Selective Functionalization of Microgels with Enzymes by Sortagging

Abstract

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.

Endogenous Nitric Oxide-Releasing Microgel Coating Prevents Clot Formation on Oxygenator Fibers Exposed to In Vitro Blood Flow

“…The introduction of epoxy groups to the microgel structure facilitates the successful attachment of microgels to PMP membranes mediated by an anchorpeptide (Scheme 1). Anchor peptides are conjugated via a free and accessible HS-group to the microgels [32,33]. At static conditions, the coating has proven successful in the prevention of protein adsorption, the sustained release of NO and subsequent mitigation of PLT-activation.…”

“…Membranes 2021, 11, x 3 of 17 microgels [32,33]. At static conditions, the coating has proven successful in the prevention of protein adsorption, the sustained release of NO and subsequent mitigation of PLTactivation.…”

Enhancing Robustness of Sortase A by Loop Engineering and Backbone Cyclization

“…Owing to the high degree of site‐specificity and the minor size (≤5 AA residues) of the sorting motif, sortase‐mediated transpeptidation, also known as sortagging, has become a widely used method for protein conjugation, [15] cyclization, [16] labelling/functionalization, [17] purification [18] and/or immobilization [19, 20] . Given to the comparably high transpeptidase activity among the sortases, [21] SaSrtA is most frequently used in sortagging applications [22] .…”

Reactive Precursor Particles as Synthetic Platform for the Generation of Functional Nanoparticles, Nanogels, and Microgels

“…The coupled peptide allows the selective site-specific immobilization of enzymes. [153] Another example for surface functionalization was reported by the group of Gallei, who prepared colloidal core-shell architectures with reactive epoxide functionalities showing low dispersity. [154] The reactive shell of epoxide functionalities was formed using semicontinuous and stepwise emulsion poly merization.…”

“…In a subsequent, step epoxy groups on the surface were used for chemical coupling of a peptide via a thiol–epoxy reaction between the epoxy groups and thiols of the cysteine residue in the peptide sequence. The coupled peptide allows the selective site‐specific immobilization of enzymes …”