title: Cloning of a novel collagenase gene from the gram-negative bacterium Grimontia (Vibrio) hollisae 1706B and its efficient expression in Brevibacillus choshinensis.
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.
authors.affiliation: Nippi Research Institute of Biomatrix, Kuwabara, Toride, Ibaraki 302-0017, Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Teramura
authors.forename: Keisuke
authors.initials: K
authors.lastname: Tanaka
authors.forename: Katsumasa
authors.initials: K
authors.lastname: Iijima
authors.forename: Osamu
authors.initials: O
authors.lastname: Hayashida
authors.forename: Koki
authors.initials: K
authors.lastname: Suzuki
authors.forename: Shunji
authors.initials: S
authors.lastname: Hattori
authors.forename: Shinkichi
authors.initials: S
authors.lastname: Irie
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D058675:Brevibacillus / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D024042:Collagen Type I / Q000378:metabolism; D017364:Collagenases / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism
chemical_list: D024042:Collagen Type I; D017364:Collagenases