来源于超嗜热古菌雅氏火球菌(Pyrococcus yayanosii)CH1耐热耐压脯肽酶的酶学性质研究

【目的】脯肽酶是一种能从二肽(Xaa-Pro)的C末端水解脯氨酸或羟脯氨酸残基的肽酶。对深海来源的雅氏火球菌(Pyrococcus yayanosii) CH1基因组中PYCH_07700基因编码的蛋白Pyprol的体外酶学性质进行研究，以期发现新型脯肽酶。【方法】在小宝岛热球菌(Thermococcus kodakarensis) TS559中异源表达Pyprol。使用二肽Met-Pro作为底物，检测重组蛋白的脯肽酶活性。【结果】Pyprol的最适温度为100 ℃，最适pH为6.0。Pyprol在与Co²⁺结合时活性最高，最适的金属离子浓度为1.2 mmol/L。与P. furiosus来源的脯肽酶Pfprol相比，Pyprol在更宽的pH范围具有活性，并且能够耐受更高浓度的金属离子。Pyprol是耐压蛋白，最适静水压为40 MPa。与常压条件下相比，40 MPa下，Pyprol在40、70和100 ℃均有更高的活性。【结论】来源于深海热液喷口的严格嗜压的超嗜热古菌P. yayanosii CH1的新型脯肽酶Pyprol具有热稳定和耐压特性。

Characterization of a thermostable and piezotolerant prolidase from the hyperthermophilic archaeon Pyrococcus yayanosii CH1

Objective] Prolidase is an enzyme that can hydrolyze proline or hydroxyproline residues from the C-terminal dipeptides (Xaa-Pro). A putative prolidase-encoding gene was identified in the genome of Pyrococcus yayanosii CH1 isolated from the deep sea. In this study, we characterized the enzymatic properties of Pyprol encoded by PYCH_07700 in vitro, aiming to find a new prolidase. Methods] Pyprol was heterologously expressed in the hyperthermophilic archaeon Thermococcus kodakarensis TS559. The dipeptide Met-Pro was used as a substrate to test the prolidase activity of the purified recombinant protein. Results] Pyprol showed the best performance at 100 °C and pH 6.0. Pyprol binding to Co²⁺ exhibited the maximum activity, and the optimal metal ion concentration was 1.2 mmol/L. Pyprol had catalytic activity in a wider pH range and can tolerate higher concentrations of metal ions than the prolidase Pfprol from P. furiosus. Pyprol was a piezotolerant protein with an optimal hydrostatic pressure of 40 MPa. It exhibited enhanced activities at 40, 70, and 100 °C under 40 MPa, compared with at the atmospheric pressure. Conclusion] Pyprol is a novel thermostable and piezotolerant prolidase of P. yayanosii CH1, which is an obligate piezophilic hyperthermophilic archaeon strain isolated from a deep-sea hydrothermal vent.