Peptide condensation activity of a neutral protease from Vibrio sp. T1800 (Vimelysin).

Condensation of Cbz-Asp and PheOMe catalyzed by a neutral protease from Vibrio sp. T1800 (Vimelysin: VLN) was studied. VLN showed a relatively higher catalytic activity of condensation and an apparently larger yield after 3 h or 24 h, in comparison with thermolysin (TLN), especially at lower pH and temperatures.VLN showed higher solvent-tolerance than TLN. TLhe apparent highest yield (25%) was obtained in 30% DMSO by using VLN; under similar conditions, TLN gave only about a half of this value. The rate of the condensation reaction per mole of enzyme (v/[E](o)) in DMSO 50% at 37 degrees C and pH 6.5 was 0.16 s(-1) for VLN and 0.047 s(-1) for TLN. In 30% ethanol VLN showed more than three-fold peptide yield than TLN after 5 h reaction. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 387-390, 1997.

Kinetic characterization of the neutral protease vimelysin from Vibrio sp. T1800.

The kinetics of the hydrolysis of dipeptide and tripeptide substrates by the recently discovered neutral protease from Vibrio species T1800 (vimelysin) were studied. In the pH dependence of the apparent second-order rate constant, the pKa2 value of vimelysin (approximately 6.5) was significantly lower than thermolysin (8.3), although the pKa1 (approximately 5.1) values were comparable (5.0). The Kcat/Km(lim) parameter for hydrolysis of Fua-Gly-PheNH2 (Fua = furylacryloyl) was more than sevenfold greater than for Fua-Gly-LeuNH2. This higher specificity for Fua-Gly-PheNH2 was deduced for both Kcat and Km parameters. Fua-Phe-PheNH2 showed the highest Kcat/Km(app) value of the six substrates studied. The discrimination between Phe/Leu at the P1' site was most evident when the P1 site was not sufficiently filled. Reflecting the characteristically high proteolytic activity of vimelysin at lower temperatures [Oda, K., Okayama, K., Okutomi, K., Shimada, M., Sato, R. & Takahashi, S. (1996) Biosci. Biotech. Biochem. 60, 463-467], the Arrhenius plot of the apparent second-order rate constant for the hydrolysis of Fua-Gly-LeuNH2 showed an inverse temperature dependence; higher reaction rates were observed at lower temperatures. This was not merely due to the pKa shift nor to thermal denaturation of the enzyme coupling, but rather to the Kcat(app) parameter, which alone showed an inverse temperature dependence. A model containing two temperature-dependent forms of the active enzyme was postulated to explain this unique temperature dependence.

Pressure-induced change in proteins studied through chemical modifications.

Pressure-induced change of two bovine proteins, alpha-lactalbumin (LA) and beta-lactoglobulin (LG), was investigated at neutral pH by means of fluorescence and CD spectroscopy. The rate and the extent of modification was considerably increased by applying high pressure during the dansylation reaction of LG, while those for LA were only moderately affected. This difference was accounted for by the structural deformation of these proteins under high pressure. The fluorescence spectrum of these proteins measured under elevated pressure, as well as their fluorescence and CD spectra after the pressure release, indicated different responses towards pressure. The structural change of LA was practically reversible up to 400 MPa, whereas that of LG lost reversibility at 150 MPa or lower. Fluorescent measurement of dansylated (prepared at atmospheric pressure) proteins, especially the energy transfer from the intrinsic Trp residue to the dansyl group, showed that the protein structure was deformed by pressure and that the energy transfer facility of the two proteins was differently affected by high pressure, probably reflecting the degree of compactness of their pressure-perturbed structures.