Comparative studies on the activities of collagenases from Grimontia hollisae and Clostridium hystoliticum in the hydrolysis of synthetic substrates

Abstract

The collagenase produced by a gram-negative bacterium Grimontia hollisae strain 1706B (Ghcol) degrades collagen more efficiently than that produced by a gram-positive bacterium Clostridium histolyticum (Chcol), which is currently the most widely used collagenase in industry [Teramura et al. (Cloning of a novel collagenase gene from the gram-negative bacterium Grimotia (Vibrio) hollisae 1706B and its efficient expression in Brevibacillus choshinensis. J Bacteriol 2011;193:3049-3056)]. Here, we compared the Ghcol and Chcol activities using two synthetic substrates. In the hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Lys-L-Pro-L-Leu-Gly-L-Leu-[N3-(2, 4-dinitrophenyl)-L-2, 3-diaminopropioyl]-L-Ala-L-Arg-NH2, Ghcol exhibited 350-fold higher activity than Chcol in the absence of CaCl2 and NaCl. The Ghcol activity markedly decreased with increasing concentrations of buffer, CaCl2 or NaCl, while the Chcol activity did not, suggesting that the Ghcol activity was sensitive to solvent components. In the hydrolysis of N-[3-(2-furyl)acryloyl]-L-Leu-Gly-L-Pro-Ala, Ghcol exhibited 16-fold higher activity than Chcol in the absence of CaCl2 and NaCl, and both enzyme activities did not decrease with increasing concentrations of buffer, CaCl2 or NaCl. pH dependences of activity revealed that the ionizable group responsible for acidic pKe may be Glu for Ghcol and Chcol, while that for alkaline pKe may be His for Ghcol and Tyr for Chcol. These striking differences suggest that the catalytic mechanism of Ghcol might be considerably different from that of clostridial collagenases.

Characterization of proteases activities in Ficus carica cultivars

“…First we analyzed the ficin and collagenase activities to hydrolyze MOCAc‐KPLGL(Dpa)‐AR (Figure 1A). This peptide has been used for the characterization of collagenase (Hatanaka et al., ; Takita et al., ). In this peptide, the fluorescent 7‐methoxycoumarin group is quenched by energy transfer to the 2,4‐dinitrophenyl group, and it emits fluorescence when the 7‐methoxycoumarin group dissociates from the 2,4‐dinitrophenyl group by the cleavage at the peptide bond between the two groups.…”

“…() reported that the collagenase produced by a gram‐negative bacterium Grimontia hollisae is classified as metalloprotease and degrades collagen more efficiently than C. histolyticum collagenase (Teramura et al., ). We reported that there was a striking difference between G. hollisae collagenase and C. histolyticum collagenase in the effect of buffers, CaCl 2 , and NaCl on activity and pH‐dependence of activity (Takita et al., ). Here we analyzed the cleavage sites of MOCAc‐KPLGL(Dpa)‐AR by G. hollisae collagenase.…”

“…We previously reported that the cleavage site of MOCAc‐KPLGL(Dpa)‐AR by C. histolyticum collagenase was Gly‐Leu (Takita et al., ). Here we analyzed the cleavage site by ficin.…”

“…M6M0414) was purchased from Nacalai Tesque (Kyoto, Japan). Collagenase from Grimontia hollisae was prepared as described previously (Teramura et al., : Takita et al., ). The cysteine protease inhibitor E‐64 was purchased from Sigma‐Aldrich (Saint Louis, MO, USA), and the serine protease inhibitor phenylmethanesulfonyl fluoride (PMSF) was purchased from Wako Pure Chemical Industries (Osaka, Japan).…”

“…The reaction was carried out as described previously (Takita et al., ; Hatanaka, Uesugi, Arima, Usuki, & Iwabuchi, ). Briefly, a 1.56 µM MOCAc‐KPLGL(Dpa)‐AR solution was prepared in 48.9 mM HEPES‐NaOH buffer (pH 7.5), 0.1 mM EDTA, 3.3 mM DTT.…”