Preparation and characterization of novel nanocombination of bovine lactoperoxidase with Dye Decolorizing and anti-bacterial activity

Abstract

Interaction between nanoparticles (NPs) and protein is particularly important due to the formation of dynamic nanoparticle-protein complex. The current study indicated that silica NPs were able to induce conformational modification in the adsorbed lactoperoxidase (LPO) which in turns degrades the synthetic dyes. The maximum degradation efficiency was recorded for the LPO modified silica NPs in the presence of H₂O₂ comparing with either free LPO or silica NPs. Degradation efficiency of crystal violet and commassie blue R250 after 6 h was assessed to be 100(%). Also, degradation efficiency of Congo red reached 90.6% and 79.3% in the presence and absence of H₂O₂, respectively, however methyl red degradation efficiency recorded 85%. The viability assay experiment indicated that the IC₅₀ value of the LPO modified silica NPs on human fibroblast cells reached 2.8 mg/ml after 48 h incubation. In addition to dye removal, the LPO modified silica NPs were able to inhibit the antibiotic resistant bacterial strains (Salmonell typhii, Staphylococcus areus, Pseudomonas aureginosa, E. coli, Proteus sp. and streptococcus sp.) at concentrations up to 2.5 mg/ml with inhibition activity about 95%. These findings emphasized that the ability of LPO for degradation of the synthetic dyes after adsorption on silica NPs besides it could be a promising agent with potent inhibitory effect targeting a wide range of multidrug resistant bacteria.

Figure 1

Purification and characterization of bovine LPO. (a) Elution profile of LPO and LF on CM-Sephadex C50 column. (b) 12% SDS-PAGE for LPO during purification steps. Lane 1 is protein marker, lane 2 is LPO eluted from CM Sephadex C50 column and lane 3 is purified LPO eluted from Sephacryl S100 column.

Figure 2

Scanning electron micrograph (a) and FTIR Spectra (b) of the prepared silica NPs (I) and the LPO modified silica NPs (II).

Figure 3

Decolorization efficiency of the LPO modified silica NPs. (a) Photographs of the decolorization samples of Congo red (I), crystal violet (II), methylene blue (III), methyle red (IV) and commassie blue R250 (V) at concentration of 500 mg/L at pH 7.0 as control (1) by the LPO modified silica NPs (2.5 mg/l) in the presence or absence of H₂O₂ (2 and 3) and by silica NPs (2.5 mg/l) in presence or absence of H₂O₂ (4 and 5). (b) Decolorization percentages of Congo red (CR), crystal violet (CV), methylene blue (MB), methyle red (MR) and commassie blue R250 (CB) at concentration of 500 mg/L by the LPO modified silica NPs (2.5 mg/l) in presence of 3.2 mM H₂O₂.

Figure 4

FTIR Spectra of Congo red dye, Methyl red, commassie blue R200, crystal violet and Methylene blue before (I) and after decolorization (II) by the LPO modified silica NPs in the presence of 3.2 mM H₂O₂ after incubation for 24 h.

Figure 5

In vitro cells viability assay against different concentrations of silica NPs and the LPO modified silica NPs using fibroblast cells.

Figure 6

Bactericidal activity of different concentrations the LPO modified silica NPs without H₂O₂ (a) and in the presence of 1.0 mM H₂O₂ (b) against Salmonell typhii, Staphylococcus areus, Pseudomonas aureginosa, E. coli, Proteus sp. and streptococcus sp. after incubation for 24 h at 37 °C.

Figure 7

Effect of the LPO modified silica NPs in the presence and absence of 1.0 mM H₂O₂ on the bacterial biofilm of Salmonell typhii, Staphylococcus areus, Pseudomonas aureginosa, E. coli, Proteus sp. and streptococcus sp. after incubation for 24 h at 37 °C.

Figure 8

Proposed reaction modes of preparation and decolorization efficiency of the LPO modified silica NPs. Silica NPs were coupled with 3-aminopropyl triethoxysilane to produce the modified silica NPs which interact with LPO through formation of covalent (amide) bond to form the LPO modified silica NPs. The LPO modified silica NPs interact with water and/or H₂O₂ in media generating the activated O-species which consequently lead to the direct oxidation of the synthetic dyes. The oxidizing environment leads to complete decolorization and mineralization of the synthetic dyes.