Chemical linkers are frequently used in enzyme immobilization to improve enzyme flexibility and activity， whereas peptide linkers， although ubiquitous in protein engineering， are much less explored in enzyme immobilization. Here， we report peptide-linker-assisted noncovalent immobilization of the bacteriolytic enzyme lysostaphin (Lst) to generate anti- Staphylococcus aureus surfaces. Lst was immobilized through affinity tags onto a silica surface (glass slides) and nickel nitrilotriacetic acid (NiNTA) agarose beads via silica-binding peptides (SiBPs) or a hexahistidine tag (His-tag) fused at the C-terminus of Lst， respectively. By inserting specific peptide linkers upstream of the SiBP or His-tag， t... More
Chemical linkers are frequently used in enzyme immobilization to improve enzyme flexibility and activity， whereas peptide linkers， although ubiquitous in protein engineering， are much less explored in enzyme immobilization. Here， we report peptide-linker-assisted noncovalent immobilization of the bacteriolytic enzyme lysostaphin (Lst) to generate anti- Staphylococcus aureus surfaces. Lst was immobilized through affinity tags onto a silica surface (glass slides) and nickel nitrilotriacetic acid (NiNTA) agarose beads via silica-binding peptides (SiBPs) or a hexahistidine tag (His-tag) fused at the C-terminus of Lst， respectively. By inserting specific peptide linkers upstream of the SiBP or His-tag， the immobilized enzymes killed >99.5% of S. aureus ATCC 6538 cells (10 CFU/mL) within 3 h in buffer and could be reused multiple times without significant loss of activity. In contrast， immobilized Lst without a peptide linker was less active/stable. Molecular modeling of Lst-linker-affinity tag constructs illustrated that the presence of the peptide linkers enhanced the molecular flexibility of the proximal Lst binding domain， which interacts with the bacterial substrate， and such increased flexibility correlated with increased antimicrobial activity. We further show that Lst immobilized onto NiNTA beads retained the ability to kill ∼99% of a 10 CFU/mL microbial challenge even in the presence of 1% of a commercial anionic surfactant， C12-14 alcohol EO 3:1 sodium sulfate， when the Lst construct contained a decapeptide linker containing glycine， serine， and alanine residues. This linker-assisted immobilization strategy could be extended to an unrelated lytic enzyme， the endolysin PlyPH， to target Bacillus anthracis Sterne cells either in buffer or in the presence of anionic surfactants. Our approach， therefore， provides a facile route to the use of antimicrobial enzymes on surfaces.
