• Fisheries and Aquatic Sciences
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• v.5 no.3
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• pp.145-149
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• 2002

The effects of pH, temperature and various inhibitors on the proteolytic activity of the cell lysate of Uronema marium were investigated using colorimetric and substrate gel electro­phoretic methods. The cell lysate of U. marinum showed proteolytic activity over a wide range of pH, and pH optima ranged from pH 5 to 7. The proteolytic activity was increased according to a rise of temperature but decreased at $40^{\circ}$. The proteolytic activity of the parasite lysate was significantly inhibited by protease inhibitors including trans-epoxysuccinyl -L-leucylamido-(4-guanidino) butane (E-64), pepstatin A, phenyl-methanesulfonyl fluoride(PMSF), and ethylenediamine-tetraacetic acid (EDTA). Preincubation of the lysate with E-64 showed the maximum inhibition of the caseionolytic activity. Four protease bands (152, 97, 67 and 40 kDa) were detected by gelatin SDS-PAGE. Significant inhibition of caseinolytic activity and complete abolition of a 152 kDa band in gelatin SDS-PAGE by EDTA indicated that the cell lysate of U. marinum had a metalloprotease Another three proteolytic bands were inhibited by E64, a cysteine protease inhibitor. Preincubation of the cell lysate with pepstatin or PMSF had no effects on the protease bands.

• Korean Journal of Plant Resources
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• v.30 no.3
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• pp.272-279
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• 2017

EP was obtained through 20% ethanol extraction of Pueraria lobata root, and the fermented form of EP, FEP, was prepared from the EP after incubating with Lactobacillus rhamnosus vitaP1. There was no significant toxicity by EP and FEP up to $1000{\mu}g/ml$ in NIH-3T3, HaCaT, and B16F10 cells. In addition to antioxidant potentials of EP and FEP determined by DPPH and ABST assays, we confirmed increase of procollagen type I and elastin synthesis by supplementation of the EP and FEP at the concentration of $50{\mu}g/ml$ using ELISA kits. The protein expression levels of matrix metalloprotease (MMP)-1, -3, and -9, those are involved in the degradation of collagen or other skin matrix proteins, were remarkably suppressed while their inhibitory protein metallopeptidase inhibitor 1 (TIMP-1) was greatly up-regulated by supplementation of the EP and FEP at a concentration of $50{\mu}g/ml$. Taken together, both EP and FEP supplementation could be involved in the suppression of the skin wrinkle formation through inhibiting degradation of collagen and stimulating the synthesis of collagen and elastin. The results showed that the anti-wrinkle potential of the EP and FEP will be a promising candidate for developing cosmeceutical compounds or products.

• 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 $55^{\circ}C$.