高压二氧化碳杀灭大肠杆菌过程中的蛋白质变化研究

Analysis of Intracellular Proteins of E. coli Inactivated by High Pressure Carbon Dioxide

摘要:

本研究采用37 ℃、10~50 MPa的高压二氧化碳（HPCD）处理大肠杆菌及其全蛋白溶液30 min，检测菌落总数、表观形态、蛋白质溶解性、蛋白质组和蛋白质二级结构的变化，研究HPCD胁迫下大肠杆菌的蛋白质变化规律。结果表明：HPCD处理压强升至30 MPa，菌落总数对数值有显著降低；经不同压强处理，菌体形态没有发生显著变化；与对照组（0.1 MPa）相比，可溶性蛋白经20 MPa的HPCD处理后含量显著降低，从1.77 mg/mL降为0.57 mg/mL，并随压强继续上升逐渐降低；30 MPa的HPCD处理菌液，经双向电泳分离到46个差异最大的蛋白点，质谱鉴定具代表性的16个点，分别涉及细胞骨架、新陈代谢、信号转换、运输与翻译等功能；蛋白溶液圆二色谱结果显示，对照组中α-螺旋占68.04%，β-折叠占0.33%，随HPCD处理压强增加，α-螺旋含量显著降低（P<0.05）。经20 MPa的HPCD处理，β-折叠含量显著增加（P<0.05），但随压强继续增加含量变化不显著。

Abstract:

The variation of proteins in E. coli after 10~50 MPa HPCD treatment at 37 ℃ for 30 min was investigated in this study. The total bacterial counts, cell structure, protein solubility, proteome and secondary structure of protein were analyzed. The results showed that the logarithm of the total bacterial counts of E. coli after HPCD treatment was significantly decreased when the pressure increased to 30 MPa. The cell structure did not change with different HPCD treatments (10~50 MPa). The soluble protein content significantly decreased from 1.77 mg/mL to 0.57 mg/mL when the extraction of E. coli proteins was treated by 20 MPa HPCD in comparison with control (0.1 MPa treatment). The content of proteins decreased as the pressure of HPCD increased. Forty-six protein spots with the greatest expression difference were isolated from the control group and 30 MPa HPCD treatment group using two-dimensional gel electrophoresis. There were 16 representative protein spots were identified by MALDI TOF/TOF mass spectrometry, which were involved in cytoskeleton structure, cell metabolism, transmission of signal and transcription. The circular dichroism results of protein solutions showed that the α-helix accounted for 68.04% and the β-sheet accounted for 0.33% in the control group. The α-helix content significantly decreased (P<0.05) as the pressure of HPCD increased, while theβ-sheet content significantly increased (P<0.05) when treated by 20 MPa HPCD, but no further change occurred when the pressure continued to increase.