• 한국잠사학회:학술대회논문집
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• 한국잠사학회 2003년도 제46회 춘계 학술연구 발표회
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• pp.56-56
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• 2003

Using gel filtration chromatography (GFC), pure separation of high molecular silk fibroin was obtained and silk fibroin microsphere particles (SFMP) could be simply made by spray dryer method. Also, some of its physicochemical properties and morphology were investigated. The average molecular weight (Mw) of pure silk fibroin protein dissolved in calcium chloride is about 61, 500 g/mol as measured by gel permeation chromatography(GPC). (omitted)

• 한국수산과학회지
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• 제41권3호
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• pp.176-181
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• 2008

To evaluate the effective use of endoprotease from squid viscera as a food processing aid, various methods of fractionating endoprotease from viscera of the Argentina shortfin squid (Illex argentinus) were evaluated. The endoprotease-positive fractions of each fractionation were fraction II (30-40%, w/w) with cold acetone, fraction IV (50-60% saturation) with ammonium sulfate, fraction UF with anion exchange chromatography, and fraction II (15-24 kDa) with gel filtration. The specific activities (approximately 25 U/mg) of the fractions using ammonium sulfate and gel filtration were higher than the others. Total azocaseinolytic activity and recovery of the positive fraction using gel filtration were 806.95U and 37.82%, respectively, and were the highest among the positive fractions. Based on the results, gel filtration was the most efficient method for fractionating endoprotease from the viscera of Illex argentinus.

• 한국식품과학회지
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• 제35권5호
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• pp.791-796
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• 2003

박피된 절편 연근의 갈변억제를 효과적으로 하기 위한 기초연구로 갈변의 주원인 효소인 polyphenol oxidase(PPO)를 분리, 정제하여 특성을 조사하였다. 박피된 절편 연근을 24시간 동안 $4^{\circ}C$에 방치하여 PPO 활성을 증가시켜 조효소액을 제조하였으며 PPO 조효소액을 acetone으로 침전시킨 후 Q-Sepharose anion-exchange column, Phenyl-Sepharose hydrophobic interaction column의 conventional column과 Mono-Q anion-exchange column, Superdex 75 gel-filtration column의 HPLC column을 이용하여 PPO 활성이 가장 높은 한 개의 PPO isoform LPIII-2를 최종 분리 정제하였다. 분리 정제된 LPIII-2의 분자량을 gel-filtration chromatography를 이용하여 측정한 결과 56kDa이었으며 SDS-PAGE를 실시한 후 silver staining한 결과 LPIII-2의 분자량은 28kDa와 26kDa으로 2개의 band를 형성하는 것으로 보아 heterodimer인 것으로 추정되었다. PPO isoform의 특성 조사를 위하여 Q-Sepharose anion-exchange chromatography를 이용하여 부분분리 정제된 2개의 isoforms(LP-II, LP-III)를 가지고 기질 특이성을 조사한 결과 LP-II의 경우 $5^{\circ}C$와 $30^{\circ}C$ 모두 catechol에 대한 기질 친화력이 높았으며 LP-III의 경우 $5^{\circ}C$와 $30^{\circ}C$ 모두에서 pyrogallol에 기질 친화력이 높았다. 그리고 pH 7에서 최적 pH를 보였으며 열안정성은 $40^{\circ}C$에서 60분간 열처리했을 때 안정하였지만 $60^{\circ}C$에서 40분, $80^{\circ}C$에서 10분간 열처리했을 때 효소가 불활성화되었다. 특이하게도 연근의 PPO는 다른 과채류의 PPO와 반응온도에 따른 특성이 달랐는데 여러 반응온도에서 효소 활성을 측정한 결과 연근 PPO isoform LP-II와 LP-III 모두 $5^{\circ}C$의 반응온도에서 높은 효소활성을 보였으며 온도가 올라갈수록 반응속도가 떨어지는 특성을 보였다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.544-547
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• 2003

Fusion $ferritin(F_H+F_L),$ an iron-binding protein, was purified from recombinant E. coli by gel filtration chromatography after two-step sonications. Unfolded ferritin was refolded by GFC with various refolding enhancing additives. 50 mM Tris-HCI(pH 7.4) buffers containing 2 M urea and additive was used in GFC. Objective was to characterize the structure change at various conditions. Molecular weight was determined using GF-HPLC and RP-HPLC was used to quantify the unfolded and refolded proteins. Activity was confirmed by iron-uptake reaction.

• BMB Reports
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• 제32권6호
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• pp.535-540
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• 1999

Two types of the thioltransferase (also called glutaredoxin) have been previously detected in the cytosolic extract of Schizosaccharomyces pombe, a fission yeast. Previously, the one with a smaller molecular mass (14kDa) was purified and characterized. In the present study, the second thioltransferase was purified. The purification procedure included ammonium sulfate fractionation (40-80%), Sephadex G-200 gel filtration, DEAE-cellulose ion-exchange chromatography, Sephadex G-50 gel filtration, and glutathione-agarose affinity chromatography. The purified enzyme showed a single band on SDS-PAGE, and its molecular mass was determined to be 23 kDa. It utilizes various compounds as substrates, including 2-hydroxyethyl disulfide. Interestingly, we found that the purified thioltransferase also contains significant glutathione S-transferase activity.

• Biotechnology and Bioprocess Engineering:BBE
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• 제10권6호
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• pp.500-504
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• 2005

Fusion ferritin (heavy chain ferritin, $F_H+$ light chain ferritin, $F_L$), an iron-binding protein, was primarily purified from recombinant Escherichia coli by two-step sonications with urea [1]. Unfolded ferritin was refolded by gel filtration chromatography (GFC) with refolding enhancer, where 50 mM Na-phosphate (pH 7.4) buffer containing additives such as Tween 20, PEG, and L-arginine was used. Ferritin is a multimeric protein that contains approximately 20 monomeric units for full activity. Fusion ferritin was expressed in the form of inclusion bodies (IBs). The IBs were initially solubilized in 4 M urea denaturant. The refolding process was then performed by decreasing the urea concentration on the GFC column to form protein multimers. The combination of the buffer-exchange effect of GFC and the refolding enhancers in refolding buffer resulted in an efficient route for producing properly folded fusion ferritin.

• BMB Reports
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• 제31권6호
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• pp.548-553
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• 1998

A minor anionic isoperoxidase named $A_{3n}$, was isolated from Korean radish (Raphanus sativus L.) root. Purification of the enzyme was accomplished by CMcellulose chromatography, DEAE-Sephacel chromatography, and Sephadex G-75 gel filtration. The enzyme was a glycoprotein with molecular weight of approximately 31,000 as determined by SDS-PAGE and 33,000 by Sepadex G-150 gel filtration, which is by far the smallest among the reported isoperoxidases. The pI value was 3.5. The optimum pH of the enzyme was 6.5 for guaiacol and $H_2O_2$, and the $K_m$ values for guaiacol and $H_2O_2$ were 13.3 mM and 1.5 mM, respectively. Kinetic studies with various substrates revealed that only A3n, unlike other isoperoxidases from radish, did not use scopoletin as a substrate and had very low $K_m$ value of 0.25 mM for ferolic acid among naturally occurring phenolic substrates.

• 한국미생물·생명공학회지
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• 제8권2호
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• pp.125-133
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• 1980

토양으로부터 분리한 포도당 이성화효소를 강하게 생산하는 방사균을 Bergey's manual 8판에 따라 동정한 결과 Streptomyces antibioticus 근록의 균주이었다. 본 균의 배양액으로부터 균체를 모아서 해사를 넣고 파쇄 하여 증류수로 추출하고 Mn-처리를 하여 핵단백질을 제거한 후 황산 ammonia 분획침전(0.5∼0.8포화), 수석, DEAE-cellulose column chromatography, DEAE-sephadex (A-50) column chromatography 및 sephadex G-200에 의한 gel filtration을 거쳐 비활성도로 약 380배, 회수율 25％ 정도로 분리 정제하였다.

• BMB Reports
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• 제29권5호
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• pp.455-461
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• 1996

A serine proteinase was purified from Acanthamoeba culbertsoni by 41~80% ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography and gel filtration chromatography. The molecular weight of the purified enzyme was estimated to be 108.0 kDa by gel filtration chromatography and 54.0 kDa by SDS-PAGE. Therefore, the purified enzyme seemed to be a dimer. Isoelectric point was 4.5. The enzyme activity was highly inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate (OFP) and phenylmethyl sulfonylfluoride (PMSF). It had a narrow pH optimum of 6.5~7.5 with a maximum at pH 7.0. These data suggested that the purified enzyme was a neutral serine proteinase. Optimal temperature was $37^{\circ}C$. It was stable for at least 16 h at $4^{\circ}C$ and $37^{\circ}C$, but it was rapidly inactivated at $65^{\circ}C$ The activity of the purified enzyme was not influenced significantly by $Mg^{2+}$, $Mn^{2+}$, $Zn^{2+}$ or $Ca^{2+}$. However, the enzyme activity was highly inhibited by $Hg^{2+}$ The enzyme degraded type I collagen and fibronectin, but not BSA, hemoglobin, lysozyme, immunoglobulin A or immunoglobulin G.

• Journal of Microbiology and Biotechnology
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• 제16권4호
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• pp.647-650
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• 2006

D-Psicose 3-epimerase (DPE) catalyzes the interconversion of D-fructose to D-psicose by epimerizing the carbon-3 position. The DPE from Agrobacterium tumefaciens was cloned and expressed in Escherichia coli. The expressed enzyme was purified by affinity chromatography on an IMAC, gel filtration chromatography on a Sephacryl S-300 HR, and anion-exchange chromatography on a RESOURCE Q. The molecular mass of the purified enzyme was estimated to be about 135 kDa by Superdex 200 gel filtration chromatography, corresponding to a homotetramer. The enzyme produced crystals suitable for X-ray diffraction to a $2.0{\AA}$ resolution at 100 K. The crystals were found to belong to the orthorhombic space group $P2_12_12_1$, with unit-cell parameters a=102.4, b=113.0, and $c=131.8{\AA}$. In addition, the calculated packing parameter $(V_m)$ was $2.79{\AA}^3/Da$, the solvent content was 55.92%, and an asymmetric unit consisted of four monomers.