• 한국식품영양과학회지
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• 제24권1호
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• pp.147-153
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• 1995

The cultured mycelial cells of Ganoderma lucidum was extracted by alkali, and then neutralized by acid. The extract was passed through the column of DEAE cellulose for more purification. The neutral fraction was concentrated and precipitated with 95% ethanol. The precipitate was lyophilized and then PSG(polysaccharides) was obtained. PSG was composed of 82.2% polysaccharide, 0.7% protein and 17.1% uronic acid. Sugar conjugates of its hydrolysates were produced using with fluorescent compound(7-amino-1,3-naphthalene disulfonic aicd : 7-AGA), and then fluorescent labeled sugar conjugates were separated by reverse phase high perfomance liquid chromatography. Hydrolysates of PSG were composed of sixteen amino acids and 95.7% glucose, 2.7% xylose, 1.6% fucose and tract amount of galactose and mannose. The immunomodulating effects of PSG on macrophage were perfomed using murine macrophage cell line ATCC TIB 71 cells. PSG augumented the phagocytic activity of TIB 71 cells against fluorescent latex beads.

• 한국동물학회지
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• 제30권2호
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• pp.167-176
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• 1987

배추흰나비의 전용기 혈림프에서 $\beta$-N-Acetylglucosaminidase를 DEAE-cellulose ion exchange 및 Sephadex G-200 column chromatography 법을 이용하여 정제하였다. 정제된 효소는 7% acrylamide gel 전기영동에서 단일 band를 나타내었으며, 당을 함유한 복합단백질이었다. 최적 pH는 5.0, 최적반응온도는 6$0^{\circ}C$이었으며, 각 온도에서 10분간 incubation한 열안정성에 있어서는 6$0^{\circ}C$까지 비교적 높은 활성을 나타내었지만 7$0^{\circ}C$ 이후에는 거의 활성을 나타내지 않았다. 또한 Hg$^2$+ 처리는 심한 효소활성의 저해 현상을 보였지만, Mn$^2$+, $Mg^2$+ 등은 활성의 증가를 나타내었으며, Km 값은 6.67$\times$10-$^3$M, pl 값은 5.8, 분자량은 4.1$\times$105 daltons 이었다.

• Archives of Pharmacal Research
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• 제21권6호
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• pp.692-697
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• 1998

GTP cyclohydrolase I catalyzing the first reaction in the biosynthesis of pterin moiety of folic acid in bacteria, was purified from Streptomyces tubercidicus by at least 203-fold with a yield of 32% to apparent homogeneity, using ammonium sulfate fractionation, DEAE-cellulose, Sepharose CL-6B, and hydroxylapatite column chromatography. The molecular weight of the native enzyme was estimated to be 230,000 daltons by gel permeation chromatography. The purified enzyme gave a single band on sodium dodesyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was apparently 58,000 daltons. These results indicate that the enzyme consists of four subunits with the same molecular weight. The $K_m$ and $V_{max}$ values for GTP of the purified enzyme were determined to be 80${\mu}$M and 90nmol/min (mg protein), respectively. The optimum pH and temperature for the enzyme reaction were pH 7.5-8.5 and $40-42^{\circ}C$, respectively. Coenzyme or metal ion was not required for the enzyme activity. The enzyme activity was inhibited by most divalent cations, while it was slightly activated by potassium ion. In case of nucleotides, CTP, GMP, GDP, and UTP inhibited enzyme activity, among which GDP exhibited the strongest inhibitory effect.

• BMB Reports
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• 제32권6호
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• pp.605-609
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• 1999

Thioltransferase, also called glutaredoxin, is a general GSH-disulfide reductase of importance for redox regulation. Previously, the protein thioltransferase, now called S-type thioltransferase, was purified and characterized from Arabidopsis thaliana seed. In the present study, a second thioltransferase, called L-type thioltransferase, was purified to homogeneity from Arabidopsis thaliana leaves. The purification procedures included DEAE-cellulose ion-exchange chromatography, Sephadex G-50 gel filtration, and glutathione-agarose affinity chromatography. The purified enzyme was confirmed to show a unique band on SDS-PAGE and its molecular weight was estimated to be 26.6 kDa, which appeared to be atypical compared with those of most other thioltransferase. It could utilize 2-hydroxyethyl disulfide, S-sulfocysteine, and insulin as substrates, and also contained dehydroascorbate reductase activity. Its optimum pH was 8.5 and its activity was greatly activated by L-cysteine. When it was kept for 30 min, it appeared to be very stable up to $70^{\circ}C$. It was activated by $MgCl_2$ and, on the contrary, inhibited by $ZnCl_2$, $MnCl_2$, and $AlCl_3$.

• Journal of Nutrition and Health
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• 제16권2호
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• pp.115-124
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• 1983

The biochemical nature of the protein constituents of six year old fresh Panax ginseng root was studied. Total protein constituents were extracted with phosphate buffer of pH 7.4, ionic strength of 0.1 and fractionated by ultrafiltration using four different membranes which cut down the materials of molecular weight of 500, 1,000, 5,000 and 10,000, respectively. Each fraction was subjected to ion exchange chromatography using DEAE - cellulose to isolate component proteins. The protein fraction larger than molecular weight of 10,000 was refractionated by the method of ammonium sulfate precipitation. The electrophoresis of the refractionated protein constituents was performed. The amino acid composition of the protein constituents was determined by gas- liquid chromatography. From the results, it could be summarized that eleven different protein constituents smaller than molecular weight of 10,000 were isolated from the fresh Panax ginseng root. At least eleven different protein constituents larger than molecular weight of 10,000 were identified from the electrophoretic patterns. These protein costituents seem to be compounded of all or some of five different subunits.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVII)
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• pp.287-292
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• 2005

The crude polysaccharides(C-CPF, C-CPM, C-CPB) derived from fruiting body, mycelia and mycelia free broth of cordyceps militaris were obtained by ethanol precipitation of hot water extracts. After a batch fermentation of C. militaris was carried out in a 5 L jar vessel, endo-polysaccharide and exo-polysaccharide were obtained. They were demonstrated as the hetero polysaccharides which were composed of glucsose, galactose and mannose by performed with HPAEC(high pH anion exchange chromatography) and conformation of random coil by its complex forming ability with congo red reagent. They were purified by ion exchange (DEAE-cellulose) and gel filtration chromatography. They were monitered by phenol-sulfuric acid method and Bradford method. The NO induction activities of crude polysaccharides and purified polysaccharides derived from mycelia free broth were enhanced rather than LPS(lipo polysaccharide) which was used as a general NO inducer. These effects presumably contibute to the antitumor activities. The homogenieties and molecular weights of polysaccharides were determined by using Sepharose CL-6B. The yield, molecular weights and NO induction activities of C-CPFN Fr.III, C-CPMN Fr.III, C-CPBN Fr.II were 0.387, 0.408 and 0.153, 127 K 210 K and 36 K, 40.79%, 88.72%, and 104.17%, respectively.

• 생명과학회지
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• 제6권4호
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• pp.250-263
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• 1996

In this study Carotenoprotein from Styela clava were extracted, and purified by ammonium sulfate fraction, Sephadex G- 200 gel chromatography and DEAE-cellulose ion exchange chromarography. The purified carotenoprotein from styela clava had absorption maxima of 487nm, 463nm and 280nm, and the carotenoid liberated from carotenoprotein had 478nm and 452nm with inflexion. One miligram of caroteno-protein contained 0.35 $\mu$g of carotenoid. The carotenoprotein had an approximate molecular weight of 398 kDa (gel filtration). SDS-PAGE showed only a single polypeptide chain with a molecular weight of 62.4 kDa. The amino acid composition of the carotenoprotein were mainly glutamic acid(11.48%), valine(10.75%), leucine (10.45%), aspartic acid(9.94%), while cysteine and tryptophan were not found. The carotenoprotein contained lipid as structure units. In the carotenoprotein, the major fatty acids were oleic acid, plamitoleic acid and palmiunsaturated fatty acids 33.6%, saturated fatty acids 18.6%. In addition, the levels of higher unsaturated fatty acids were high as much as 30.8% of the total fatty acids. Carotenoid was extracted from the carotenoprotein by acetone. Thin layer chromatography showed only carotenoid to be present. Its chemical reactivities and spectroscopic properties were studied and elucidated as astaxanthin.

• Journal of Plant Biology
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• 제38권3호
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• pp.251-258
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• 1995

The soluble acid invertase ($\beta$-D-fructofuranoside fructohydrolase, EC 3.2.1.26) was isolated and characterized from the hypocotyls of mung bean (Phaseolus radiatus L.). The enzyme was purified to apparent homogeneity by consecutive step using diethylaminoethyl (DEAE)-cellulose anion exchange, Concanavalin (Con) A affinity and Sephacryl S-300 chromatography. The overall purification was about 148-fold with a yield of about 15%. The finally purified enzyme exhibited a specific activity of about 139 $\mu$mol of glucose produced mg-1 protein min-1 at pH 5.0 and appeared to be a single protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nondenaturing PAGE. The enzyme had the native molecular weight of 70 kD and subunit molecular weight of 70 kD as estimated by Sephadex G-200 chromatography and SDS-PAGE, respectively, suggesting that the enzyme was composed of a monomeric protein. On the other hand, the enzyme appeared to be a glycoprotein containing N-linked high mannose oligosaccharide chain on the basis of its ability to bind to the immobilized C on A. The enzyme had a Km for sucrose of 1.8 mM at pH 5.0 and maximum activity around pH 5.0. The enzyme showed highest enzyme activity with sucrose as substrate, but the activity was slightly measured with raffinose and cellobise. No activity was measured with maltose and lactose. These results indicate the soluble acid invertase is a $\beta$-fructofuranosidase.

• Journal of Microbiology and Biotechnology
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• 제1권2호
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• pp.90-95
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• 1991

A ${\alpha}-galactosidase{\;}({\alpha}-D-galactoside$ galactohydrolase; EC 3.2.1.22) was purified from the culture filtrate of Penicillium purpurogenum by DEAE-cellulose column chromatography, gel filtration of Bio gel p-l00, and subsequent SP-Sephadex C-25 chromatography. The final preparation thus obtained showed a single band on polyacrylamide disc-gel and SDS-polyacrylamide gel electrophoresis. The molecular weight and isoelectric point were determined to be 63,000 and pH 4.0 by SDS-polyacrylamide gel electrophoresis and isoelectric focusing, respectively. The galactosidase exhibited maximum activity at pH 4.5 and $55^{\circ}C$, and was stable between pH 2 and 5, and also stable up to $40^{\circ}C$. The enzyme activity was not affected considerably by treatment with other metal compounds except mercuric chloride and silver nitrate. Copra galactomannan was finally hydrolyzed to galactose, mannose and mannobiose through the sequential actions of the purified galactosidase and mannanase from the same strain. The enzyme hydrolyzed melibiose and raffinose, but not lactose.

• Journal of Applied Biological Chemistry
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• 제42권2호
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• pp.71-74
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• 1999

The crude enzyme prepared from the culture supernantant of Arthrobacter sp. S37 was purified by Phenyl Toyopearl column chromatography. Six endo-inulinases were detected by activity staining on native PAGE and named Inu I to Inu VI. Endo-inulinase were further purified by DEAE cellulose column chromatography and band slicing. Inu II~VI produced mainly inulotriose (F3) and inulotetraose (F4) as well as a small amount of inulobiose (F2) and fructose in contrast to Inu I producing F3, F4 and F5 from inulin. The N-terminal amino acid sequence of native and six CNBr-cleaved fragment of Inu VI were determined. No homology was found in amino acid sequences between Inu VI and other fructan hydrolase including invertase reported.