• Title/Summary/Keyword: Collagenase

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Purufication and Characterization of Extracellular Collagenase from Vibrio mimicus (Vibrio mimicus 가 생산하는 collagenase의 정제 및 특성)

  • 김용태;김세권
    • Journal of Life Science
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    • v.6 no.4
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    • pp.241-249
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    • 1996
  • A collagenase was isolated from the culture filtrate of Vibrio mimicus (ATCC 33658). The enzyme was purified to homogeneity by ammonium sulfate precipitation and DEAE-Sephadex A-50 chromatography, which an activity recovery of 22%. The molecular weight of the purified enzyme was estimated to be 42 kDa by SDS-polyacrylamide gel electrophoresis and gel filtration, indication a monomer structure. The optimum pH and temperature od the enzyme for insoluble collagen (Type I) were around 7.75 and 28$\circ$C, respectively. Some chelating agents and serine protease inhibitor inactivated the enzyme, but L-cysteine and histidine did not affect the activity. The amino acid composition indicated that the collagenase contained high amounts of amino acid residues of glycine and alanine. The K$_{m}$ and R$_{cat}$/K$_{m}$ values for the collagenase, using insoluble collagen (type I) as substrate, were 2.86 mg/ml and 972.28 U/mg-protein, respectively.

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Cloning and Expression of a Collagenase Gene from the Marine Bacterium Vibrio vulnificus CYK279H

  • Kim, Bong-Jo;Kim, Hak-Ju;Hwang, Sun-Hee;Bae, Seoung-Kwon;Ha, Soon-Duck;Kim, Jong-Deog;Kong, Jai-Yul
    • Journal of Microbiology and Biotechnology
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    • v.8 no.3
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    • pp.245-250
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    • 1998
  • A gene encoding an extracellular collagenase from the marine bacterium Vibrio vulnificus CYK279H was cloned into E. coli JM83 using the multicopy plasmid vector pUC19. The cloned strain of recombinant E. coli showing collagenase activity had an insert fragment of 3.5 kb and was named E. coli JM83/pKCL 279H. The cloned strain produced two different collagenase during cultivation. These enzymes, named collagenase-I and -II, were purified from the culture supernatant. SDS-PAGE indicated that collagenase-I had a molecular weight of 41 kDa and collagenase-II had a weight of 37 kDa. The N-terminal amino acid sequence of collagenase-I from the cloned strain, E. coli JM83/pKCL279H was determined and was not found to be similar to any other known collagenases. The optimum pH and temperature of the purified collagenase-I were 7.8 and $37^{\circ}C$, respectively.

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Purification and Characterization of Bacillus subtilis JS-17 Collagenase. (Bacillus subtilis JS-17이 생산하는 Collagenase의 정제 및 특성)

  • Lim Kyoung-Suk;Son Shung-Hui;Kang Ho Young;Jun Hong-Ki
    • Journal of Life Science
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    • v.15 no.4 s.71
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    • pp.657-663
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    • 2005
  • Collagenases are generally defined as enzymes that are capable of degrading the polypeptide backbone of native collagen under conditions that do not denature the protein. An extracellular collagenase-producing bacterial strain was isolated from kimchi and identified to be Bacillus subtilis JS-17 through morphological, cultural, biochemical characteristics and 16S rDNA sequence analysis. Optimum culture condition of Bacillus subtilis JS-17 for the production of collagenase was $1.5\%$ fructose, $1\%$ yeast extract, $0.5\%\;K_2HPO_4,\;0.4\%\;KH_2PO_4,\;0.01\%\;MgSO_4\cdot7H_2O,\;0.01\%\; MnSO_4\cdot4H_2O,\;,0.1\%$ citrate and $0.1\%\;CaCl_2$. The production of collagenase was optimal at $30^{\circ}C$ for 72 hr. A collagenase was isolated from the culture filtrate of Bacillus subtilis JS-17. The enzyme was purified using Amberlite IRA-900 column chromatography, Sephacryl S-300 HR column chromatography and DEAE-Sephadex A-50 column chromatography The purified collagenase has an specific activity 192.1 units/mg. The molecular weight of the purified enzyme was estimated to be 28 kDa by SDS-PACE. The purified collagenase has $100\%$ activity up to $55^{\circ}C$.

Characterization of a Collagenase-1 Inhibitory Peptide Purified from Skate Dipturus chilensis Skin (홍어류(Dipturus chilensis) 껍질로부터 분리 정제된 collagenase-1 저해 펩타이드의 특성)

  • Park, Sung-Ha;Lee, Jung-Kwon;Jeon, Joong-Kyun;Byun, Hee-Guk
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.44 no.5
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    • pp.456-463
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    • 2011
  • We attempted to isolate a collagenase-1 inhibitory peptide from skate Dipturus chilensis skin protein. The protein from skate skin was digested by various enzymes (alcalase, ${\alpha}$-chymotrypsin, neutrase, papain, pepsin, and trypsin) to produce a collagenase-1 inhibitory peptide. The collagenase-1 inhibitory activity of the peptides obtained was measured by gelatin digestion assay. Among the six hydrolysates, pepsin hydrolysate exhibited the highest collagenase-1 inhibitory activity. The peptide showing strong collagenase-1 inhibitory activity was purified by Sephadex G-25 gel chromatography and HPLC using an octadecylsilyls (ODS) column. The amino acid sequence of purified collagenase-1 inhibitory peptide was identified to be Asn-Leu-Asp-Val -Leu-Glu-Val-Phe (961 Da) by quadrupole time of flight (Q-TOF) and electrospray ionization mass spectrometry (ESI-MS) mass spectroscopy. The $IC_{50}$ value of purified peptide was 87.0 ${\mu}M$. Moreover, the peptide did not exhibit cytotoxic effects on human dermal fibroblast cell lines.

AUTORADIOGRAPHIC STUDY OF THE COLLAGENASE - INFLUENCE ON THE RAT PERIODONTIUM AFTER EXPERIMENTAL TOOTH MOVEMENT (실험적(實驗的) 치아이동후(齒牙移動後) 교원효소(膠原酵素) 투여(投與)가 치근막(齒根膜) 섬유(纖維)의 변화(變化)에 미치는 영향(影響)에 관(關)한 자기방사법적(自己放射法的) 연구(硏究))

  • Hong, Sung Joon;Suhr, Cheong Hoon
    • The korean journal of orthodontics
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    • v.20 no.2
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    • pp.227-245
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    • 1990
  • The purpose of this study was to analyze the reorganization of periodontal ligament after collagenase treatment with autoradiography. The author compared the collagenase-treated experimental group and no-treated experimental group with control group. Fourty eight Sprague-Dawley rats were divided into nine groups, including normal control and immediate group. Closed coil springs were used between the upper incisors and the first molars with 100 grams. Collagenase and $^3H-proline$ were adminstered and the samples were sacrificed and sectioned. After being dipped into the NTB-3 emulsion the samples were analyzed with light microscope under H/E stain. Data were analyzed by t-test and ANOVA. The results were as follows: 1) Generally collagenase-treated groups got more $^3H-proline$ uptake than no-treated groups. 2) Compared with normal control group, collagenase-treated group had the same $^3H-proline$ uptake in amount at 21th day. 3) Among cemento-enamel junction, middle, apex areas, cementa-enamel junction area of collagenase-treated group arrived at normal control level earlier than no-treated group. 4) Cemento-enamel junction area had the most $^3H-proline$ incorporation amount in no-treated group, but apex area had the most in collagenase group.

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Inhibitory Effect of Green Tea Extract on Collagenase Activity and Growth of Fish Pathogenic Bacteria (녹차 추출물을 이용한 어병세균의 collagen 분해효소 및 생육 억제)

  • Park, Sun-Mee;Park, Soo-Il;Huh, Min-Do;Hong, Yong-Ki
    • Journal of fish pathology
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    • v.12 no.2
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    • pp.83-88
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    • 1999
  • Green tea extract has been suggested to possess properties of collagenase inhibition and antimicrobial effect to fish pathogenic bacteria. Most fish pathogenic bacteria showed collagenase activities of 0.08-0.7 unit/ml. Among them, Edwardsiella tarda produced large amount of collagenase in the ST medium at $25^{\circ}C$ after 16 hrs. Biosynthesis of the enzyme from E. tarda was decreased to 1/3 by addition of 0.08-0.8 mg/ml of tea extract in the culture medium. The collagenase activity was inhibited almost 100% in the reaction mixture by 0.2 mg/ml of the extract. Then, the minimal inhibition concentration against E. tarda growth appeared as 8 mg/ml of the tea extract in the culture medium.

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Inhibitory Effect on Melanin Formation, Collagenase and Elastase Activity by synthesized Coenzyme $Q_{10}$ Derivatives (세포내 멜라닌 생성 및 Collagenase와 Elastase에 대한 Coenzyme $Q_{10}$ 유도체들의 억제활성)

  • Choi, Won-Sik;Jang, Do-Yoen;Nam, Seok-Woo;Eo, Jin-Yong;Lee, Kyoung-Ju
    • Applied Biological Chemistry
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    • v.51 no.3
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    • pp.164-170
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    • 2008
  • Coenzyme $Q_{10}$ and six derivatives of coenzyme Qn were synthesized and tested for their inhibitory effects on melanogenesis occurred in murine melanoma (B16/F1) cells and on collagenase/elastase activities as well. As the result, synthetic coenzyme Qn showed a potent inhibitory effect on melanin formation, collagenase and elastase activities in all tested concentrations. Among these synthetic compounds, coenzyme $Q_1$ and coenzyme $Q_2$ potentially inhibited melanin formation and elastase activity when compared to other coenzyme Qn derivatives. For the collagenase activities, all coenzyme Qn derivatives inhibited 80-85% of controls. As compared, coenzyme Qn derivatives exhibited strong inhibitory activities with the decrease of isoprenoid unit number of coenzyme Qn derivatives except for collagenase activity. For the inhibition of collagenase activity, moiety of benzoquinone might be considered as the active functional group. Taken together, coenzyme $Q_1$ and coenzyme $Q_2$ might be used for functional cosmetics.

Inhibition of Growth and Collagenase Activity of the Extract from Salvia miltiorrhiza against Microorganisms Causing Periodontal Diseases (단삼(Salvia miltiorrhiza) 추출물의 치주질환유발 세균의 생육억제 및 Collagenase 저해 활성)

  • 민응기;김용해;금상일;한영환
    • Korean Journal of Microbiology
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    • v.40 no.2
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    • pp.111-114
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    • 2004
  • This study was carried out to evaluate the inhibition of growth and collagenase activity of Salvia miltiorrhiza Bunge against microorganisms causing periodontal diseases. The ethanol extract of Salvia miltiorrhiza showed sig-nificant growth inhibition against microorganisms causing periodontal diseases. Ethanol extract was further fractionated with organic solvents in the order of hexane, chloroform and ethyl acetate. Among the fractions tested, the hexane fraction showed the highest cell growth inhibition. The minimal inhibitory concentration (MIC) of the extract against C. curvus, C. rectus, E. corrodens, F nucleatum, P. gingivalis, P. intermedia and W. succinogenes were 200, 50, 50, 250, 150, 250 and 200 ${\mu}g$/ml, respectively. The inhibition of collagenase activity by organic solvent fractions were higher than that of minocycline, and the inhibition ratio of collagenase activity was $88.2{\pm}2.1$ % in the chloroform fraction.

Inhibition of Collagenase by Anti-inflammatory Synthetic Flavones

  • Park Hae-Il;Sin Bo-Young;Kim Hyun-Pyo
    • Biomolecules & Therapeutics
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    • v.14 no.1
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    • pp.36-39
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    • 2006
  • Some flavones/flavonols were previously found to inhibit collagenase. To establish a therapeutic potential for skin inflammation, twenty-three synthetic flavone derivatives were examined for their inhibitory potential against collagenase from Clostridium histolyticum. From the results, it was found that most of them having various hydroxyl, methoxyl, methylsulfuryl and/or chloro substitution(s) on A- and B-rings were not efficient collagenase inhibitors. Among the synthetic flavones tested, only two synthetic derivatives, 3',4'-dihydroxyflavone and 5-hydroxy-4'-methoxyflavone, weakly inhibited bacterial collagenase (13-29% inhibition at 50-100 ${\mu}M$).

In Vitro Effects of Female Sex Hormones on Collagenase Activity of Gingival Fibroblast and Periodontal Ligament Fibroblast (여성 호르몬의 변화가 치은 섬유아세포와 치주인대세포의 교원질 분해 효소의 활성에 미치는 영향)

  • Sin, Ji-Yearn;Lee, Chul-Woo;Han, Soo-Boo
    • Journal of Periodontal and Implant Science
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    • v.29 no.1
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    • pp.31-40
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    • 1999
  • Many factors may affect periodontal changes during the physiologic conditions of woman(e.g. puberty, menstrual cycle, pregnancy, menopause). Recently many research has focused on the immunological changes of host, but the exact mechanism is not clear. Collagen is a major constituent of periodontium, and collagenase specifically digests the collagen and plays a role in destruction of periodontal tissue. So, I suppose that it participates with the cytokines in the inflammation of gingiva and vascular response during the changes of female sex hormones. Because there are some evidences of the existence of the receptors of estrogen and progesterone in the gingiva, it may be a target tissue of female sex hormones. In this experiment, gingival fibroblast and periodontal ligament cell were cultured in the presence of various concentrations of estrogen or progesterone corresponding to the menstrual cycle and pregnancy. Collagenase activity of the supernatant of culture media was determined by Spectrophotometric collagenase assay. The enzyme activity was calculated by the % decrease of the coated collagen. 1. The estrogen at both concentrations had no effect on the activity of collagenase of the gingival fibroblast. 2. The progesterone had some effect on the collagenase activity of the gingival fibroblast at low and high concentration of menstrual cycle, and elevated the enzyme activity at all range of pregnancy concentrations. 3. In periodontal ligament cells, estrogen elevated the enzyme activity at the early pregnancy concentration and progesterone elevated at the concentration just before menstruation. In this experiment, pregesterone elevated the collagenase activity of gingival fibroblast and periodontal ligament cells. But the mechanism of the up-regulation of the enzyme activity was not confirmed. The more experiments of direct effect of progesterone on gingival at the molecular level(e.g. northern blot analysis) can reveal the exact mechanism.

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