• Microbiology and Biotechnology Letters
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• v.20 no.5
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• pp.559-564
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• 1992

To increase the efficency of utilizing cellulosic biomass, a potent xylanase producing bacteria was isolated and identified as Bacillus sp. N-25. Extracellular xylanase from Bacillus sp. N-25 was partially purified by ammonium sulfate precipitation, DEAE-Sephadex A-25 and Sephadex G-IOO column chromatographies. The xylanase was single fraction on chromatography and was true xylanase without cellulase activity. The enzyme was stable at pH 6-8 and 80% activity was remained at $50^{\circ}C$ for 30 min, but was inhibited by $Hg^{2+}$, $Ag^{2+}$, and $Mn^{2+}$. From the fact that the major end product was xylose, we suggested that the enzyme is an exo-xylanase which may be a prime candidate for industrial use.

• Microbiology and Biotechnology Letters
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• v.22 no.1
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• pp.73-79
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• 1994

The optimum cultural temperature and time for the pullulanase production by Bacillus cereus subsp. mycoides were 35$\circ $C and 48 hrs, respectively. The addition of egg albumin and casein to the basal medium increased the enzyme production. The enzyme was purified by ammonium sulfate fractionation and DEAE-cellulose column chromatography. specific activity of the purified enzyme was 82.37 U/mg protein and yield of theenzyume activity was 62.1%. The purified enzuyme showed a single band on ployacrylamide disc gel electrophoresis and its molecular weight was estimated to be 66.,000 by SDS-polyacrylamide disc gel electrophoresis. The isoelcular point for the purified enzyme was pH 5.0. The optimum temperature and pH were 50$\circ $C and pH 6.5, respectively. The purified enzyme was stable below 40$\circ $C and in the pH range of 6.5~10.0 The pullulanase activity was greatly inhited by Ag$^{+}$, Hg$^{2+}$ and EDTA, and its heat stability was increased by the addition of Ca$^{2+}$. The tydrolysis product with the enzyme on pullulan was maltotriose.

• Korean Journal of Microbiology
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• v.31 no.3
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• pp.237-244
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• 1993

Aspartate aminotransferase (ASAT) (L-aspartate : 2-oxyoglutarate, EC 2.6. 1. 1.) from Streptomyces fradiae NRRL 2702 has been purified by acetone precipitation, DEAE-cellulose, hydroxyapatite, and preparative electrophoresis (Prep cell), of which the last was the most effective step in the purification of ASAT. The molecular mass was estimated to be 54,000 dalton by SDS-PAGE and 120,000 dalton by gel filtration chromatography. Preparative isoelectric focusing of purified ASAT resulted in one polypeptide band with a pI of 4.2, showing homogeneity and indicating that the enzyme is composed of two identical subunits. The enzyme was specific for L-aspartate as an amino donor ; the $K_{m}$ values were determined to be 2.7 mM for L-aspartate, 0.7 mM for 2-oxoglutarate, 12.8 mM for L-glutamate, and 0.15 mM for oxaloacetate. The enzyme was relatively heat-stable, having maximum activity at 55.deg.C, and it had a broad pH optimum ranging from 5.5 to 8.0. The activity of the purified enzyme was not inhibited by ammonium ions. This paper reports the first purification and characterization of the aspartate aminotransferase from a species of Streptomyces.s.

• Proceedings of the Korean Society of Postharvest Science and Technology of Agricultural Products Conference
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• 2007.11a
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• pp.27-31
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• 2007

This study relates to low and medium molecular weight isoflavone-${\beta}$-D-glucan produced by submerged liquid culture of Agaricus blazei, a method of producing the isoflavone-B-D-glucan using autolysis enzyme of Agaricus blazei mycelia, and use of the isoflavone-B-D-glucan for anti-cancer and immunoenhancing effect. In acordance with one aspect of the present study, it deals with a method of producing isoflavone-${\beta}$-D-glucan, which comprises the followings; 1) culturing and separating mushroom mycelia, 2) producing low-medium molecular weight isoflavone-${\beta}$-D-glucan from high molecular weight one. The cytotoxicity on human cnacer cell line (Caco-2, MCF-7), the expression of Cyclin D, Bcl-2, Bax protein, p21 protein, p53 protein in MCF-7 cells assessed by SDS-PAGE and immunoblotting, and other immuno related factors such as TNF-${\alpha}$ and IL-1B activities were examined. Structural identification of isoflavone-${\beta}$-D-glucan which showed cytotoxicity against cancer cell and immunoenhancing effects was carried by separation with DEAE-cellulose column chromatography, TLC, HPLC, IR, NMR. Clinical test for the cancer patients (n=119) for 6 month was carried out, and immunoenhancing factors (NK. cell number, ratio of T4/T8) were checked. We concluded the identified isoflavone-${\beta}$-D-glucan has immuno enhancing effects and could be useful for cancer chemoprevention.

• BMB Reports
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• v.29 no.4
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• pp.335-342
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• 1996

Porcine liver cysteinesulfinic acid decarboxylase was purified approximately 460-fold by means of ammonium sulfate fractionation and sequential column chromatographic separation with Sephadex G-100, DEAE-cellulose and hydroxylapatite. The enzyme has a flat pH profile with maximum activity occurring between pH 6.0 and 7.6. Pyridoxal 5'-phosphate must be present in all buffers used for purification procedures in order to stabilize the enzyme. Addition of sulfhydryl reagents such as 2-mercaptoethanol are also necessary to maintain maximum enzyme activity throughout purification. The absorption spectrum shows that cysteinesulfinic acid decarboxylase is a pyridoxal 5' -phosphate-containing protein. The major absorption is at 280 nm with two smaller absorption regions, one at 425 nm which is ascribed to a Schiffs base between pyridoxal phosphate and protein, and another at 325 nm which is thought to be due to the interaction of 2-mercaptoethanol with the Schiffs base. A number of divalent cations tested did not affect enzyme activity with the exception of mercury, copper, and zinc which are inhibitory. The partially purified enzyme has an apparent $K_m$ of 0.94 mM for cysteinesulfinate. Cysteic acid is a competitive inhibitor of the enzyme with a $K_i$ of 1.32 mM. The molecular weight of the enzyme was estimated to be about 79,600 by using Sephadex G-200 column chromatography.

• Preventive Nutrition and Food Science
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• v.2 no.1
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• pp.71-76
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• 1997

Pullulanase was produced from the Klebisella pneumonias NFB_320 with the conmposition of 0.1% pullualn 1.5% yeast extract, 0.2% $K_2$HPO$_4$ and 0.02% MgSO$_4$.7$H_2O$(pH5.5). The optimum temperature for activity of the pulluanase was 3$0^{\circ}C$ and the highest yield of the enzyme was obtained after cell growth at 3$0^{\circ}C$ for 18hr, and maintained until 24hr cultivation. The pullulanase was successively purified 52.6 folds with 7.8% yield by acetone precipitation. DEAE-cellulose column chromatography and gel fitrations. The purified enzyme hydrolyzed pullulan into maltotriose exclusively. Chemical and physical properties of purified pullulanase from Klebisella pneumonias NFB-320 were examined. The optimum pH and temperature for enzyme activity were 5.0 and 6$0^{\circ}C$, respectively. The enzyme was stable between pH4 and 7, and up 5$0^{\circ}C$. The effect of mo-dification on the rate of enzyme reaction was studies with various chemicals and metal ions. The enzyme has been found to be inactivated by I$_2$ and N-bromosussinimide(NBS), which probably indicated the involve- ment of tryptophan residues in the active center of the enzyme.

• Microbiology and Biotechnology Letters
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• v.22 no.5
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• pp.485-490
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• 1994

A restriction endonuclease, PsyI, has been isolated from Pseudomonas syringae pv. pha- seolicola, and its catalytic properties have been studied. This enzyme was purified through strepto- mycin sulfate and ammonium sulfate fractionation, phosphocellulose Pll, DEAE-cellulose, hydroxy- apatite and Sephadex G-100 column chromatography. It's molecular weight was about 50,000 dalton as determined by 7.5% polyacrylamide gel electrophoresis containing 0.1% SDS. In catalytic proper- ties, PsyI shows stable at wide ranges of pH between 7.0 and 10.0, of temperature between 30$\circ$C and 37$\circ$C, and its thermal stability is between 25$\circ$C, and 45$\circ$C, at the presence Of 10 mM MgCl$_{2}$-PsyI essentially require Na salt for enzyme reaction, is rather inhibited in the high Na salt concent- ration. The presence of 2-mercaptoethanol is absolutely required for the enzyme activity. This endonuclease, PsyI was determined to be an isoschizomer of SalI from the results of the restriction mapping and DNA sequencing.

• Korean Journal of Pharmacognosy
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• v.27 no.3
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• pp.224-230
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• 1996

To find pharmacologically active hybrids among the inter-order protoplast fusants of Lentinula edodes and Ginoderma lucidam the antitumor test was performed and the fusant P22 was selected among them. The hot water and alkaline extracts from the cultured mycelia of P22 were purified and separated into four fractions by DEAE-cellulose anion exchange chromatography. When a dose of 20 mg/kg/day of each fractions was injected into ICR mice by i.p., the tumor inhibition ratio of Fr. IV against solid sarcoma 180 was the higher than any other fraction. Fr. IV was a protein-bound polysaccharide which was composed of 69. 12% polysacchafide and 9.76% protein and the molecular weight of Fr. IV was $6.7{\times}10^4$ dalton.

• YAKHAK HOEJI
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• v.43 no.1
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• pp.68-76
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• 1999

Phenoxazinone synthase catalyzes the oxidative condensation of two molecules of substituted o-aminophenol to the phenoxazinone chromophore of actinomycin. Mutant strain, Streptomyces sp. V-8-M-1 producing higher phenoxazinone synthase, was obtained from Streptomyces sp. V-8 by treatment of N-methyl-N'-nitro-N-nitrosoguanidine. The phenoxazinone synthase was purified from extract of mutant strain of Streptomyces sp. V-8-M-l by successive steps of streptomycin sulfate, ammonium sulfate precipitation. DEAE-cellulose and Sephadex G-200 column chromatography. Molecular weight of the enzyme was 360,000 daltons. The enzyme was composed of octamer of a single subunit of 45,000 daltons. The Km value and Vmax value for 3-HAA were $14.9{\;}{\mu}M$ and 9.5 mg/U, respectively. The optimal pH and temperature for the enzyme activity were 9.0 and $25~30^{\circ}C$, respectively. Treatment of the enzyme with group specific reagents, phenylglyoxal, p-hydroxymercury-benzoate, Nbromosuccinimide, 5.5'-dithiobis-nitrobenzoic acid and ethylmaleimide resulted in loss of enzyme activity, which shows arginine and cysteine residues are at or near the active site.

• Archives of Pharmacal Research
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• v.13 no.4
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• pp.330-337
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• 1990

The crude polysaccharide from Panax ginseng prepared by hot water extration and precipiation with ethanol was further fractionated into neutral and acidic fractions by DEAE- cellulose ion exchange chromatography. The chemical compositions were 85.0% carbohydrorate and 15.0% protein for the neutral fraction, and 28.4% carbohydrate, 10.0% protein and 29.0% uronic acid for the acidic fraction. The acidic fraction was more effective in increasing of the ratio of spleen to body weight, the number of antibody secreting cells to SRBC and phagocytic activity of reticuloendothelial system, as well as antitumor activity against the solid form of sarcoma 180 in ICR mice than the neutral fraction. All polysaccharide fractions were mitogenic to cultured spleen cells of C57BL/6 mice. However, FA was different from FN in the co-mitogenicities with lectin mitogens. Both crude and acidic fractions potentiated remarkably the mitogenic activity of PHA-P or LPS in dose-dependent manner but neutral fraction enhanced only that of LPS. Three polysaccharide fractions had no effect on that of Con A. These results suggest that the acidic fraction may stimulate B and Td cells as well as macrophages while the neutral fraction may simulate only B cells and macropages.