Abstract:

The use of carbonic anhydrase (CAs) to capture CO₂ is more in line with the concept of sustainable development, but it is urgent to reduce the cost of its separation and purification and enhance its survivability in complex environments. Herein, the ferritin with the capability of self-assembling and forming multimers was used as the tag, and the carbonic anhydrase (SazCA) from Sulfurihydrogenibium azorense was linked to the ferritin by a rigid linker. The intracellular difficultly soluble active aggregates were formed during gene expression, which facilitate the purification of SazCA and the activity recovery was as high as 84.8%. The recombinant CAs were obtained after 30 min of sonication of the active aggregates, which was incubated at 50℃ for 50 days without detectable activity lost, and the half-life in pH=9.0 buffer were 150 days. The difficultly soluble active aggregates can spontaneously re-solubilize and formed nano-scale CAs from the micron-scale CAs. The activity of the nano-scale CA was 10-fold higher than that of the micron-scale one. Encouragingly, they showed significantly improved thermal stability compared with wild-type CAs, with a half-life of (211±22) h at 80℃. More importantly, they had a half-life of up to (40.8±2.2) h in the ionic liquid [N1111][Gly] (pH=11.64) with the concentration of 15% (mass), which may have great potentials in the combined uptake and regeneration of CO₂ by subsequent ionic liquids and CAs. Finally, the molecular mechanism of difficultly soluble reactive aggregate formation was explored and the electrostatic interaction was found to be one of the important factors. The results demonstrated that ferritin was a novel tag that both greatly simplifies the preparation process and substantially improves its stability, laying a solid foundation for industrial CO₂ capture by CAs.

Key words: CO₂ capture, ferritin, carbonic anhydrase, oligomerization, biocatalysis, enzyme stability, ionic liquid

摘要：

利用碳酸酐酶（CAs）捕集CO₂更符合可持续发展的理念，但亟需降低其分离纯化的成本和增强在复杂环境的生存能力。以铁蛋白（Ferritin）为标签，经linker把CAs与之相连，在胞内表达形成微米级难溶活性聚集体，经低速离心实现酶高效分离，酶活回收率达84.8%，活性聚集体超声30 min后，50℃孵育50 d酶活基本不变，在pH=9.0的缓冲液中半衰期为150 d。难溶活性CAs聚集体可转变为可溶性纳米CAs，其活力提升10倍以上，80℃时半衰期为（211±22）h。在15%（质量）离子液体［N1111］［Gly］（pH=11.64）中半衰期达（40.8±2.2）h，可用于后续离子液体和重组CAs联合吸收和再生CO₂。静电作用是难溶活性CAs聚集体形成的重要原因之一。研究结果表明，CAs融合Ferritin后，既能极大简化制备过程，又能大幅提高稳定性，为酶法捕集CO₂奠定了基础。

关键词: 二氧化碳捕集, 铁蛋白, 碳酸酐酶, 寡聚化, 生物催化, 酶稳定性, 离子液体