Immobilization to Positively Charged Cellulose Nanocrystals Enhances the Antibacterial Activity and Stability of Hen Egg White and T4 Lysozyme

Abstract

Antibacterial bionanostructures were produced from cellulose nanocrystals (CNC) with immobilized lysozyme from hen egg white (HEW) and T4 bacteriophage, respectively. The nanocrystals were prepared from microcrystalline cellulose by ammonium persulfate oxidation with a yield of 68% and having an average size of 250 nm and low polydispersity index. HEW lysozyme (HEWL) and T4 lysozyme (T4L) were immobilized to CNC by different mechanisms including adsorption and covalent coupling to carbodiimide-activated carboxylate groups and to glutaraldehyde-activated aminated CNC (Am-CNC), respectively. The effect of immobilization on the enzymatic activity (both lytic and hydrolytic) and antibacterial activity of the lysozymes was studied using different methods. Am-CNC-lysozyme conjugates retained the highest lytic activity, 86.3% and 78.3% for HEWL and T4L, respectively. They also showed enhanced bactericidal activity with high potency against Gram-positive as well as Gram-negative bacteria in a relatively shorter time as compared to the free enzymes and resulted in extensive cellular damage, as shown by transmission electron microscopy. The enhanced antibacterial activity was correlated with the increase in zeta potential of Am-CNC-lysozyme conjugates. The immobilized lysozyme preparations further exhibited enhanced storage stability at 4 and 22 °C.

Insights on the Structure, Molecular Weight and Activity of an Antibacterial Protein–Polymer Hybrid

“…Our activity tests (Figure ) suggest the PEGylated T4L can be great potential antibacterial materials. Herein PEG protects the protein surface from the environment and T4L performs antibacterial functions . Our structural findings provides indications for optimizing the activity.…”

“…The Micrococcus lysodeikticus cells have been an effective platform to assess the antibacterial function of lysozymes . We, therefore, also employ the same approach to evaluate the activity on a total of 7 hybrids.…”

“…The Micrococcusl ysodeikticus cellsh ave been an effective platform to assess the antibacterial functiono fl ysozymes. [64,65] We, therefore, also employ the same approach [47,48] to evaluate the activity on at otal of 7h ybrids. The general trend follows our expectation:t he more PEG attached per T4L, the higher the activity loss;t he longer the attached PEG, the higher the activity loss.…”

“…Herein PEG protects the protein surface from the environment and T4L performs antibacterialf unctions. [64,65] Our structuralf indingsp rovides indications for optimizing the activity.F irst, PEG is ag ood candidate for preparing hybrids since it does not disturb the protein surface (but rather stays away). It caused only minor structuraldisruption to T4L which is not the primary reasonf or the activity loss.…”

Protein‐Based Nanoparticles for the Delivery of Enzymes with Antibacterial Activity

“…Similarly, Abouhmad et al showed that lysozyme can be covalently linked to the surface of amine‐functionalized cellulose nanocrystals. The positive charge of the nanocrystal is beneficial for the overall antibacterial activity . A drawback of the system is the lack of lysozyme protection, since it is fully exposed on the surface of the particles.…”