• Food Science and Preservation
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• v.12 no.5
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• pp.489-495
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• 2005

Polyphenol oxidase (PPO) from Burdock (Arctium lappa L.) was purified and characterized. Purification of polyphenol oxidase was achieved by ammonium sulfate precipitation, Phenyl-sepharose CL-4B hydrophobic chromatography and Sephadex G-100 gel filtration chromatography. The molecular mass of the purified PPO was estimated to be 30 kDa by SDS polyacrylamide gel electrophoresis. In a substrate specificity, maximum activity was achieved with chlorogenic acid, followed by catechol and catechin. Whereas, there was low activity with hydroquinic acid, resorcinol or tyrosine. The optimum pH and temperature for enzyme activity were 7.0 and 35$\circC$ with catechol, respectively. The enzyme was most stable at pH 7.0 while unstable at acidic and alkaline pH. The enzyme was stable when heated to 40$\circC$. But heating at 50$\circC$ for more than 30 min caused 50% loss of activity. Ascorbic acid, L-cystein and $Cu^{2+}$ inhibited the activity of pholyphenol oxidase.

• Applied Biological Chemistry
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• v.22 no.1
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• pp.10-17
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• 1979

The saponins isolated form the herb of Panax ginseng C.A. Meyer were investigated as compared with ginseng root saponins. By adopting DEAE cellulose ion exchange chromatography the pure saponins were isolated from Korean ginseng roots and leaves. The ginseng root and leaf saponins showed some differences in the pattern of the two-dimensional thin layer chromatogram. The ratio of panaxadiol to panaxatriol in the saponins was 1.7 in the roots and 3.5 in the leaves. Infra-red spectrum of ginseng leaf saponins isolated by liquid chromatography was identical with that of root saponins.

• Journal of Microbiology
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• v.39 no.4
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• pp.279-285
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• 2001

Vibrio parahaemolyticus is a halophilic bacterium associated with seafood gastroenteritis. An unusual strain of Kanagawa-positive urease producing Vibrio parahaemolyticus O1:K1 was isolated from the environment and identified . A polymerase chain reaction assay revealed that this strain harbored both the tdh and the genes. The urease from this strain was studied. Maximum urease production was induced in LB medium containing 0.2% urea, 0.5% glucose, 2% NaCl and pH 5.5 with 6h of culti-vation at 37$\^{C}$ under aeration. Purification of urease was achieved by the process of whole cell lysate, 65% ammonium sulphate precipitation, DEAE-cellulose ion exchange column chromatography, Sepharose CL-6B gel filtration and oxirane activated Sepharose 6B-urea affinity chromatography with 203 fold purification and 2.2% yield. Analysis of the purified enzyme by SDS-PAGE demonstrated the presence of the subunits with a molecular weight of 85kDa, 59kDa, 41kDa and the molecular weight for the native enzyme by nondenaturing PAGE and gel filtration chromatography was 255kDa. The purified urease was stable at pH 7.5 and the opeimal pH in HEPES buffer was 8.0 The enzyme was stable at 60$\^{C}$ for 2 h with a residual activity of 32% . The addition of 10$\mu$M if NiCl$_2$maintained stability for 30 min. The Km value of the purified enzyme was 35.6 mM in urea substrate. The TD$\_$50/(median toxic dose) of the purified urease was 2.5$\mu\textrm{g}$/ml on human leukemia cells.

• YAKHAK HOEJI
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• v.35 no.6
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• pp.473-482
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• 1991

Protein methylase II (S-adenosyl-L-methionine:protein carboxyl-O-methyltransferase; EC 2.1.1.24., PM II) was purified from chicken pancreas by subcellular fractionation, DEAE-cellulose chromatography, QAE-Sephadex A-50 chromatography, Sephadex G-75 chromatography, and Sephadex G-75 rechromatography. The purified PM II gave a single band upon polyarcrylamide gel electrophoresis both in the presence of SDS and in Tris glycine buffer without SDS. The pI value of purified PM II was identified as 5.7 on isoelectric focusing gel. Properties and activities of PM II were studied and the following results were obtained. 1) PM II from chicken pancreas was purified approximately 221-fold with a yield of 1.3%. 2) The purified PM II appear constituted of a single polypeptide chain of a molecular weight 46,800 daltons. 3) Hemoglobin exhibited the highest of methyl-accepting activity among the substrates tested. 4) The purified PM II has a $K_m$ of $4.67{\times}10^{-6}M$ and a $V_{max}$ of 37.5 pmoles of $methyl-^{14}C/min./mg$ enzyme for $SAM^{-14}CH_3$ as methyl donor in the presence of histone type II-As. 5) It is found that S-adenosyl-L-homocysteine is a competitive inhibitor for PM II with $K_i$ value of $3.23{\times}10^{-5}M$.

• Applied Biological Chemistry
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• v.31 no.4
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• pp.331-338
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• 1988

Acetone powder was prepared from raw arrowroots and the polyphenol oxidases of crude enzyme prepared from acetone powder were identified 5 isoenzymes by staining with catechol containing 0.05% phenylene diamine. The crude enzyme was passed through the columns of ion exchangers and gel permeation to fractionate the polyphenol oxidases. The main fraction of polyphenol oxidase appeared to be purified by 94-fold, with the activity yield of 45.4%, and its molecular weight was determined as 38,500 by poly acrylamide gel electrophoresis. The optimal pH and temperature for the enzyme activity were pH 7.5 and $50^{\circ}C$, respectively. The purified enzyme showed a high affinity for catechol and pyrogallol. The Michaelis constant for catechol was calculated to be 16.67mM according to the Lineweaver-Burk method. The enzyme activity was strongly inhibited by L-ascorbic acid, sodium bisulfite, EDTA and KCN, and totally inhibited, by $Fe^{3+}$ at a concentration of 1mM. However the enzyme was activated by $Zn^{2+}$ approximately 1.7 times at the same concentration.

• Microbiology and Biotechnology Letters
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• v.16 no.3
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• pp.238-245
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• 1988

Studies were made on the regulation of chorismate mutase and prephenate dehydratase of a species of Intrasporangium, a phenylalanine producing Actinomycete isolated from soil. Two distinctly regulated species of chorismate mutase, designated CM I and CM IIwere resolved by DEAE Cellulose and DEAE Sephadex A 50 chromatography. The activity of CM II was inhibited by L-tyrosine, whereas that of CM I appeared to be unregulated. Single species of prephenate dehydyatase was also separated in the same purification steps. The activity of the enzyme was strongly feedback inhibited by L-phenylalanine, but by L-tyrosine or L-methionine it was rather slightly stimulated. Synthesis of chorismate mutase was not influenced by the presence of phenylalanine, tyrosine or tryptophan, whereas prephenate dehydratase was found to be subject to strong feedback repression by L-phenylalanine. The rate of repression was 94% at the concentration of 1mM L-phenylalanine but the repression was completely offset by the presence of 5mM tyrosine. The critical regulatory site of the phenylalanine terminal biopathway was, therefore, proved to be the second reaction which was catalyzed by the L-phenylalanine inhibitable and repressible prephenate dehydratase.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.25 no.5
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• pp.432-442
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• 1992

To identify the histological site of synthesis of yolk protein precursor, vitellogenin, by immunocytochemical method in the freshwater crab Eriocheir japonicus, we purified the yolk protein, vitellin, from crude egg extracts, and prepared the anti-rabbit serum against vitellin. Then, the site of vitellogenin synthesis was demonstrated by immunotytochemical method with PAP(peroxidase-antiperoxidase) reaction using the rabbit antiserum aganist vitellin. Female specific serum protein was identified in female serum by immunoelectrophoresis and Ouchterlony's immunodiffusion test for mature male and female sera. Based on the immunoelectrophoresis and Ouchterlony's diffusion test for mature male and female sera and crude egg extracts using antiserum against vitellogenic female serum absorbed with male serum, the female specific serum protein was identified as vitellogenin, detected in female serum only. The major yolk protein, vitellin, was purified from the crude egg extracts by DEAE-cellulose ion exchange chromatography, followed by sepharose CL-4B gel filteration chromatography. The molecular weight of vitellin was estimated to be about 245,000 dalton by sepharose CL-4B gel filteration chromatography. from the results of immunological analysis for vitellin, it was found that the vitellin antiserum contained the antibody against vitellogenin. In the results of immunocytochemical reaction by PAP method with the rabbit antiserum against vitellin, the vitellogenic oocytes and the hepatopancreas of mature female showed positive PAP reaction, but not in follicle cells and previtellogenic oocytes nf ovary, muscle of female and mature male hepatopancreas. Therefore, it showed that the hepatopancreas of mature female is the site of vitellogenic synthesis.

• Journal of Life Science
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• v.12 no.4
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• pp.490-495
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• 2002

Hemolysin (VFH) of V. fluvialis, which is a pathogenic bacteria, causing watery diarrhea with vomiting, abdominal croup, was purified. V. fluvialis was cultivated in BHI medium and the culture supematant was precipitated by ammonium sulfate. The protein was purified by chromatographies on columns of DEAE-cellulose and Mono-Q. Molecular weight of the purified VFH was estimated as 79kDa by SDS-PAGE. The optimal temperature for a maximum hemolytic activity was at around 35$^{\circ}C$ and the activity was decreased at 4$0^{\circ}C$ Cytotoxicity of VFH was also investigated using RTG-2 cell line. LDH assay study showed that 50$\mu\textrm{g}$/m1 of VFH release 80% of total cellular LDH (lactate dehydrogenase) from RTG-2 cell and microscopic observation also showed the morphological change of cell.

• The Journal of the Korean Society for Microbiology
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• v.15 no.1
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• pp.71-75
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• 1980

Antisera to ox red blood cell were prepared by intraperitoneal hypermultiple injections without adjuvant in outbred white rabits. Purified IgG fraction from these rabbits anti-ox red blood cell antiserum for T subset assay was obtained by precipitation with 50% saturated ammonium sulphate followed by DEAE-cellulose chromatography and Sephadex G-150 gel filtration. These purified IgG fraction was compared with Cappel company standard IgG fraction for $T_G$ subpopulation assay. We used home-made IgG fraction and obtained favorable results in $T_G$ subopulation assay as Cappel company standard IgG fraction.

• Microbiology and Biotechnology Letters
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• v.23 no.4
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• pp.432-438
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• 1995

The intracellular alginase from Vibrio sp. AL-145 was purified by ion chromatography on DEAE-Cellulose column, Q-Sepharose column, and gel filtration on Sephadex G-100 column. The optimum pH and temperature for the activity of the purified intracellular enzyme were 8.0 and 37$\circ$C, respectively. The enzyme was stable at the pH range of 7.5-8.5, and at 30$\circ$C for 30 min. The molecular weight of the intracellular enzyme was estimated to be about 23, 000 daltons by SDS-polyacrylamide gel electrophoresis. NaCl was required for enzyme activity and the optimum concentration was 0.5 M. The activity of intracellular enzyme was inhibited by Co$^{2+}$, Hg$^{2+}$, Zn$^{2+}$, 0-phenanthroline, $\rho$-CMB, EDTA and iodoacetate, and stimulated by Ca$^{2+}$, L-cysteine and 2-mercaptoethanol. This enzyme was an alginase specifically degrading alginic acid.