融合自组装双亲短肽对重组过氧化氢酶酶学性质的影响

Effect of fusing a self-assembling amphipathic peptide on the enzymatic properties of recombinant catalase

【目的】利用融合自组装双亲短肽策略对源自枯草芽孢杆菌(Bacillus subtilis)的过氧化氢酶KatA进行改性,以强化重组过氧化氢酶在工业中的应用适应性。【方法】将自组装双亲短肽S1vw通过连接肽PT-linker融合在KatA的N端,构建重组质粒pHT254-S1vw-PT-katA,将其与携带天然酶基因的pHT254-katA分别转入枯草芽孢杆菌WB800N中进行分泌表达,之后将分离纯化得到的纯酶进行酶学性质研究。【结果】成功构建出工程菌并将胞外粗酶液通过乙醇沉淀、DEAE阴离子交换层析、疏水层析和凝胶过滤层析4步纯化,最终获得电泳纯的重组酶蛋白。酶学性质研究结果显示,融合酶S1vw-PT-KatA和天然酶KatA的最适反应温度均为30℃,最适反应pH值均为11.0。然而,融合酶在pH 12.0下孵育30 min的相对酶活为77.3%,是相同处理条件下天然酶相对酶活的14.9倍,在65℃和70℃下孵育30 min的相对酶活分别为19.8%和17.5%,是相同处理条件下天然酶相对酶活的1.8倍和1.7倍。此外,融合酶在4℃储存14 d后相对酶活为88.6%,而天然酶仅具有44.3%的相对酶活。同时,融合酶的k_(cat)/K_(m)提高到天然酶的2.3倍。【结论】融合自组装双亲短肽S1vw提高了重组过氧化氢酶KatA的pH稳定性、温度稳定性、储存稳定性和催化效率,不仅获得了催化效率和应用适应性改良的重组酶蛋白,为针对过氧化氢酶的进一步分子改造提供了策略参考和实验依据,而且促进了其在工业上的规模化制备和应用。

[Objective] In this study, we fused a catalase Kat A from Bacillus subtilis with a self-assembling amphipathic peptide to improve its adaptability in industrial production. [Methods] S1 vw-PT-kat A and kat A were cloned into p HT254 to yield the constructs p HT254-S1 vw-PT-kat A and p HT254-kat A, respectively, which were separately introduced into B. subtilis WB800 N for expression. The recombinant enzymes were then purified and characterized. [Results] The purified enzymes were acquired from the extracellular crude extract of the engineering bacteria through a four-step procedure consisting of ethanol precipitation, DEAE anion exchange chromatography, hydrophobic chromatography, and gel filtration chromatography. The fused enzyme S1 vw-PT-Kat A and natural enzyme Kat A exhibited maximum activity at 30 ℃ and p H 11.0. However, the relative activity of the S1 vw-PT-Kat A incubated at p H 12.0 for 30 min was 77.3%, which was 14.9 times that of Kat A under the same conditions. The relative activities of S1 vw-PT-Kat A incubated at 65 ℃ and 70 ℃ for 30 min were 19.8% and 17.5%, respectively, which were 1.8 and 1.7 times that of Kat A. The relative activity of S1 vw-PT-Kat A stored at 4 ℃ for 14 days was 88.6%, while that was only 44.3% for Kat A. Meanwhile, the k_(cat)/K_(m) value of S1 vw-PT-Kat A was 2.3 times that of Kat A. [Conclusion] Fusing with a self-assembling amphipathic peptide S1 vw can improve the p H stability, thermostability, storage stability, and catalytic efficiency of recombinant Kat A. This finding provides a potential strategy for the modification, large-scale production, and application of catalase.