• IMMUNE NETWORK
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• 제10권6호
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• pp.230-238
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• 2010

Background: The MK1 strain, a novel bacterial isolate from soft-rotten tissue of the Neungee mushroom, produces copious amounts of exopolysaccharide (EPS) in a dextrose minimal medium. This study examined the molecular characteristics and immunomodulatory activity of MK1 EPS. Methods: The EPS in the culture supernatant was purified by cold ethanol precipitation, and characterized by SDS- PAGE/silver staining and Bio-HPLC. The immunomodulatory activities of the EPS were examined using the mouse monocytic cell line, RAW 264.7 cells. Results: The molecular weights of the purified EPS were rather heterogeneous, ranging from 10.6 to 55 kDa. The EPS was composed of glucose, rhamnose, mannose, galactose, and glucosamine at an approximate molar ratio of 1.00 : 0.8 : 0.71 : 0.29 : 0.21. EPS activated the RAW cells to produce cytokines, such as TNF-${\alpha}$ and IL-$1{\beta}$, and nitric oxide (NO). EPS also induced the expression of co-stimulatory molecules, such as B7-1, B7-2 and ICAM-1, and increased the phagocytic activity. The macrophage-activating activity of EPS was not due to endotoxin contamination because the treatment of EPS with polymyin B did not reduce the macrophage-activating activity. Conclusion: The EPS produced from the MK1 strain exerts macrophage-activating activity.

• BMB Reports
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• 제39권6호
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• pp.671-676
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• 2006

The holoenzyme of protein phosphatase (PP) from tulip petals was purified by using hydrophobic interaction, anion exchange and microcystin affinity chromatography to analyze activity towards p-nitrophenyl phosphate (p-NPP). The catalytic subunit of PP was released from its endogenous regulatory subunits by ethanol precipitation and further purified. Both preparations were characterized by immunological and biochemical approaches to be PP2A. On SDS-PAGE, the final purified holoenzyme preparation showed three protein bands estimated at 38, 65, and 75 kDa while the free catalytic subunit preparation showed only the 38 kDa protein. In both preparations, the 38 kDa protein was identified immunologically as the catalytic subunit of PP2A by using a monoclonal antibody against the PP2A catalytic subunit. The final 623- and 748-fold purified holoenzyme and the free catalytic preparations, respectively, exhibited high sensitivity to inhibition by 1 nM okadaic acid when activity was measured with p-NPP. The holoenzyme displayed higher stimulation in the presence of ammonium sulfate than the free catalytic subunit did by protamine, thereby suggesting different enzymatic behaviors.

• Journal of Microbiology and Biotechnology
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• 제3권4호
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• pp.298-302
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• 1993

Pullulan was produced from starch hydrolysate with Aureobasidium pullulans SH8646. We could measure the correct amount of pullulan produced without the interference of starch from the culture supernatant by using a bacterial $\alpha$-amylase treatment and ethanol: acetone (1:1) precipitation. When 5% acid-hydrolyzed starch was used as a carbon source, the dry cell weights obtained were similar irrespective of DE values of starch hydrolysates. The dry cell weights of those on the starch hydrolysate media prepared with 0.1 N HC1 treatment, were slightly higher (9.5~10.5 g/l) than those on the starch hydrolysate media prepared with 1.0 N HCl (8.5~9.5 g/l). And among the starch hydrolysates showing DE values lower than 50, maximum pullulan production of 15 g/l was obtained at DE 30~40 starch hydrolysate but those showing DE values higher than 50, the pullulan production was increased with the increase of the DE value of starch hydrolysates. From the media containing 5%, 10%, and 15% starch hydrolysate (DE 25, 45, and 75), about 20~34% pullulan yield was obtained and the maximum pullulan yield of 34% (17g/l) was obtained from 5% DE 75 starch hydrolysate. The pullulan yields from starch hydrolysate media were much lower than those from glucose, maltose, maltotriose, and sucrose media.

• 한국식품과학회지
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• 제13권1호
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• pp.67-73
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• 1981

Streptomyces chibaensis로부터 분비하는 이눌라아제를 일연(一連)의 에탄올 침전, DEAE-cellulose 칼럽크라마토그래피 및 Sephadex-200 gel frltration 방법에 의(依)해서 120배 분리정체하였고 정제된 효소의 최적 pH는 6.5이고 pH $5.0{\sim}9.0$ 범위내에서는 비교적 안정성을 보였다. 효소는 $Mn^{++},\;Mg^{++}$ 및 $Ca^{++}$의 존재하에서는 약간 활성화되었고, $Hg^{++}\;Ag^{++}$의 존재하에서는 강한 비활성화를 보였다. 정제된 효소는 endo 형(型)으로 Km 값은 $4.54{\times}10^{-4}M$이었다.

• 한국식품영양학회지
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• 제7권3호
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• pp.159-168
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• 1994

From the hot water extract of bracken(Pteridium aquilinum var. latiusculum), a Korean win edible plant, anti-complementary acidic polysaccharides were Isolated. Crude polysaccharide fraction(HPA-1) was obtain ed by methanol reflux, ethanol precipitation, dialysis, and lyophilization. HPA-1 contained 81.80% of total sugar, 30.40% of uronic acid, and 15.60cA of protein. HPA 1 was purified consecutively by cetavlon fractionation and chromatography including ion exchange nth DEAE-Sepharose CL 6B and gel permeation with Sephadex G-100 and Sepharose CL-6B. HPA-2- IVa and HPA-Va-2 were nearly homogeneous on HPLC and had 500,000 and 560,000 daltons of molecular weights, respectively. HPA-2-Wa consisted of fucose, galacturonic acid, and glucuronic acid at the molar ratio of 1.40 : 0.97 : 1.88. HPA-2-Va 2 was composed of rhamnose, galactose, and galacturonic acid at the molar ratio of 1.00 : 1.38 : 1.39. The polysaccharides were found to activate the C3 component both In the presence and In the absence of Ca2+ through the crossed-immunoelectrophoresis suggesting that those Involved in both classical and alternative complement pathway.

• Mycobiology
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• 제36권1호
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• pp.50-54
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• 2008

This study examined the chemical composition of A. blasiliensis and the chemical structural properties of an immuno-stimulating polysaccharide. The amino acids, free sugars, and organic acids by HPLC and fatty acids by GC were analyzed. The immuno-stimulating substance from A. blasiliensis was extracted with hot water and purified by ethanol precipitation. It underwent ion exchange chromatography on DEAE-cellulose and gel filtration on Toyopearl HW 65F. Through GP-HPLC, the substance was found to be homogeneous. Its chemical structure was determined by $^{13}C-NMR$. Fatty acids, organic acids, and sugar alcohol composition consisted exclusively of linoleic acid, fumaric acid and mannitol, respectively. The amino acids were mainly glutamic acid, glycine, and arginine. By $^{13}C-NMR$ analysis, the immuno-stimulating substance was identified as ${\beta}-(1{\rightarrow}3)\;(1{\rightarrow}6)$-glucan, composed of a backbone with $(1{\rightarrow}3)$-linked D-glucopyranosyl residues branching a $(1{\rightarrow}6)$-linked D-glucopyranosyl residue. The ${\beta}$-glucan from A. blasiliensis showed pronounced immuno-stimulating activity on the antibody-production ability of B-lymphocytes by the hemolytic suspension assay. In these results, A. blasiliensis was estimated to have potent pharmacological properties and potential nutritional values.

• Journal of Microbiology
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• 제43권6호
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• pp.555-560
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• 2005

Laccase is one of the ligninolytic enzymes of white rot fungus Trametes versicolor 951022, a strain first isolated in Korea. This laccase was purified 209-fold from culture fluid with a yield of $6.2\%$ using ethanol precipitation, DEAE-Sepharose, Phenyl-Sepharose, and Sephadex G-100 chromatography. T. versicolor 951022 excretes a single monomeric laccase showing a high specific activity of 91,443 U/mg for 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as a substrate. The enzyme has a molecular mass of approximately 97 kDa as determined by SDS-PAGE, which is larger than those of other laccases reported. It exhibits high enzyme activity over broad pH and temperature ranges with optimum activity at pH 3.0 and a temperature of $50^{\circ}C$. The $K_m$ value of the enzyme for substrate ABTS is $12.8{\mu}M$ and its corresponding $V_{max}$ value is 8125.4 U/mg. The specific activity and substrate affinity of this laccase are higher than those of other white rot fungi, therefore, it may be potentially useful for industrial purposes.

• 한국미생물·생명공학회지
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• 제20권3호
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• pp.311-315
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• 1992

Coriolus versicolor KFCC 30388 균을 액체배양하여 배양 6일째 균사체 생성이 가장 우수하였으며, 건조 균사체량은 17.2g/l을 얻었다. 균사체들로부터 항암성 단백다당체를 분리하여 in vitro에서 plate assay 방법으로 human cancer cell lines 3종에 대한 항암성을 조사한 결과, 0.1mg/ml의 survival rate를 나타내어 항암성을 확인하였다.화학적 분석 결과 단벡다당체는 polysaccharide 42.2%, protein 10.5%로 이루어졌으며, 구성 단당류는 D-glucese, L-glucese, galatose, mannose, xylose 등 으로 되어 있었다.

• Journal of Applied Biological Chemistry
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• 제43권4호
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• pp.235-239
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• 2000

A polysaccharide with antithrombin activity in Manda$^{(R)}& (PAM) was purified via procedures comprising three major steps, i.e. fractional precipitation with ethanol, anion exchange chromatography, and gel permeation chromatography. PAM showed a symmetrical peak on size exclusion HPLC, as assessed by refractive index, and behaved as a single band on cellulose acetate electrophoresis. The average molecular mass was estimated to be 222 kDa by gel filtration. PAM was found to be a sulfated heteropolysaccharide that contains sulfate group (20.5%, w/w) and uronic acid moiety (7.1 %, w/w) in addition to neutral sugar consisting of fucose, xylose, mannose, galactose, and glucose in a molar ratio of 1.00 : 0.35 : 0.28: 0.22 : 0.15. This polysaccharide appeared to inhibit blood coagulation via the intrinsic pathway in a dose-dependent pattern. The clotting of fibrinogen by thrombin was also significantly mitigated by the presence of PAM.

• Journal of Microbiology and Biotechnology
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• 제19권6호
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• pp.566-572
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• 2009

Lentinus edodes is a well-known edible and medicinal mushroom used in Oriental cultures. Recently, L. edodes has attracted a lot of attention owing to its antifungal activity, antibacterial activity, antiviral activity, hepatoprotective effect, antitumor activities, and immunomodulatory and cytotoxic effects. In this study, the water-soluble crude polysaccharides, CPF and CPB, which were obtained from the fruiting body and culture cell-free broth of L. edodes by hot-water extraction and ethanol precipitation, were fractionated by DEAE cellulose and Sepharose CL-6B column chromatography, resulting in six polysaccharide fractions, CPFN-G-I, CPFN-G-II, CPFN-G-III, CPFA-G, CPBN-G, and CPBA-G Among these fractions, CPFN-G-I, CPBN-G, and CPBA-G were shown to stimulate the functional activation of macrophages including NO production, cytokine expression, and phagocytosis.