超氧化物歧化酶和过氧化氢酶融合蛋白的设计、表达及稳定性研究

Design, Expression, and Stability Evaluation of a Superoxide Dismutase-catalase Fusion Protein

摘要:

【目的】设计一种超氧化物歧化酶（SOD）和过氧化氢酶（CAT）酶活力和稳定性均较高的融合蛋白。【方法】以AcSOD11和FeCAT蛋白质为研究对象，设计3种融合蛋白AcSOD11-（GGGGS）*3-FeCAT、FeCAT-（GGGGS）*3-AcSOD11和FeCAT-（EAAAK）*3-AcSOD11，分别命名为AcSOD11-G3-FeCAT、FeCAT-G3-AcSOD11和FeCAT-E3-AcSOD11，并在大肠杆菌BL21（DE3）中进行可溶性表达，然后对3种融合蛋白进行热稳定性和pH稳定性检测。【结果】3种融合蛋白的SOD比酶活无显著差异，而CAT比酶活存在显著差异，FeCAT-G3-AcSOD11的CAT比酶活最高（531 U/mg），FeCAT-E3-AcSOD11的最低，表明（GGGGS）*3柔性肽连接有利于保持CAT的酶活力。抗逆性研究发现，FeCAT-G3-AcSOD11中CAT和SOD的高温和酸碱耐受性都较强。【结论】该研究成功构建了3种融合蛋白，得到一种SOD和CAT酶活力和稳定性均较高的融合蛋白FeCAT-G3-AcSOD11，为SOD和CAT的联合应用提供了理论依据。

Abstract:

[Objective] This study aims to design a fusion protein with high activities and high stability of superoxide dismutase (SOD) and catalase (CAT).[Method] Using AcSOD11 and FeCAT proteins as the research subjects, three fusion proteins of SOD and CAT were designed: AcSOD11-(GGGGS)*3-FeCAT, FeCAT-(GGGGS)*3-AcSOD11, and FeCAT-(EAAAK)*3-AcSOD11, which were named AcSOD11-G3-FeCAT, FeCAT-G3-AcSOD11, and FeCAT-E3-AcSOD11, respectively. Soluble expression of these three fusion proteins was achieved in Escherichia coli BL21(DE3). The thermal stability and pH stability of the three fusion proteins were then assessed.[Result] The three fusion proteins showed no significant difference in the specific activity of SOD, while they had significant differences in the specific activity of CAT. The specific activity of CAT was the highest (531 U/mg) in FeCAT-G3-AcSOD11 and the lowest in FeCAT-E3-AcSOD11, indicating that the flexible (GGGGS)*3 peptide linker was favorable for maintaining CAT enzyme activity. Stress resistance studies demonstrated that both CAT and SOD in FeCAT-G3-AcSOD11 exhibited strong tolerance to high temperatures and varying pH conditions.[Conclusion] This study successfully constructed three fusion proteins and obtained FeCAT-G3-AcSOD11, a highly stable fusion protein with high enzyme activities of both SOD and CAT, providing a theoretical foundation for the combined application of SOD and CAT.