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KMID : 0613820030130030308
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Journal of Life Science
2003 Volume.13 No. 3 p.308 ~ p.313
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Isolation and Characterization of Recombinant Vibrio parahaemolyticus Collagenase
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Kim Soo-Kwang
Jun In-Joon
Lee Jae-Won
Cha Jae-Ho
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Abstract
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The collagenase gene from Vibrio parahaemolyticus 04 was subcloned into an expression vector pET-29b. The recombinant collagenase was expressed in Escherichia coli BL21(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℃. The thermostability measurement of the enzyme indicated that the enzyme was stable up to 55℃, but the activity was diminished quickly above 60℃.
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KEYWORD
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collagenase, collagen, Vibrio parahaemolyticus
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