• Korean Journal of Fisheries and Aquatic Sciences
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• v.29 no.6
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• pp.797-804
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• 1996

Two trypsins were purified from shrimp hepatopancreas through ammonium sulfate fractionation, Q-Sepharose ionic exchange, benzamidine Sepharose-6B affinity, and Sephacryl S-300 gel chromatography. Both enzymes had a single polypeptide chain with a molecular weight (M.W.) of 32 kDa by sodium dodecylsulfate polyacrylamide gel electrophoresis (SOS-PAGE), although trypsin A and B were estimated to be a molecular weight of 27.2 and 22.8 kDa, respectively, using Sephacryl S-300 gel filtration. Both trypsins had similar amino acid compositions and rich in glycine, valine, alanine, aspartic acid, and glutamic acid, but low in methionine and basic amino acids. Both enzymes were completely inactivated by soybean trypsin inhibitor (SBTI), phenylmethylsulfonyl fluoride (PMSF), tosyl-L-lysine chloromethyl ketone (TLCK), benzamidine, leupeptin, however, not affected by tosyl-L-phenylalanine chloromethyl ketone (TPCK) and pepstatin.

• BMB Reports
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• v.29 no.6
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• pp.540-544
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• 1996

Ferritins from germinated pumpkin seeds were isolated by ammonium sulfate precipitation (0.55 saturation), ion-exchange chromatography on DEAE-cellulose, and gel filtration chromatographies on Sephacryl S-300 and Sephadex G-100. Pumpkin ferritin contains less iron than soybean ferritin. Pumpkin ferritin cross-reacted with anti-soybean ferritin antiserum made in rabbit, and showed two distinct antibody reactive bands, both of equal intensity. The pumpkin ferritins corresponding to the two bands were separable by centrifugation in a sucrose gradient (20~50%). The molecular weights of the native pumpkin ferritins based on the estimation of sucrose gradient centrifugation, gel filtration on Sephacryl S-300 and non-denaturing polyacrylamide gel electrophoresis appeared to be: 530~580 KD (the large molecular weight pumpkin ferritin) and 330-360 KD (the small molecular weight pumpkin ferritin) The large molecular weight pumpkin ferritin contains less iron. Both pumpkin ferritins cross-reacted with anti-soybean ferritin antibody with a spur formation suggesting partial antigenic recognition.

• Korean Journal of Microbiology
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• v.25 no.4
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• pp.309-317
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• 1987

It was known that normal-haemolymph from the 3rd instar larvae of Lucillia illustris contain a lysozyme (or lysozyme-like substance) with bactericidal activity to fram positive bacteria, and the bactericidal activity of injured-haemolymph was increased significantly after injuring the body wall. To elucidate the defence mechanism of insect against the nonpathogenic bacteria, the immune-haemolymph against Escherichia coli K-12 was prepared after immunization. The bactericidal activity between injured and immune-haemolymph was compared, and it was revealed that the immune-haemolymph showed higher titer of bactericidal activity to fram positive bacteria as well as to Escherichia coli. The bactericidal substance from the immune-haemolymph was purified through a successive chromatographies on Sephacryl S-300 and CM-Sepharose CL-6B, and it was characterized as a basic protein in nature with heat stable property at acidic conditions.

• Korean Journal of Food Science and Technology
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• v.20 no.6
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• pp.780-786
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• 1988

In order to investigate the most efficient and rapid method for the purification of enterotoxin A from Staphylococcus aureus M 7/1, various methods such as ion-exchange chromatography on Amberlite, and CM-cellulose. gel filtration on Sephadex G-50, 75, 100 and Sephacryl, and fast protein liquid chromatography (FPLC) were applied and compared in terms of purity and speed. Although ion-exchange chromatography on Amberlite resin was good enough to remove other materials in culture medium from enterotoxin, and convenient, and fast method, the purity of this method was less than 70%. However. carboxymethyl ion-exchange column showed to be better purity than that of Amberlite method. The yields of these two methods were about 70% and 75%, respectively. When gel filtration methods on Sephadex G-50, 75, 100 and Sephacryl were applied, the purity was about 90%. Fast protein liquid chromatography was found to be the most efficient method in terms of purity (97%) and speed. The combined method, gel filtration after CM-cellulose column (stepwise elution) treatment can be also used as a efficient method particularly for the purification of large volume of sample.

• Korean Journal of Microbiology
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• v.30 no.6
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• pp.564-569
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• 1992

A green sulfur bacterium, Chlorobium limicola f. thiosulfatophilum NCIB 8327, was grown in modified Pfennig's medium including glu1amate as a nitrogen source. Glutamine synthetase was isolated through a series of ultracentrifugation. DEAE-Sepharose CL-6B ion exchange chromatography. Sephacryl S-300 gel permeation chromatography, and preparative HPLC. The recovery and purification fold of the enzyme were 2% and 46.3. respectively. The isolated enzyme was homogeneous on UV-Visible spectrum and polyacrylamide gel electrophoretogram. The relative molecular mass of the native enzyme was estimated to be 280,000 by gel permeation chromatography. The enzyme consisted of ten subunits with relative similar molecular mass. 30.000. which was estimated by SDS-polyacrylamide gel electrophoresis. The optimal temperature and pH of the enzyme were $30^{\circ}C$ and 7.0. Km values were 27.9 mM for L-glutamine and 0.92 mM for hydroxylamine-HCr. The enzyme activity was inhibited by alanine. glycine. and tryptophan considerably, but was not affected by asparagine, lysine. leucine. and valine.

• Korean Journal of Microbiology
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• v.25 no.3
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• pp.221-228
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• 1987

The extracellular adenine deaminase from Nocardioides sp. J-275L was purified by the following techniques: ammonium sulfate fractionation, DEAE-Cellulose, DEAE-Sephadex A-50 column chromatography, and Sephacryl S-200 superfine gel filtration. The enzyme was partially purified about 3889.5-fold with about 5.2% yield by these procedures. The molecular weight of the enzyme was 39,000 by a calibrated Sephacryl S-200 superfine column chromatography. The enzyme was stable at pH 7.5 and up to $40^{\circ}C$. Glycerol was effective on the stabilization of the enzyme during storage. The optimum pH and temperature of the enzyme were around pH 7.5 and $40^{\circ}C$, respectively. The apparent Michaelis constant Km of the enzyme for adenine was $7.4\times 10^{-5}$M. The purine analogues, 6-chloropurine, 2,6-diaminopurine, 6-bromopurine, 4-aminopyrazolo [3.4-d]pyrimidine, and 8-azaadenine were substrates for the enzyme. 6-Dimethylaminopurine was a competitive inhibitor of the enzyme. The enzyme was inhibited by 1mM of $Cu^{2+}, Fe^{3+}, Pb^{2+}, Hg^{2+}$, and $Ag^{+}$, and 1mM of $\alpha$,$\alpha$'-dipyridyl, pentachlorophenol, and pCMB.

• Journal of Microbiology
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• v.40 no.1
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• pp.26-32
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• 2002

From the culture supernatant of the psychrotrophic strain of Bacillus amyloliquefaciens an extracellular serine protease was purified to apparent homogeneity by successive purification steps using QAE-Sephadex, SP-Sephadex and Sephacryl S-100 column chromatography. The pretense is monomeric, with a relative molecular mass of 23,000. It is inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride, but not by EDTA. The enzyme is most active at pH 9-10 and at $45^{\circ}C$, although it is unstable at $60^{\circ}C$.

• Korean Journal of Microbiology
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• v.29 no.2
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• pp.92-96
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• 1991

The soluble cytochrome c-551 of photosynthetic bacterium, Rhodopseudomonas gelatinosa ATCC 17013 was purified through a sequene of four step chromatography including CM-cellulose ion-exchange chromatography, DEAE-Sephacel chromatography, Sephacryl s-200 gel permeation chromatography, and HPLC (SP-5PW). The molecular weight of the purified cytochrome c-551 was 14, 600 Da, and this protein shows the absorption peak at 551 nm, 522 nm, and 417 nm as the reduced form, and at 412 nm as the oxidized form. The cytochrome c-551 seems to be a substrate for the terminal oxidase in the electron transport chain.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1993.04a
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• pp.45-45
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• 1993

괄루근으로부터 분리된 다당류에 대하여 continuous gel electrophoresis, SDS-Polyacrylamide gel eletrophoresis, ion exchange column chromatography, Hydroxyapatite column chromatography 및 Gel filtration등의 방법을 이용하여 다음과 같은 결과를 얻었다. 1) 황산암모늄 분별침전법에 의한 렉틴의 정제도는 초추출물의 4.85배이며 DEAE Sephadex A-50 column chromatography법에 의한 정제도는 24.17배로 나타났고, 마지막 정제단계인 Sephacryl S-200 gel filtration에 의한 정제도는 47.34배로 나타났다. 2) 정제된 렉틴의 분자량은 60,000da1ton으로 나타났다. 3) 사람의 혈액형에 따른 응집효과는 90-100％로 특이성은 없었다.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1993.04a
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• pp.136-136
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• 1993

immunotoxin으로 쓰일 수 있는 리보솜 불활성화 단백질 (RIP, ribosome-inactivating protein)을 보리 씨앗에서 분리하였다 이 단백질은 분자량이 약 30,000 kDa 정도되고, pl가 9.0 보다 높다. 이러한 성질을 이용하여 Na-phosphate 완충용액으로 추출하고, 황산암모늄 60-80％ 포화로 분획화하였다. 다음 CM-cellulose를 이용한 이온 교환 크로마토그래피, Sephacryl S-200 HR 컬럼을 이용한 gel filtration을 하여 순수히 분리하였고, 이를 전기영동하여 확인하였다.