Peptide-Inspired One-Step Synthesis of Surface-Functionalized Fe₃O₄ Magnetic Nanoparticles for Oriented Enzyme Immobilization and Biocatalytic Applications

Abstract

Immobilization of enzymes on magnetic nanoparticles (MNPs) is an effective way to ensure their recycling. However, MNPs are usually prepared and modified step by step before immobilization as the conditions are too harsh to maintain the three-dimensional structures and functions of the target enzymes. Herein, peptide-inspired magnetic nanocomposites (K5C@Fe3O4) were mildly prepared, where the proteins in the composites could functionalize efficiently for immobilization of the target dual enzymes without prior purification. The one-pot preparation of the K5C@Fe3O4 was achieved by the SpyCatcher-fused elastin-like polypeptides (ELPs-SpyCatcher, K5C) for biomimetic mineralization of the precursors at 30 °C without vacuum. Then, K5C@Fe3O4 were adopted to immobilize and purify the SpyTag-tagged target dual enzymes (xylanase–SpyTag–lichenase, X–T–L) directly from the crude cell lysate. Characterizations of the composites and activity assay of the dual enzymes confirmed that the X–T–L was successfully immobilized by a spontaneous reaction between SpyCatcher and SpyTag, with high immobilization yield (>90%), immobilization efficiency (>70%), and activity recovery (>70%). Besides, the immobilized dual enzymes showed excellent catalytic efficiency and reusability (>65%, 10 times). The peptide-inspired MNPs represent a bioactive hybrid material for biocatalytic applications and will shed light on covalently oriented enzyme immobilization. They will have exciting potentials in functional materials.

Remote Activation of Enzyme Nanohybrids for Cancer Prodrug Therapy Controlled by Magnetic Heating

“…Moreover, our proposed encapsulating strategy may avoid the problems that arise from unfavored orientations of enzymes bound directly to MNPs. This last approach has faced challenges in terms of substrate accessibility and the extent of enzyme remote activation. − Consequently, an encapsulation method may provide versatility and even expand the range of therapeutic enzymes that can be remotely activated.…”

Site-Specific and Tunable Co-immobilization of Proteins onto Magnetic Nanoparticles via Spy Chemistry

“…Likewise, the RFP-SpyCatcher loadings on MNPs were 14.9 ± 0.3 μg-protein/mg-MNP and 16.2 ± 0.1 μg-protein/mg-MNP, respectively. In comparison, it was reported that SpyCatcherfunctionalized MNPs had a loading of 0.7 μg-enzyme/mg-MNP in a previous study that used Spy chemistry for enzyme co-immobilization from cell lysates, 34 suggesting the high loading capacity of our platform for protein co-immobilization from cell lysates. Notably, each protein loading on MNPs accounted for 75−90% of those achieved from co-immobilization using pure proteins, suggesting the high efficiency of the MNP platform to co-immobilize two different proteins from cell lysates in a one-pot system.…”

“…31,33 More recently, immobilization of dual SpyTagfused enzymes on SpyCatcher-functionalized MNPs from cell lysates has been reported. 34 However, the enzyme loading was low (∼0.7 mg/g) and the ratio of each enzyme loading on MNPs could not be tuned because two enzymes were fused to the N and C termini of one SpyTag. Also, the materials reported in the study were prone to aggregation, which limits their colloidal stability in high ionic strength solutions.…”

“…Spy chemistry is a highly specific reaction between SpyTag peptide and the SpyCatcher domain and has been exploited for site-specific protein co-immobilization on the polymeric SpyCatcher scaffold, silica nanoparticles, and polyhydroxyalkanoate beads, circumventing the poor spatial arrangement of immobilized proteins. Spy reaction at 1:1 molar stoichiometry also enables a tunable protein co-immobilization on the supporting surface by varying SpyTag:SpyCatcher molar ratios. , More recently, immobilization of dual SpyTag-fused enzymes on SpyCatcher-functionalized MNPs from cell lysates has been reported . However, the enzyme loading was low (∼0.7 mg/g) and the ratio of each enzyme loading on MNPs could not be tuned because two enzymes were fused to the N and C termini of one SpyTag.…”

Enhanced Stability of β-Agarase Immobilized on Streptavidin-Coated Fe3O4 Nanoparticles: Effect of Biotin Linker Length

“…50 Feng and co-workers 51 used 3-(aminopropyl)triethoxysilanecoated MNPs to immobilize aminoacylase, and the immobilized enzymes exhibited more favorable thermal and pH stability than the free enzymes. Zhou et al 52 immobilized dual enzymes onto peptide-inspired MNPs, enhancing their thermal stability.…”