• Journal of Life Science
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• v.13 no.3
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• pp.308-313
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• 2003

The collagenase gene from Vibrio parahaemolyticus 04 was subcloned into an expression vector pET-29b. The recombinant collagenase was expressed in Escherichia coli BL2l(DE3) and partially purified by Hi-Trap affinity and Sephacryl S-100 size exclusion chromatographies. The recombinant enzyme was purified by 43.7-fold and the yield was 73%. SDS-PAGE revealed that the molecular weight of the enzyme was approximately 35 kDa. Substrate specificity study of the enzyme displayed that the enzyme showed the highest activity with the type I collagen and the synthetic peptide, Z-GPGGPA, indicating that the enzyme was indeed a collagenase. The enzyme showed broad pH optimum around pH 6-12 and was stable between pH 5.5 and 11.5. The optimum temperature for the type I collagen degradation was $35^{\circ}C$. The thermostability measurement of the enzyme indicated that the enzyme was stable up to $55^{\circ}C$, but the activity was diminished quickly above $60^{\circ}C.$

• Food Science of Animal Resources
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• v.36 no.5
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• pp.665-670
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• 2016

The objectives of this study were conducted to characterize pepsin-soluble collagen (PSC) extracted from bones (PSC-B), skins (PSC-S), and tendons (PSC-T) of duck feet and to determine their thermal and structural properties, for better practical application of each part of duck feet as a novel source for collagen. PSC was extracted from each part of duck feet by using 0.5 M acetic acid containing 5% (w/w) pepsin. Electrophoretic patterns showed that the ratio between α₁ and α₂ chains, which are subunit polypeptides forming collagen triple helix, was approximately 1:1 in all PSCs of duck feet. PSC-B had slightly higher molecular weights for α₁ and α₂ chains than PSC-S and PSC-T. From the results of differential scanning calorimetry (DSC), higher onset (beginning point of melting) and peak temperatures (maximum point of curve) were found at PSC-B compared to PSC-S and PSC-T (p<0.05). Fourier transform infrared spectroscopy (FT-IR) presented that PSC-S and PSC-T had similar intermolecular structures and chemical bonds, whereas PSC-B exhibited slight difference in amide A region. Irregular dense sheet-like films linked by random-coiled filaments were observed similarly. Our findings indicate that PSCs of duck feet might be characterized similarly as a mixture of collagen type I and II and suggest that duck feet could be used for collagen extraction without deboning and/or separation processes.

• Applied Biological Chemistry
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• v.36 no.3
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• pp.163-171
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• 1993

The collagens from filefish (Novoden modestus) and cod (Gadus macrocephalus Tilesius) skin were isolated and their physicochemical properties were investigated. Glutamic acid, hydroxyproline, valine and phenylalanine in the filefish skin collagen (FSC) were presented at higher levels than those of cod skin collagen (CSC), but the contents of glycine, proline and serine were contrary. The content of essential amino acids of FSC (265 residues/1000 residues) was higher than CSC (229 residues). The solubilities of both collagens were the lowest at pH 7.0, but precipitously increased at acid zone(below pH 5.0). FSC has lower viscosity than CSC. Furthermore, while the viscosities of both collagens were the lowest at pH 7.0, the viscosities of FSC and CSC were the highest at pH 4.0 and pH 2.0, respectively. The denaturation temperature of $FS(25^{\circ}C)$ was higher than $CSD\;(17^{\circ}C)$. The free hydrophobic residue contents of FSC and CSC tended to increase till $60^{\circ}C,\;and\;50^{\circ}C$ respectively, and to decrease thereafter. Hydration capacities of both collagens were the lowest at pH 7.0, and CSC had the superior hydration capacity to FSC. In addition, emulsifying and emulsifying stability of CSC was also superior to FSC.

• Parasites, Hosts and Diseases
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• v.30 no.3
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• pp.191-200
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• 1992

A proteolytic enzyme was purified from the tissue extract of spargana (plerocercoids of Spirometra erinacei) by DEAE-Trisacryl M ion exchange chromatography and thiopropyl-sepharose affinity chromatography resulted in a 21-fold purification. The proteinase activity was assayed with a synthetic fluorescent substrate, carbobensoxy-phenylalanyl-7-amiso-4-trifluoromethyl-coumarin. SDS-polyacplamide gel electrophoresis of the purified materials revealed a single 28,000 dalton band. Inhibitor profiles of the band indicated that it belonged to cysteine endopeptidases. It exhibited identical pH curves with optimum at pH 5,5, and 50% activity from pH 4.7 to 8. It could completely degrade collagen chains to three identical products. It also showed some activity on hemoglobin. Furthermore, the band on immunoblots was reactive to the sera of sparganosis patients. These results suggest that the proteolytic enzyme belongs to cysteine proteinase which plays a role in the tissue penetration. Also it may be used as the antigen for diagnosis of active sparganosis.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1997.04a
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• pp.69-69
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• 1997

In the screening of tropical medicinal plants using PAE receptor binding assay, the ether extract of Ardisia crispa showed the potent antagonistic activity. Ardisia crispa have been used to heal the scurf, earache, orchitis, fever and diarrhoea, cough and given to the mother after childbirth to ‘wash out dirty blood’ in Malaysia. By means of activity guided isolation, compound AC7-1 was isolated as the potent PAF antagonist. In this study, antiplatelet effects of compound AC7-1 were examined in vitro platelet aggregation assay using the chronolog aggregometer. Compound AC7-1 inhibited PAF-, collagen-, ADP-, thrombin-induced platelet aggregation in human, rabbit and rat platelet rich plasma. In vitro rabbit platelet aggregation, the IC$\_$50/ value of compound AC7-1 was 5 ${\times}$ 10$\^$-6/ M against PAF(5 ${\times}$ 10$\^$-7/M)-induced aggregation. The IC$\_$50/ values of AC7-1 on PAF-induced platelet aggregation increased with increase of the concentration of PAF used. This result suggested the competitive nature of the AC7-1 antagonism. In vitro rat platelet aggregation, the IC$\_$50/ values of AC7-1 on collagen-, ADP-induced platelet aggregation were 4 ${\times}$ 10$\^$-6/ M, 2 ${\times}$ 10$\^$-5/ M, respectively. Also in vitro human platelet aggregation, AC7-1 potently inhibited both the primary phase and secondary phase of thrombin-induced aggregation.

• The Journal of the Korean dental association
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• v.54 no.4
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• pp.284-295
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• 2016

The aim of this study was to evaluate the effect of Epigallocatechin-3-Gallate (EGCG) on the alveolar bone metabolism in a collagen-induced arthritis (CIA) model in mice to enhance the understanding of rheumatoid arthritis (RA)-associated alveolar bone loss. Following the induction of CIA in animals (mice, n=16), mandibles were retrieved for micro-computed tomography (micro-CT) and isolation of alveolar bone cells (ABCs). In vitro osteogenic potentials of ABCs were evaluated and the mRNA expression of downstream effector genes was assessed. CIA was successfully induced in all animals, and micro-CT data showed that alveolar bone loss was significantly increased in the CIA group while the treatment of EGCG prevented the alveolar bone resorption. Osteogenesis by ABCs was significantly increased in the CIA+EGCG group in vitro. The analysis of mRNA expressions showed that osteoclastogenesis-associated genes were increased in CIA group while bone protecting genes were upregulated in EGCG treated group. The results demonstrate that EGCG downregulated the alveolar bone resorption in a CIA model in mice, and upregulation of bone protecting genes appear to be involved. Further studies are warranted.

• Journal of the Korea Organic Resources Recycling Association
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• v.2 no.2
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• pp.31-37
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• 1994

For a microbiological study of composting process, screening and assay method for biopolymer degrading enzymes and microorganisms were developed and for the study of the possibility of composting in anaerobic state, distribution of sulfate reducing bacteria which plays a final role in anaerobic degradation was investigated. Substrates used for the development of assay methods for biopolymer degradation are ${\beta}-glucan$, xylan, dextran, CMC(carboxy methly cellulose), casein, and collagen. These substrates were made insoluble by a cross-linking agent and linked with dye to make chromogenic substrates. ${\beta}-glucan$ and xylan substrates could substitute congo-red method for screening of polymer degrading microorganisms without damaging the colonies. Sulfate reducing bacteria contained in the sample sludge showed preference to lactic acid, propionic acid, butyric acid and formic acid and could use acetic acid and valeric acid.

• Parasites, Hosts and Diseases
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• v.36 no.2
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• pp.133-142
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• 1998

A 40 kDa cysteine protease was purified from the crude extract of adult worms of GMnnophalloines seoi by two consecutive steps: Sephacryl S-200 HR and DEAE- Sephacel chromatography. Enzyme activities were completely inhibited by cysteine protease inhibitors, L-lorans-epoxysuccinylleucylamido (4-guanidino) butane (E-64) and iodoacetic acid, strongly suggesting that the purified enzyme belongs to the cysteine family of proteases. The enzyme was maximally acive at pH 4.5 in 0.1 M of buffer, and its activity was greatly potentiated in the presence of 5 mM dithiothreitol. The protease degraded macromolecules with differential capabilities : it degraded extracellular matrix proteins, such as collagen and fibronectin, with a stronger activity against collagen than fibronectin . However, the enzyme digested hemoglobin and human immunoglobulins only slightly. leaving them nearly intact after an overnight reaction. Our results suggest that the cysteine protease of G. seoi adults is potentially significant in the nutrient uptake from the host intestine.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.31 no.3 s.52
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• pp.227-235
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• 2005

Skin aging is not a disease nor an abnormal phenomenon but a collection of degenerative changes with age, characterized by skin dryness, wrinkle formation, and loss of skin elasticity. The skin wrinkles are caused by either genetically predisposed factors or environmental factors such as UV irradiation or physical/chemical stimulus. The histological manifestations of wrinkles are changes in both amount and integrity of elastic and collagen fibers. Here we report the isolation and characterization of 3 active compounds, prangenidin, 8-hydroxybergapten, and xanthotoxol from Angelica dahurica root. The anti-wrinkle activities of these compounds were also investigated.

• Journal of Food Hygiene and Safety
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• v.13 no.4
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• pp.355-359
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• 1998

Platelet activation is originated by the intracellular or/and extracellular $Ca^{2+}$. Agonist-induced $Ca^{2+}$ entry through a plasma-membrane pathway has been reported repeatedly, but the mechanisms has proven harder to elucidate. Recently, a number of natural products have been isolated from medicinal plants and marine organisms and have proved to be useful chemical tools for resolving the mechanism of cellular functions. In an attempt to understand the mechanism of platelet activation in Bupleuri Radix, we have studied some aspects of the isolation of active components and their dependence of external $Ca^{2+}$> on platelet activation. Acetone extract of Bupleuri Radix has the most activity on platelet activation and it's active components were identified as saikosaponin a and d. Their optimal concentration was respectively $20\;\mu\textrm{g}/ml$ and $5\;\mu\textrm{g}/ml$ and their platelet activation was not dependent on external $Ca^{2+}$>, whereas optimal concentration of each agonist was arachidonic acid ($10\;\mu\textrm{M}$), collagen ($10\;\mu\textrm{M}/ml$), thrombin (0.1 unit/mi), PAF (5 nM), PMA ($5\;\mu\textrm{M}$), ionophore A23187 ($2\;\mu\textrm{g}$) and their dependence of external $Ca^{2+}$> on platelet activation appeared to thrombin > collagen $\geq$ PAF > PMA > arachdonic acid> ionophore A23187. These results suggest that saikosaponin is different from each agonists in the dependence of external $Ca^{2+}$ on platelet activation.