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.

Fig. 1.

Sequencing strategy for the collagenase gene inserted into pCC1BAC-2. The thick line represents the collagenase gene inserted into pCC1BAC-2 plasmid. The thin line indicates probe region used for cloning. The lower arrows indicate the direction of sequence determinations, starting from specific primers.

Fig. 2.

DNA sequence and deduced amino acid sequence of Grimontia hollisae collagenase gene. The N-terminal amino acid sequences of the 74- and 60-kDa collagenases are indicated by box 1. Numbered boxes indicate biochemically identified peptides as follows: boxes 2, 3, and 4, lysyl endoprotease-digested fragments; box 5, trypsin-digested fragment; boxes 6 and 7, V8 protease-digested fragments. The zinc metalloprotease HEXXH consensus motif is underlined. The SD site is indicated by dotted line. The putative transcriptional terminator sequence is indicated by arrows.

Fig. 3.

Amino acid sequence comparison of Grimontia hollisae collagenase with homologous collagenase. (A) The amino acid sequences from G. hollisae (the present study), Vibrio parahaemolyticus (NP_797719), and Vibrio alginolyticus (CAA44501) were aligned by using the CLUSTAL W2 program. Identical residues among the three sequences are indicated by asterisks. (B) Schematic representation of the domain architecture of G. hollisae (the present study), V. parahaemolyticus (NP_797719) and V. alginolyticus (CAA44501). Pre, signal peptide; pro, putative pro-domain; PKD, polycystic kidney disease-like domain; PPC, pre-peptidase C-terminal domain.

Fig. 4.

Analysis of recombinant collagenase purified from Brevibacillus culture media. (A) Purified recombinant collagenase was analyzed by SDS-PAGE using a reducing 7.5% gel. Lane 1, molecular weight marker; lane 2, culture medium from Brevibacillus (carrying pNY326-Col2); lane 3, purified collagenase. (B) Real-time gelatin zymography using a nonreducing 10% gel. Lane 1, molecular weight marker; lane 2, SDS-PAGE of purified collagenase; lanes 3 and 4, gelatin zymogram of purified collagenase after 2 h (lane 3) or 19 h (lane 4) of incubation. (C) Inhibition assay using real-time gelatin zymography. Lane 1, SDS-PAGE of purified collagenase; lanes 2 to 6, gelatin zymogram of purified collagenase in the absence (lane 2) or presence of the inhibitors EDTA (lane 3), o-phenanthroline (lane 4), NEM (lane 5), and PMSF (lane 6).