• Korean Journal of Microbiology
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• v.32 no.4
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• pp.285-290
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• 1994

About 500 bacterial and fungal strains from a wide variety of natural habitats were screened for a new type II restriction endonuclease. Among the 500 species, we selected one species that produced a new restriction endonuclease. This strain has an optimum temperature of $30^{circ}C$ for growth. Morphological, cultural, and physiological characteristics were examined for identification of the isolated strain J-482. This strain was found to belong to the genus Alcaligenes. The restriction endonuclease was named as AspJI and partially purified from Alcaligenes sp. J-482 by DEAE-Sephadex A-50 column chromatography and gel filtration. Most of other nucleases were removed by the purification steps. The AspJI has a substrate specificity to ${lambda}$ DNA, pBR322 and Adenovirus-2 DNA. For its maximal activity, the isolated enzyme requires $MgCl_2$, which should be at least 12.5 mM and it does not need any other cofactors. It is maximally active in the absence of NaCl and is completely inactivated at 100 mM NaCl. The pH and temperature optima for activity were pH 7.5 and $37^{circ}C$, respectively. The DNA fragments generated by digesting ${lambda}$ DNA, pBR322, and Adenovirus-2 DNA with AspJI were the same as that produced by AatII. This suggests that AspJI is an isoschizomer of AatII.

• KSBB Journal
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• v.10 no.5
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• pp.589-597
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• 1995

Protein-bound polysaccahrides(PBP) were isolated, purified, and characterized from the sawdust media after harvesting the fruit body of Flammulina velutipes. The yield of the crude PBP(Fr.CA) extracted from the sawdust media, was 0.367% relative to the original sawdust media. The total sugar and protein contents of Fr.CA were 19.8% and 23.8% respectively. Using the membrane filtration, the fraction of which the molecular weight is over 300 kDa(Fr.A) was isolated from the Fr.CA and the yield was 44.6% relative to the Fr.CA. This result indicates that high molecular PBP is the dominant components of the Fr.CA. The Fr.A was separated into three fractions (Fr.A-1, Fr.A-2 and Fr.A-3) whose yields are 5.8%, 8.5% and 13.2% respectively. These fractions were further purified using gel filtration, obtaining a single peak in each fraction that considered as pure PBP Among them, the yield of Fr.A-1-${\alpha}$ was 20.9% relative to the Fr.A-1, and the molecular weight was 800 kDa. Monosaccharide components such as glucose, galactose, mannose, xylose and fucose could be detected all fractions by a HPLC analysis. Especially in the Fr.A-1-${\alpha}$ fraction, the content of glucose and galactose appeared to be high.

• Microbiology and Biotechnology Letters
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• v.31 no.4
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• pp.377-382
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• 2003

Purification of methioninase resulted in a yield of 69%, and SDS-PAGE analysis of the purified product revealed a single band of approximately 43 kDa in molecular weight. in vitro experiments with cancer cells incubated in methionine-free media demonstrated an increase in $^{11}$ C-methionine uptake to 25.8$\pm$1.1% at 6 hr, 31.8$\pm$0.8% at 24 hr, and 62.2$\pm$0.6% at 48hr, compared to controls. Treatment of the cancer cells with purified methioninase showed no decrease in survival after a 2 hr incubation with 0.01 U/ml, but survival of RR1022 cells decreased 30% after 24 to 48 hr incubation. SKOV-3 cells showed a 5% and 14% decrease in survival with 0.1 and 1 U/ml methioninase after 24 hr. After 48hr survival decreased 15% and 24% with 0.1 and 1 U/ml methioninase. Measurements of $^{11}$ C-methionine uptake in RR1022 cells demonstrated no change at 2 hr, but a 13.7$\pm$4.7% and 40.7$\pm$2.6% increase in uptake at 24 and 48 hr, respectively. SKOV-3 cells also showed no change at 2 hr, but had a 17.7$\pm$7.2% and 38.9$\pm$4.9% increase in $^{11}$ C-methionine uptake after 24 hr and 48 hr treatment with methioninase, respectively. $^{11}$ C-methionine PET imaging revealed clear visualization of both the tumors and contralateral infectious lesions. Administration of rMET appeared to result in a slight increase in tumor:nontumor contrast on $^{11}$ C-methionine PET images. Injection of purified methioninase also produced PET images where tumor uptake was higher than that of infectious lesions.

• Korean Journal of Pharmacognosy
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• v.24 no.4
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• pp.267-281
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• 1993

To find antitumor components from higher fungi, the mycelia of Collybia confluens (Pers. ex Fr.) Kummer were cultured in artificial media. For efficient production of the mycelia, the influences of various modifications of culture conditions were examined. A water-soluble protein-bound polysaccharide fraction, Fr. A, was obtained from the mycelia by hot water extraction. When Fr. A was purified and fractionated by DEAE-cellulose and Sepbadex G-200 gel filtration chromatographies into four fractions which were designated B, C, C-I and C-II. The tumor inhibition ratios of these fractions ranged from 46% to 75% against the solid forms of sarcoma 180 in ICR mice at doses of 20 and 50 mg/kg/day when given intraperitoneally. Especially, Fr. C which was named Collyban(CB) exhibited a marked life-prolonging effect of the mice against ascitic forms of sarcoma 180 at a dose of 50 mg via i.p. administration. To extend spectra of the antitumor activities and eliminate the effects of allograft rejection, the characterization of antitumor effects of CB was performed in syngeneic host-tumor systems. It did not show any antitumor activity against L1210 murine leukemia in $CD_2Fl$ mice but prolonged their life span against ascitic forms of $MM_{46}$ carcinoma in $C_3H/He$ mice. Also it exhibited antitumor activity against human cervical cancer HeLa cells that were xenografted into nude mice having BALB/c genetic backgrounds by the i.p. injection at a dose of 100 mg/kg/day. In order to characterize the antitumor components, CB was examined by chemical analysis. It was acidic protein-bound polysaccharides composed of 31% polysaccharide, 27% protein and 3% hexosamine. CB was fractionated into two fractions, Fr. C-I(M.W.: 500 Kd) and Fr. C-II(M.W.:30 and 8 Kd) by Sephadex G-200 gel filtration chromatography.

• Korean Journal of Food Science and Technology
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• v.53 no.3
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• pp.304-312
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• 2021

The present study aimed to develop new physiologically active ingredients from Angelica gigas. The polysaccharides purified from A. gigas, AGE-2c-I, showed potent anti-complementary activity in a dose-dependent manner. C₃ activation products were identified through crossed immuno-electrophoresis using anti-human C₃ antibodies and the anti-complementary activity of AGE-2c-I under Ca⁺⁺-free conditions suggests that AGE-2c-I may induce complementary activation via both alternative and classical pathways. In addition, AGE-2c-I augmented the production of various cytokines, such as interleukin (IL)-6, IL-10, IL-12, and tumor necrosis factor-α, by peritoneal macrophages. Furthermore, intravenous (i.v.) administration of AGE-2c-I dose-dependently enhanced natural killer cell cytotoxicity against YAC-1 lymphoma. In experimental lung metastasis, prophylactic i.v. administration of AGE-2c-I inhibited lung metastasis by 58% at 100 ㎍/mouse. From the above results, we suggest that AGE-2c-I purified from A. gigas has potent immune system-stimulating activities, and is a potentially promising food ingredient beneficial to human health.

• The Journal of the Korean Society for Microbiology
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• v.21 no.1
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• pp.151-161
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• 1986

In order to develop sensitive and sepcific assay methods for E. coli heat labile enterotoxin(LT) hybridoma cell lines secreting LT specific monoclonal antibody were obtained. LT was purified from cell lysate of E. coli O15H11. The steps included disruption of bacteria by French pressure, DEAE Sephacel ion exchange chromatography, Sephadex G200 gel filtration, and second DEAE Sephacel ion exchange chromatography, successively. Spleen cells from Balb/c mice immunized with the purified LT and $HGPRT^{(-)}$ plasmacytomas, $P3{\times}63Ag8.V653$ were mixed and fused by 50% (w/v) PEG. Hybrid cells were grown in 308 wells out of 360 wells, and 13 wells out of them secreted antibodies reacting to LT. Among these hybridoma cell 1G8-1D1 cell line was selected since it had produced high-titered monoclonal antibody continuously. By using culture supernatant and ascites from 1G8-1D1 cells the monoclonal antibody was characterized, and an assay system for detecting enterotoxigenic E. coli was established by double sandwich enzyme-linked immunosorbent assay (ELISA). The following results were obtained. 1. Antibody titers of culture supernatant and ascites from 1G8-1D1 hybridoma cells were 512, and 102, 400, respectively by GM1-ELISA and its immunoglobulin class was IgM. 2. The maximum absorption ratio of 1G8-1D1 cell culture supernatant to LT was 90% at $300\;{\mu}g/ml$ of LT concentration. LT concentration shown at 50% absorption ratio was $103.45{\mu}g$ and the absorption ratio was decreased with tile reduction of LT concentration. This result suggests that monoclonal antibody from 1G8-1D1 hybridoma cell bound with LT specifically. 3. The reactivities of 1G8-1D1 cell culture supernatant to LT and V. cholerae enterotoxin(CT) were 0.886 and 0.142(O.D. at 492nm) measured by the GM1-ELISA, indicating 1G8-1D1 monoclonal antibody reacted specifically with LT but not with CT. 4. The addition of 0.1ml of ascites to 0.6mg and 0.12mg of LT decreased the vascular permeability factor to 41% and 44% respectively, but it did not completely neutralize LT. 5. By double sandwich ELISA using monoclonal antibody, as little as 75ng of the purified LT per ml could be detected. 6. The results by assay of detecting LT in culture supernatants of 14 wild strains E. coli isolated from diarrhea patients by the double sandwich ELISA were almost the same level as those by reverse passive latex agglutination.

• Applied Biological Chemistry
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• v.39 no.6
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• pp.494-500
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• 1996

Uptake of hen metal ions by water dropwort (Oenanthe stolonifera DC.) and its cadmium-binding protein were studied to probe for good method to remove heavy metal contaminants from environments. The plant was cultured in the culture medium (pH 7.0) containing the various concentrations of $Cd^{2+}$, $Cr^{3+}$ or $Pb^{2+}$, for 3 and 7 days. The residual heavy metals deposited in roots linearly increased as the metal ions concentration increased up to 17 ppm for $Cd^{2+}$, 20 ppm for $Cr^{3+}$ and 50 ppm for $Pb^{2+}$. Above these concentrations, the plant growth was inhibited and the uptake rates of the metal ions decreased. The heavy metals absorbed by the plant were mostly deposited in roots. In particular, the residual concentration of lead in roots was about four times higher than those of cadmium and chromium. When cultured in the medium containing 20 ppm of each metal ion, 80% of cadmium, 90% of cromium and 96% of lead were deposited in roots out of the total residual metal ions in the plant. These values correspond to 6.1 mg of cadmium, 5.2 mg of chromium and 23.6 mg of lead per one gram of roots tissue on a dry weight basis. A cadmium-binding protein was partially purified by extraction, gel filtration and DEAE-Cellulose chromatography from water dropworts that was grown in the medium containing 20 ppm $Cd^{2+}$. The purified protein was a single band on SDS- and non-denaturing- polyacrylamide gel electrophoresis. Its molecular mass was estimated to be ca. 5,000 dalton by gel filteration. Analysis of amino acid composition of the protein indicated that it had a typical amino acid composition of heavy metal-binding protein in that it contained 27% of acidic amino acids and 9.9% of cysteine. However, it is likely that the protein is a new plant metal-binding protein, since its amino acid composition is somewhat different from those of phytochelatins that have been known so far.

• Korean Journal of Agricultural Science
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• v.21 no.2
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• pp.122-132
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• 1994

Bacteriostatic activity of secretory immumoglobulin A (SIgA) in human and bovine colostrums on enterotoxigenic type Salmonella was tested in the tissue culture medium. SIgA was used in $0.1{\sim}5.0mg/m{\ell}$ concentration with or without the addition of egg lysozyme tested for theirs bacteriostatic activites. 1. Bovine SIgA rich fraction with a large amount of $IgG_1$-dimer could be prepared from bovine colostrum of Holstein cows by anion exchange chromatography using DEAE-Sephadex A-50 and gel filtration on Sephadex G-200 and Sepharose 6B. 2. Human SIgA appeared to be the most bacteriostatic effect for all varieties of Salmonella in a range of $0.5{\sim}1.0mg/m{\ell}$ Bovine SIgA showed a marked bacteriostatic effect increased by increasing the concentration. Bovine IgG had not show bacteriostatic effect against both enterotoxigenic type Salmonella. Egg lysozyme as well as bovine SIgA also showed a marked bacteriostatic effect increased by increasing the concentration. 3. When the growth inhibition of human SIgA was tested by adding egg lysozyme with time interval, egg lysozyme showed bacteriostatic effect as compared with control. But human SIgA and adding with lysozyme showed slight the bacteriostatic effect. 4. When the growth inhibition of bovine SIgA was tested by adding egg lysozyme with time interval, all treatment against S. enteritidis showed bacteriostatic effect as compared with control In the case of S. typhinwrium, egg lysozyme showed a marked bacteriostatic effect as compared with control.

• Parasites, Hosts and Diseases
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• v.24 no.2
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• pp.177-186
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• 1986

This study was designed to evaluate the partially purified antigens which were fractionated from crude extract of Paragonimus westermani and to monitor the enzyme-linked immunosorbent assay (ELISA) in experimental cat paragonimiasis during the course of infection as well as before and after chemotherapy. Crude extract of 6-month-old adult P. westermani was fractionated to 5 antigens by successive applications of ammonium sulfate precipitation, ion exchange chromatography and gel filtration. And the cats, 10 in each group, were infected with 60, 30, 15, and 5 metacercariae, then the half of each group was treated with praziquantel 2 times in one day of 100mg per kilogram of weight on 150 days after the infection. Sera were collected every 10 days. ELISA was performed with the concentration of $2{\mu}g/ml$ antigen, 100 times diluted sera and 1,000 times diluted alkaline phosphatase conjugated anti-cat IgG. The results were as follows: 1. Absorbance by ELISA with proteins precipitated by differential concentration of ammonium sulfate was the highest at $51{\sim}65%$ precipitate (PA2), followed by $0{\sim}50%$ precipitate (PAl), $66{\sim}80%$ precipitate (PA3), and $81{\sim}90%$ precipitate (PA4). Unprecipitated protein over 90% ammonium sulfate (PA5) showed the lowest antigenicity. 2. Fractionation of PA1, PA2, and PA3 through the DEAE-cellulose column did not differentiate the antigenic proteins. 3. By passing through the Sephadex G-200 column, PA1 and PA2 were fractionated to high molecular weight proteins and those of low molecular weight which showed high absorbance by ELISA (PA1-I, II and PA2-I, II). But PA3 was shown to have a fraction of high molecular weight proteins (PA3-I) which showed high antigenicity. 4. SDS-polyacrylamide gel electrophoresis of PA1-I, P A1-II, PA2-I, PA2-II, PA3-I, and crude extract was performed. Fraction PA1-I was composed of proteins which had the molecular weight of 270 kilodaltons(KD) to 196 KD; of them 220KD protein was major band. Fraction PA2-I was composed of $255{\sim}225\;KD$, and PA3-I, $255{\sim}240\;KD$, respectively. Fraction PA1-II and fraction PA2-II consisted of 30 KD proteins. 5. Absorbance by ELISA began to increase within $10{\sim}20$ days after the infection and reached the highest on $140{\sim}180$ days, then made plateau thereafter. 6. Absorbance by ELISA decreased after praziquantel treatment. In 60 metacercariae infection group, the absorbance had been decreasing, but remained within the positive range during observation period, while those of 30, 15, and 5 metacercariae infection groups turned to negative range. 7. Fraction PA1-II showed the highest antigenicity in ELISA, then fraction PA2-I, fraction PA1-I, fraction PA2-II, fraction PA3-I and crude extract followed. In early phase of infection, the absorbance of fraction PA1-II showed more rapid increase than those of the other fractions and it came to positive range at $20{\sim}30$ days after infection.

• Applied Biological Chemistry
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• v.14 no.1
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• pp.59-97
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• 1971

As a study on the cellulase of Myriococcum albomyces the culture media for enzyme formation and properties of its crude preparation were investigated and the crude enzyme preparation was further fractionated. The results are summarized as follows: 1. Wheat bran solid culture produced stronger activities of cellulase than rice bran or defatted soy bean meal solid culture. 2. Shaking culture with wheat bran, rice bran or defatted soy bean meal produced higher cellulase activities than solid culture with the corresponding media. 3. The enzyme formation was higher at $45^{\circ}C$ than at $37^{\circ}C$ or $50^{\circ}C$ regardless of the kind of culture medium. 4. The formation of CMCase activity was more promoted by organic nitrogen source than inorganic nitrogen source. 5. The formation of cellulase activities were increased 1.5 to 3.0-fold by adding CMC, Avicel or cellulose powder as an inducer into 5% wheat bran basal medium. 6. Cellulase production using a tank culture procedure with addition of CMC or Avicel as an inducer was the highest at fifth day and thereafter decreased slightly. 7. The crude enzyme preparation showed pH optimum in 4.0 to 4.5, and pH stability in the range of 3.5 to 8.0. Optimum temperature for the activity was $65^{\circ}C$ which was higher than among other cellulases and it was stable at $60^{\circ}C$ for 120 minutes. 8. Dialyzed crude enzyme was activated by $Ca^{++}$ and $Mg^{++}$, but inhibited by $Hg^{++}$, $Cu^{++}$ and $Ag^{+}$. 9. Four different types of cellulase, i. e., fraction I, fraction II-a, fraction II-b, and fraction III were purified from the culture filtrate of Myriococcum albomyces through a sequence of ammonium sulfate fractionation, and elution chromatography on DEAE-Sephadex A-25, Amberlite CG-25 type 2 and hydroxyapatite columns. 10. These four cellulase fractions were showed to be homogenous by electrophoresis and ultracentrifugation and also gave a typical ultraviolet absorption spectrum of protein. 11. Four purified fraction showed different specificity toward substrates, fraction I has a stronger activity toward Avicel, cellulose powder, and gauze than that of other cellulase fractions. Fraction II-a had a powerful activity toward cellobiose but it was almost inactive agaisnt fibrous cellulose contrary to fraction I. On the contrary, the main component fraction II-b had a fairly higher activity on CMC and Avicel. Activity of fraction II-b toward cellobiose was about one-third of that of fraction II-a and activity on Avicel was lower than that of fraction I. Fraction III had a more powerful activity in decreasing viscosity of CMC. 12. Final hydrolysis products of fibrous cellulose by each fraction were cellobiose and glucose. Whereas oligosaccharides were predominant in the early stage of hydrolysis, prolonged reaction produced more glucose than cellobiose. Fraction I and fraction II-a acted synergically on Avicel. 13. Optimum pH for the activities of cellulase fraction I, fraction II-a, fraction II-b and fraction III were found to be 5.5, 5.0, 4.0 and $4.0{\sim}4.5$, respectively. These fractions were found to be stable in the range of pH $3.0{\sim}7.5$. 14. Optimum temperature for the activities of fraction I, fraction II-a, fraction II-b, and fraction III were $50^{\circ}C$, $55^{\circ}C$, $60^{\circ}C$ and $55^{\circ}C$, respectively. No less of activity was found by heating 120 minutes at $55^{\circ}C$ and fraction II-a was more stable than the others at $60^{\circ}C$. 15. Fraction I and fraction II-b were activated by $Ca^{++}$ and $Mg^{++}$ but inhibited by $Hg^{++}$ and $Ag^{+}$.