In this work we explored an enzyme-mediated method for selective and efficient decoration of aqueous microgels with biomolecules. Poly(N-vinylcaprolactam) (VCL) microgels with varied amounts of glycidyl methacrylate (GMA) as comonomer incorporated in the microgel shell were synthesized and characterized in regard to their size, swelling degree, and temperature-responsiveness in aqueous solutions. The surface of the PVCL/GMA microgel containing 5 mol % glycidyl methyacrylate was modified by grafting of a specific recognition peptide sequence (LPETG) for Sortase A from Staphylococcus aureus (Sa-SrtA). Sortase-mediated conjugation of the enhanced Green Fluorescent Protein (eGFP) carrying a N-terminal triglycine tag to LPETG-modified microgels was successfully performed. Conjugation of eGFP to the microgel surface was qualitatively proven by confocal microscopy and by fluorescence intensity measurements. The developed protocol enables a precise control of the amount of eGFP grafted to the microgel surface as evidenced by the linear increase of fluorescence intensity of modified microgel samples. The kinetic of the sortase-mediated coupling reaction was determined by time-dependent fluorescence intensity measurements. In summary, sortase-mediated coupling reactions are a simple and powerful technique for targeted surface functionalization of stimuli-responsive microgels with biomolecules.

Poly(N-vinylcaprolactam-co-2-methoxyethyl acrylate) core–shell microgels as imaging/diagnostic system.

Enzyme immobilization has been widely used to improve the stability and recyclability of enzymes in industrial processes. In this work, a sortase-mediated and therefore selective covalent immobilization strategy (sortagging) for enzymes on microgels (GelZyms) was investigated. Aqueous microgels were synthesized from poly(N-vinylcaprolactam)/glycidyl methacrylate (PVCL/GMA) and tagged with the sortase A recognition peptide sequence (LPETG) or its nucleophilic counterpart-tag (GGG). General applicability and selective immobilization were confirmed by subsequent sortagging of five different enzymes (Bacillus subtilis lipase A (BSLA), Yersinia mollaretii phytase (Ym-phytase), Escherichia coli copper efflux oxidase (CueO laccase), cellulase A2, and Bacillus megaterium monooxygenase P450 BM3). The latter was performed directly from the cell lysate to ensure cost-effective immobilization. All five immobilized enzymes were catalytically active and could be recycled (e.g., laccase CueO and monooxygenase P450 BM3 F87A; >55% residual activity after six cycles). Application potential was demonstrated by using CueO decorated microgels for bleaching of the synthetic dye indigo carmine.