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http://dx.doi.org/10.5352/JLS.2003.13.3.308

Isolation and Characterization of Recombinant Vibrio parahaemolyticus Collagenase  

차재호 (부산대학교 자연과학대학 미생물학과)
김수광 (부산대학교 자연과학대학 미생물학과)
전인준 (부산대학교 자연과학대학 미생물학과)
이재원 (부산대학교 자연과학대학 미생물학과)
Publication Information
Journal of Life Science / v.13, no.3, 2003 , pp. 308-313 More about this Journal
Abstract
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 $60^{\circ}C.$
Keywords
collagenase; collagen; Vibrio parahaemolyticus;
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