Modular Assembly of Unique Chimeric Lytic Enzymes on a Protein Scaffold Possessing Anti-Staphylococcal Activity

Abstract

Lytic enzymes have been considered as potential alternatives to antibiotics. These enzymes, particularly those that target Gram-positive bacteria, consist of modular cell wall-binding and catalytic domains, which can be shuffled with those of other lytic enzymes to produce unnatural chimeric enzymes. In this work, we report the in vitro shuffling of two different modular domains using a protein self-assembly methodology. Catalytic domains (CD) and cell wall-binding domains (BD) from the bacteriocin lysostaphin (Lst) and a putative autolysin from Staphylococcus aureus (SA1), respectively, were genetically site-specifically biotinylated and assembled with streptavidin to generate 23 permuted chimeras. The specific assembly of a CD (3 equiv) and a BD (1 equiv) from Lst and SA1, respectively [CDL–BDS (3:1)], on a streptavidin scaffold yielded high lytic activity against S. aureus (at least 5.6 log reduction), which was higher than that obtained with either native Lst or SA1 alone. Moreover, at 37 °C, the initial rate of cell lysis was over 3-fold higher than that with free Lst, thereby revealing the unique catalytic properties of the chimeric proteins. In vitro self-assembly of functional domains from modular lytic enzymes on a protein scaffold likely expands the repertoire of bactericidal enzymes with improved activities.

PhaLP: A Database for the Study of Phage Lytic Proteins and Their Evolution

“…Natural domain recombination driven by horizontal transfer events has played a major role in shaping the existing diversity of phage lytic proteins [ 11 , 42 ]. Analogously, synthetic domain recombination is a proven protein engineering approach to create enzybiotics with increased activity, altered host specificity or other improved properties [ 11 , 42 , 43 , 44 , 45 ]. While plausibly all modular combinations have been tested in nature, those recombination events that resulted in modular combinations with the highest fitness to exert the biological function during phage replication have been retained throughout natural evolution (survival of the fittest).…”

Pickering Emulsion-Based Microreactors for Size-Selective Interfacial Enzymatic Catalysis

“…Therefore, it is of great importance to immobilize enzymes on solid carriers to improve their stabilities and achieve their facile recoveries during industrial applications (Duan et al, 2019). To date, significant efforts have been devoted to immobilize enzymes onto various carriers and composite materials, such as hydrogels (Jo et al, 2019), mesoporous silica (Popat et al, 2011;Li et al, 2019), magnetic nanoparticles (Desai and Pawar, 2020), polymers (Kuiper et al, 2008), and proteins (Kim et al, 2019).…”

Targeted Therapeutic Strategies in the Battle Against Pathogenic Bacteria

“…As enzyme preparations, they are relatively safe to the host and easy to control. Also, through genetic engineering technology, catalytic domains and binding domains of different lysins can be connected to form chimeric lysins with high bactericidal activities ( Kim et al, 2019 ). Considering its rich resources, phage lysin is regarded as an ideal agent with potential and distinction in preventing and controlling bacterial infections.…”