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  

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

作　　者：张欢欢 陈柔珂[1] 徐俊 ZHANG Huanhuan;CHEN Rouke;XU Jun(State Key Laboratory of Microbial Metabolism,School of Life Sciences and Biotechnology,Shanghai Jiao Tong University,Shanghai 200240,China;School of Oceanography,Shanghai Jiao Tong University,Shanghai 200240,China)

机构地区：[1]上海交通大学生命科学技术学院微生物代谢国家重点实验室,上海200240 [2]上海交通大学海洋学院,上海200240

出　　处：《微生物学报》2024年第5期1494-1505,共12页Acta Microbiologica Sinica

基　　金：国家重点研发计划(2020YFA0906800);国家自然科学基金面上项目(41976085,42276091)

摘　　要：【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Co^(2+)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。[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℃ and pH 6.0.Pyprol binding to Co^(2+)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℃ 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.

关 键 词：超嗜热古菌 雅氏火球菌 脯肽酶 热稳定 耐压 

分 类 号：Q936[生物学—微生物学]