A pH-Responsive and recyclable homogeneous carbonic anhydrase with 24-polymeric fusion protein: Simple preparation...

Enzymes are efficient green biocatalysts. However, the complex preparation process, poor stability and non-reusability have hindered their applications. Although the chemically/genetically modification could enhance recyclability, there existed limited strategies to solve all the above problems in one step currently. A novel strategy, namely, the peptide based recyclable homogeneous biocatalyst, was proposed with the ancestral carbonic anhydrase (AncCA19) as an example. Ferritin was fused to the C-terminal of AncCA19 (AncCA19F) to obtain the enzyme-ferritin fusion proteins (EFFPs). They could self-assemble into micron-sized AncCA19F (msAncCA19F) in host cells and achieve low-speed centrifugal separation/purification, which streamlined the purification procedures and improved the yield. Interestingly, the msAncCA19F could spontaneously dissolve into nano-sized AncCA19F (nsAncCA19F) in vitro as pH increased, which functioned as homogeneous biocatalysts with improved performances - the experimental half-life (Et₁₂) increased by 9-fold in artificial seawater and by 2-fold in 25% N-methyldiethanolamine (MDEA). The nsAncCA19F could spontaneously aggregate into msAncCA19F as pH decreased, which functioned as recyclable immobilized biocatalysts: with negligible activity loss (<10%) after 10 cycles. The strategy not only realizes the time-effective and scalable preparation and simple recycling of enzymes, but also improves their stability. The mechanism of AncCA19F with improved performances and their responsive to the variation of pH were also addressed.