RESUMEN
We characterized a protease of the M4 family from the cold-adapted Vibrio sp. Pr21 that was isolated from seawater at 320-m deep in Sagami Bay, Japan, and named it as PR protease based on the strain name Pr21. The PR protease had activities at 10-60 °C and 0.1-350 MPa, with the optimal temperature and pressure at 40 °C and 250 MPa. The mutant 10C9 (Q301P) obtained by error-prone PCR had higher activities than the wild-type enzyme at 10-60 °C, and the Q301P mutation contributed to the increase of the activity. The specific activity value of 10C9 was also higher than that of the wild-type enzyme at 0.1-200 MPa, but the specific activity ratios (1.28-1.59) of 10C9/wild-type enzyme at 50-200 MPa at 30 °C were smaller than those at 10-60 °C (1.73-4.39) at 0.1 MPa. The catalytic efficiency value of 10C9 was lower than that of the wild-type enzyme at 200 MPa. The homology models of PR protease suggested that the side chain of Q301 was hydrogen-bonded with the carbonyl oxygen atom of the main chain of N234 in the wild-type enzyme, and P301 had no contact with N234 in 10C9. The break of the hydrogen bond in 10C9 might strengthen the increase of the flexibility of the ß-sheet near the substrate binding pocket under high-temperature conditions, whereas the flexibility of the ß-sheet in 10C9 might be moderately increased compared to that in the wild-type enzyme under high-pressure conditions.
