Cloning of a Novel Collagenase Gene from the Gram-Negative Bacterium Grimontia (Vibrio) hollisae 1706B and Its Efficient Expression in Brevibacillus choshinensis

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Abstract
The collagenase gene was cloned from Grimontia ( Vibrio ) hollisae 1706B, and its complete nucleotide sequence was determined. Nucleotide sequencing showed that the open reading frame was 2,301 bp in length and encoded an 84-kDa protein of 767 amino acid residues. The deduced amino acid sequence contains a putative signal sequence and a zinc metalloprotease consensus sequence, the HEXXH motif. G. hollisae collagenase showed 60 and 59% amino acid sequence identities to Vibrio parahaemolyticus and Vibrio alginolyticus collagenase, respectively. In contrast, this enzyme showed <20% sequence identity with Clostridium histolyticum collagenase. When the recombinant mature collagenase, which consisted of 680 amino acids with a calculated molecular mass of 74 kDa, was produced by the Brevibacillus expression system, a major gelatinolytic protein band of ∼60 kDa was determined by zymographic analysis. This result suggested that cloned collagenase might undergo processing after secretion. Moreover, the purified recombinant enzyme was shown to possess a specific activity of 5,314 U/mg, an ∼4-fold greater activity than that of C. histolyticum collagenase.

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