Bio-catalysis by enzymes on solid surfaces has been implemented in several practical applications. However, the current methods for efficient enzyme immobilization with retained activity need further development. Herein, a simple, rapid, and economical, bio-affinity-based approach was developed for the direct immobilization with high activity recovery of the Bacillus subtilis catalase (CAT), recombinantly expressed in Escherichia coli. Silaffin-3-derived pentalysine cluster (Sil3K) from Thalassiosira pseudonana and its mutant variant (penta-arginine peptide; Sil3R) were used for the first time in the non-covalent immobilization of the recombinant enzyme on silica particles. The fusion proteins CAT-Sil3K and CAT-Sil3R were selectively loaded from the cell lysates onto the silica surface. Unexpectedly, the Lys-based tag (Sil3K) was the superior to Arg-based tag (Sil3R) or tag-less system for the high recovery of CAT activity upon immobilization; an 8.4-fold and 1.5-fold increase in the catalytic activity was observed for CAT-Sil3K compared with the tag-less CAT and CAT-Sil3R, respectively. Furthermore, the CAT-Sil3K immobilized on silica particles exhibited improved thermal, pH and storage stabilities, and retained 72% of the initial activity after five reaction cycles. Moreover, CAT-Sil3K was released with approximately 85% recovery and 91% purity, in a biologically active form when free lysine solution was used as the eluent. Our data proved that Sil3K-tag, 12-mer peptide, can be a highly promising silica-affinity tag for effective enzyme immobilization with preserved activity. Additionally, the novel findings obtained here may open a new route not only for cost-effective enzyme immobilization approaches but also for high recovery of enzyme activity.
Keywords: Affinity tag; Catalase immobilization; Silaffin-3.
Copyright © 2020 Elsevier B.V. All rights reserved.