. The yields of partially purified fucoidans were $2.51\%$ for H. fusiformis, and $65\%$ for S. fulvellum, respectively. The partially purified fucoidans were separated into 3 fractions by DEAE-Sephadex A-25 ion exchange column chromatography and the major fractions were refractionated with fractional preripitation with ethanol. $60-70\%$ ethanol precipitated fraction(P-70) of H. fusiformis and $60-65\%$ ethanol precipitated fraction(P-65) of S. fulvellum turned out to be homogeneous by cellulose acetate electrophoresis and gel filtration chromatography. The molar ratios of fucose, galactose, and sulfate In the purified fucoidans were 1 : 0.66 2.74 for H. fusiformis and 1 : 0.24. 1.46 for 5. fulvellum. The averaged molecular weights of the purified fucoidans from H. fusiformis and S. fulvellum were 26,000 and 105,000, respectively.