• KSBB Journal
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• v.19 no.4
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• pp.295-300
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• 2004

A marine bacterium for producing an collagenolytic protease was isolated from the southern sea of Korea and identified as Vibrio vulnificus and named as Vibrio vulnificus CYK279H. This strain producing an collagenolytic protease was showed high activity toward collagen and gelatin as substrate. The optimum initial pH, NaCl, and temperature for cell growth and protease production was 7.5, 2.0% and 25$^{\circ}C$, respectively. Optimization for collagenolytic protease production was composed of 0.3% D-galactose, 0.6% yeast extract, 4.0% gelatin, 0.2% (NH$_4$)$_2$SO$_4$, and 0.2 mM ferric citrate in artificial sea water. The maximum protease production was required gelatin and yeast extract. The collagenolytic protease production by Vibrio vulnificus CYK279H reached a maximum of 73 unit/l after the cultivation for 18 h under the optimized medium.

• BMB Reports
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• v.35 no.2
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• pp.165-171
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• 2002

A serine collagenolytic protease was purified from the internal organs of filefish Novoden modestrus, by ammonium sulfate, ion-exchange chromatography on a DEAE-Sephadex A-50, ion-exchange rechromatography on a DEAE-Sephadex A-50, and gel filtration on a Sephadex G-150 column. The molecular mass of the filefish serine collagenase was estimated to be 27.0 kDa by gel filtration and SDS-PAGE. The purified collagenase was optimally active at pH 7.0-8.0 and $55^{\circ}C$. The purified enzyme was rich in Ala, Ser, Leu, and Ile, but poor in Trp, Pro, Tyr, and Met. In addition, the purified collagenolytic enzyme was strongly inhibited by N-P-toluenesulfonyl-L-lysine chloromethyl ketone (TLCK), diisopropylfluorophosphate (DFP), and soybean trypsin inhibitor.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.6
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• pp.593-598
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• 2005

A collagenolytic enzyme, produced by Vibrio vulnificus CYK279H, was purified by ultrafiltration, dialysis, Q-Sepharose ion exchange and Superdex-200 gel chromatography. The enzyme from the supernatant was purified 13.2 fold, with a yield of 11.4%. The molecular weight of the purified enzyme was estimated by SDS-PAGE to be approximately 35.0kDa. The N-terminal sequence of the enzyme was determined as Gly-Asp-Pro-Cys-Met-Pro-Ile-Ile-Ser-Asn. The optimum temperature and pH for the enzyme activity were $35^{\circ}C$ and 7.5, respectively. The enzyme activity was stable within the pH and temperature ranges 6.8-8.0 and $20{\sim}35^{\circ}C$, respectively. The purified enzyme was strongly activated by $Zn^{2+},\;Li^{2+},\;and\;Ca^{2+}$, but inhibited by $Cu^{2+}$. In addition, the enzyme was strongly inhibited by 1, 10-phenanthroline and EDTA. The purified enzyme was suggested to be a neutral metalloprotease.

• Restorative Dentistry and Endodontics
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• v.33 no.2
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• pp.148-161
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• 2008

The purpose of this study was to evaluate the effect of chlorhexidine (CHX) on microtensile bond strength (${\mu}TBS$) of dentin bonding systems. Dentin collagenolytic and gelatinolytic activities can be suppressed by protease inhibitors, indicating that MMPs (Matrix metalloproteinases) inhibition could be beneficial in the preservation of hybrid layers. Chlorhexidine (CHX) is known as an inhibitor of MMPs activity in vitro. The experiment was proceeded as follows: At first, flat occlusal surfaces were prepared on mid-coronal dentin of extracted third molars. GI (Glass Ionomer) group was treated with dentin conditioner, and then, applied with 2 % CHX. Both SM (Scotchbond Multipurpose) and SB (Single Bond) group were applied with CHX after acid-etched with 37% phosphoric acid. TS (Clearfil Tri-S) group was applied with CHX, and then, with adhesives. Hybrid composite Z-250 and resin-modified glass ionomer Fuji-II LC was built up on experimental dentin surfaces. Half of them were subjected to 10,000 thermocycle, while the others were tested immediately. With the resulting data, statistically two-way ANOVA was performed to assess the ${\mu}TBS$ before and after thermo cycling and the effect of CHX. All statistical tests were carried out at the 95 % level of confidence. The failure mode of the testing samples was observed under a scanning electron microscopy (SEM). Within limited results, the results of this study were as follows; 1. In all experimental groups applied with 2 % chlorhexidine, the microtensile bond strength increased, and thermo cycling decreased the micro tensile bond strength (P > 0.05). 2. Compared to the thermocycling groups without chlorhexidine, those with both thermocycling and chlorhexidine showed higher microtensile bond strength, and there was significant difference especially in GI and TS groups. 3. SEM analysis of failure mode distribution revealed the adhesive failure at hybrid layer in most of the specimen. and the shift of the failure site from bottom to top of the hybrid layer with chlorhexidine groups. 2 % chlorhexidine application after acid-etching proved to preserve the durability of the hybrid layer and microtensile bond strength of dentin bonding systems.