• Journal of Food Hygiene and Safety
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• v.3 no.2
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• pp.75-81
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• 1988

Various methods such as lel-mtration on Sephadex G-SO, 75, 100 Sephacry, and Ultro gel, and lon-exchanle chromatoaraphy on Amberilte and carboxymethyl (CM)-cellulose, and Fast Protein liquid Chromatolraphy (FPLC) were applied for the purification of enterotoxin B from Staphylococcus aureus ATCC 14458 and compared one another. lon-exchanle chromatography on Amberllte resin was good enough to remove non-entrotoxln materials in culture, convlnient to use and fast although tbe purity was less tban 70%. However, CM-cellulose showed to be better purity and yield tban those of Amberilte resin. The yields of these two resins for ion-exchange cbromatograpby were about 70% and 75%, respectively. When the gel-filtration methods on Sepbadex G-50, 75, 100, Sepbacryl, and Ultro lei were applied, the purities were about 90%. FPLC was found to be tbe most efficient metbod in terms of purity (96%) and speed. For the purification of sample with large volume, particularly, tbe combined metbod, gel-mtration after Amberlite can be also used efficiently. Tbe purified toxin was found to be identical to type B enterotoxin used for reference standard by Oucbterlony immunodiffusion test.

• Korean Journal of Food Science and Technology
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• v.20 no.6
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• pp.856-861
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• 1988

A new method developed for simultaneous purification of enterotoxin A and C from Staphylococcus aureus strain L 350/1 consisted of chromatography on carboxymethyl (CM)-cellulose using a buffer of variable pH, gel filtration on Ultro gel, and fast protein liquid chromatography(FPLC) using a buffer of variable pH. The enterotoxin A and C were purified by three steps: batchwise adsorption from culture supernatant on Amberlite CG-50; chromatography on CM-cellulose using a buffer of constant pH and molarity; and gel filtration on Sephadex G-75. The purified enterotoxin appeared homogeneous by gel diffusion and polyacrylamide gel electrophoresis. Upon treatment with CM-cellulose using a elution of variable pH, enterotoxin A and C were so close that they were not separated completely. After elution from gels, the enterotoxins appeared as a single peak at the same position. Gel filtration gave a reaction of complete identity to enterotoxin A and C in Ouchterlony immunodiffusion. In FPLC using a CM-cellulose, enterotoxin A and C were simultaneously separated at pH 8.6 and 6.8. When each fraction was performed to gel immunodiffusion, at peak of enterotoxin A and C were not detected each other. In a method of elution by pH-gradient was to be more efficient as a simultaneous separation method in terms of speed, yields and simplicity. The purified toxin A and C were identical to type A and C reference enterotoxin on both disc electrophoresis and Ouchterlony gel diffusion.

• Applied Biological Chemistry
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• v.33 no.1
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• pp.79-86
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• 1990

Cyclodextrin hydrolase from Bacillus stearothermophilus KFCC 21203 was purified and the properties of the purified enzyme were investigated. The enzyme was purified 15 folds with 77 % recovery by ammonium sulfate fractionation, DEAE-cellulose chromatography, and Ultro AcA 34 gel filtration. The specific activity and the molecular weight of the enzyme were 1.30 units/mg protein and about 29,500, respectively, The maximum activity of the enzyme was shown at $55^{\circ}C$ and pH 5.5. However, stable temperature and pH were $40^{\circ}C$ and $5.0{\sim}8.0$, respectively. The Km value for ${\gamma}-cyclodextrin$ was $3.78{\times}10^{-3}$ M. The degradation activity of the enzyme was selectively high for ${\gamma}-cyclodextrin$, and very low for ${\beta}-cyclodextrin$, but not for ${\alpha}-cyclodextrin$. The decomposed products of ${\gamma}-cyclodextrin$ were mainly glucose and maltose, and a little mlatotriose. The activity of the enzyme was very high for amylose, potato starch, corn starch, amylopectin and maltooligomer, and relatively high for glycogen and dextrin. The decomposed products of them were mainly glucose and maltose.

• Journal of the Korean Society of Food Science and Nutrition
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• v.21 no.1
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• pp.84-90
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• 1992

Peroxidase in the fruit of Malus sieboldii (Regel) Rehder was partially purified by DEAE-cellulose column chromatography and Ultro-AcA 54 gel filtration. The optimum pH of peroxidase was 4.5 and optimum temperature was $80^{\circ}C$. The enzyme was stable at pH 5.0 and below $30^{\circ}C$, and inactivated by heat treatment at $80^{\circ}C$ for 15min. In the presence of 30mM $H_{2}O_2$ Km value on o-phenylenediamine as substrate was 1.65mM, and in the presence of 10mM o-phenylenediamine Km value on $H_{2}O_2$ was 7.97mM. L-Ascorbic acid and sodium L-ascorbate greatly inhibited the enzyme activity and among several metal ions $Mn^{2+}$ only increased the activity at 5mM.

• Korean Journal of Agricultural Science
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• v.24 no.2
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• pp.275-282
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• 1997

The prcx.iuction of extracellular 1,4-${\beta}$-glucanase by Cellulomonas sp. CS1-1 on microcrystalline cellulose, sigmacell was maximal after 5-day cultivation as 280 units/mL, which was three times higher than the level produced on carboxymethyl cellulose. A carboxymethyl cellulase containing the carbohydrate of 8.2% was purified from the culture filtrate by successive procedures of column chromatographies. Purification factor was calculated as 22-folds with the specific carboxymethyl cellulase activity of 31.9 units/mg. The molecular weight and isoelectric point of the purified enzyme were 54,000 and pI 5.4, respectively. The optimal pH and temperature were 6.0 and $45^{\circ}C$, and the enzyme was stable between pH 6.5 and 7.5 and below $50^{\circ}C$. The estimated Km and Vmax were 10 mg/mL and $6.25{\mu}mol/min$ for carboxymethyl cellulose and 30.3 mg/mL and $2.85{\mu}mol/min$ for sigmacell, respectively. The enzyme was partially inhibited by $Ag^+$, $Zn^{+{+}}$, $Fe^{+{+}}$ and EDTA, while completely inhibited by $Cd^{+{+}}$ and $Hg^{+{+}}$ at 1 mM concentration.