• Proceedings of the Korean Society of Sericultural Science Conference
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• 2003.04a
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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)

• Korean Journal of Fisheries and Aquatic Sciences
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• v.41 no.3
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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.

• Korean Journal of Food Science and Technology
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• v.35 no.5
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• pp.791-796
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• 2003

Polyphenol oxidase isoforms were purified from the lotus roots using 50% acetone precipitation, conventional chromatographies of Q-Sepharose and hydrophobic interaction, and high performance liquid chromatographies of Mono-Q and Superdex 75 gel-filtration. Molecular mass of a purified PPO isoform (LPIII-2) was determined to be 56 kDa using gel-filtration chromatography. The active form of LPIII-2 appeared to bea heterodimer, as purified LPIII-2 on SDS-PAGE gel showed two bands that were determined to be 28 kDa and 26 kDa. To further characterize PPO, partially purified PPO isoforms (LP-II, LP-III) were obtained from Q-Sepharose anion-exchange chromatography. In substrate specificity, the partially purified PPO isoform LP-II showed a high affinity to catechol, while LP-III showed a high affinity to pyrogallol. The optimum pH of LP-II and LP-III was pH 7.0. Interestingly, the partially purified PPO isoforms showed high activities at low temperatures $(0{\sim}5^{\circ}C)$, and as temperatures rose, the activities decreased. Both PPO isoforms were stable at $40^{\circ}C$ and were inactivated by incubation at $60^{\circ}C$ for 40 min.

• 한국생물공학회:학술대회논문집
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• 2003.10a
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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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• v.32 no.6
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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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• v.10 no.6
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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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• v.31 no.6
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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.

• Microbiology and Biotechnology Letters
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• v.8 no.2
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• pp.125-133
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• 1980

A microorganism which produced D-glucose isomerase was identified to be similar to Streptomyces antibioticus on the morphological, cultural and physiological characteristics except the spore chain and the utilization of sucrose. D-xylose grown cells of Streptomyces sp. strain K-17 were disrupted by grinding with sea sand. D-glucose isomerase was partially purified with the fractionation by ammonium sulfate, Mn-treatment, DEAE-cellulose column chromatography, DEAE-sephadex (A-50) column chromatography and gel filtration of sephadex G-200. The enzyme was purified about 380 fold with 25 ％ recovery.

• BMB Reports
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• v.29 no.5
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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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• v.16 no.4
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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.