定点突变提高过氧化氢酶热稳定性和催化效率

Enhanced Thermo-Stability and Catalytic Efficiency of Catalase by Site-Directed Mutagenesis

DOI：10.3969/j.issn.1673-1689.2020.03.014

中文关键词: 过氧化氢酶 定点突变 热稳定性 催化效率

英文关键词: catalase,site-directed mutagenesis,thermo-stability,catalytic efficiency

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中文摘要:

过氧化氢酶催化过氧化氢分解形成水和氧，主要作用是保护细胞免受活性氧(ROS)的损伤，尽管其在工业上被广泛使用，但因热稳定的不足限制了它的使用前景。本研究以Bacillus pumilusML413来源的血红素过氧化氢酶(KatX2)为研究对象，采用定点突变技术提高其热稳定性及催化效率。利用 PoPMuSiC algorithm软件计算并预测了其潜在突变位点的折叠自由能，根据预测的结果，选择了其中自由能最低的3个位点（K117V, K117I 和 K117M）进行突变。结果发现，对K117位点进行突变，可以有效提高KatX2的热稳定性，突变体K117V在60 ℃下半衰期比野生型KatX2提高38 min，催化效率较突变前提高31.7%。根据结构分析发现，该点突变并未改变KatX2的二级结构。突变体K117V具有良好的工业应用潜力。

英文摘要:

Catalase catalyzes the breakdown of hydrogen peroxide to form water and oxygen. The main role of this enzyme is to protect cell damage against reactive oxygen species（ROS）. Although it is widely used industrially，it is sensitive to high temperature，which limits its use. In this work，a thermo-stable heme-catalase（KatX2） was constructed with high catalytic efficiency from Bacillus pumilus ML413 through site-directed mutagenesis and using PoPMuSiC algorithm to predict and calculate folding free energy of highly potential residues for mutation. Lys117 was selected and genetically engineered to enhance KatX2 thermostability. From the set of mutants，the K117V recombinant mutant showed significant improvement in thermo-stability with the increase of half-life at 60 ℃ by 38 minutes compared to the wild-type. Furthermore，the catalytic efficiency of K117V mutant was increased by 31.7% compared to that of the wild-type. Interestingly，no change of secondary structure was observed after structural analysis. Therefore，this highly stable KatX2 could be used as a potential industrial biocatalyst.

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