Gene cloning and characterization of a Bacillus vietnamensis metalloprotease.
A Bacillus vietnamensis metalloprotease (BVMP) with high affinity toward collagen was isolated and purified from the culture supernatant of Bacillus vietnamensis 11-4 occurring in Vietnamese fish sauces. The BVMP gene was cloned and its nucleotide and coded amino acid sequences determined. BVMP consists of 547 amino acid residues, with the zinc-binding sites conserved in common metalloproteases. It shares 57% amino acid identity with thermolysin originating from Bacillus thermoproteolyticus. The three-dimensional structure of BVMP was deduced by computer-aided modeling with the use of the known three-dimensional thermolysin structure as a template. Like thermolysin, BVMP cleaved the oxidized insulin B-chain at the peptide bonds involving the N-terminal sides of hydrophobic and aromatic amino acids. BVMP also showed high hydrolytic activity toward gelatin, collagen, casein, and elastin, especially toward the skeletal proteins at increased NaCl concentration. The high activity was found to be due to enhanced affinity to the substrates. Kinetical data on BVMP indicated that the Km values for the hydrolysis of Cbz-GPGGPA as a collagen model decreased as the concentration of added NaCl increased. Some contribution of this enzyme during the aging of fish sauces at high salt concentrations can thus be expected.
Department of Nutritional Science, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan.
Mihwan
M
Kim
Yoshitaka
Y
Nishiyama
Kiyoshi
K
Mura
Chiyoko
C
Tokue
Soichi
S
Arai
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005396:Fish Products / Q000382:microbiology*; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D045726:Metalloproteases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D004269:DNA, Bacterial; D007328:Insulin; D009842:Oligopeptides; C033157:N-carbobenzoxyglycyl-prolyl-glycyl-glycyl-prolyl-alanine; D045726:Metalloproteases