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 β-sheet near the substrate binding pocket under high-temperature conditions, whereas the flexibility of the β-sheet in 10C9 might be moderately increased compared to that in the wild-type enzyme under high-pressure conditions.

我们从冷适应的弧菌中鉴定了M4家族的蛋白酶sp。从日本相模湾深320米的海水中分离出的Pr21，根据菌株名称Pr21命名为PR蛋白酶。PR蛋白酶在10–60°C和0.1–350 MPa时具有活性，最佳温度和压力在40°C和250 MPa时。通过易错PCR获得的突变体10C9（Q301P）在10–60°C时具有比野生型酶更高的活性，并且Q301P突变有助于活性的增加。10C9的比活度值在0.1–200 MPa时也比野生型酶高，但10C9 /野生型酶在30°C时50–200 MPa的比活度比（1.28–1.59）为小于在0.1 MPa下10–60°C（1.73–4.39）的温度。在200 MPa时10C9的催化效率值低于野生型酶。PR蛋白酶的同源性模型表明，野生型酶中Q301的侧链与N234主链的羰基氧原子氢键结合，而P301在10C9中不与N234接触。10C9中氢键的断裂可能会增强高温条件下靠近底物结合袋的β-折叠的柔韧性的增加，而10C9中的β-折叠的柔韧性可能会比高温下的温和增加。高压条件下的野生型酶。