Hisham Radwan Ibrahim

Bactericidal action of lysozymes attached with various sizes of hydrophobic peptides to the C‐terminal using genetic modification

The genetic modification of lysozyme was attempted to improve the bactericidal activity against Gram‐negative bacteria E. coli. The different lengths of hydrophobic peptides were attached to the C‐terminus of the hen egg white lysozyme to investigate the most effective length of the hydrophobic peptides for killing bacteria. The oligonucleotides encoding Phe‐Val‐Pro (H3), Phe‐Phe‐Val‐Ala‐Pro (H5) and Phe‐Phe‐Val‐Ala‐Ile‐Ile‐Pro (H7) were fused to the C‐terminus Leu 129 of lysozyme cDNA. The reconstructed cDNAs were inserted into the yeast expression vector. The hydrophobic peptide‐fused lysozymes were secreted in the yeast carrying the reconstructed cDNA. Although the hydrophobic peptide‐fused lysozymes retained 75–80% lytic activity of the wild‐type protein, the bactericidal action to E. coli was greatly increased with the length of hydrophobic peptides. These results suggest that the hydrophobic peptides play an important role in killing Gram‐negative bacteria. To elucidate the role of catalytic domain in bactericidal action of the hydrophobic fusion lysozyme (H5‐Lz), the mutant hydrophobic lysozyme (H5/E35A‐Lz) whose glutamic acid was substituted with alanine at the position 35 was constructed to diminish the catalytic activity. The mutant hydrophobic lysozyme (H5/E35A‐Lz) was greatly lost the bactericidal action to E. coli, suggesting that not only the length of hydrophobic peptide fused to C‐terminus but also the catalytic domain is important for the bactericidal action of the hydrophobic peptide‐fused lysozyme.

Triclosan-lysozyme complex as novel antimicrobial macromolecule: A new potential of lysozyme as phenolic drug-targeting molecule

A novel anti-infection strategy to alleviate antibiotic-resistance problem and non-specific toxicity associated with chemotherapy is explored in this study. It is based on utilizing a bacteriolytic enzyme (lysozyme) as a carrier to allow specific targeting of a potential phenolic antimicrobial drug (triclosan) to microbial cells. Lysozyme (LZ) was complexed, via electrostatic and hydrophobic condensation at alkaline pH, to various degrees with triclosan (TCS), a negatively charged phenolic antimicrobial that inhibits bacterial fatty acid synthesis. Fluorescence and absorbance spectra analysis revealed non-covalent association of TCS with the aromatic residues at the interior of LZ molecule. The conjugation greatly promoted the lytic activity of LZ as the degree of TCS derivatization increased. The complexation with LZ turned TCS into completely soluble in aqueous solution. TCS-LZ complexes showed significantly enhanced bactericidal activity against several strains of Gram-positive and Gram-negative bacteria compared to the activity of TCS or LZ alone when tested at the same molar basis. Strikingly, TCS-LZ complex, but not LZ or TCS alone, exhibited unique specificity to scavenge superoxide radicals, generated by the natural xanthine/xanthine oxidase coupling system, without affecting the catalytic function of oxidase. This finding is the first to describe that the membrane disrupting function of lysozyme can be utilized to specifically target antimicrobial drug(s) to pathogen cells and heralding a fascinating opportunity for the potential candidacy of TCS-LZ as novel antimicrobial strategy for human therapy.

Improvement of foaming property of egg white protein by phosphorylation through dry-heating in the presence of pyrophosphate.

Egg white protein (EWP) was phosphorylated by dry-heating in the presence of pyrophosphate at pH 4 and 85 degrees C for 1 d, and the foaming properties of phosphorylated EWP (PP-EWP) were investigated. The phosphorus content of EWP increased to 0.71% as a result of phosphorylation. To estimate the foaming properties of EWP, the foams were prepared by 2 methods: bubbling of the 0.1% (w/v) protein solution and whipping of the 10% (w/w) protein solution with an electric mixer. The foaming power, which was defined as an initial conductivity of foam from 0.1% (w/v) protein solution, was a little higher in PP-EWP than in native EWP (N-EWP), and the foaming stability of PP-EWP was much higher than that of dry-heated EWP (DH-EWP) and N-EWP. The microscopic observation of foams from the 10% (w/w) solution showed that the foams of PP-EWP were finer and more uniform than those of N- and DH-EWP. Although there were no significant differences in the specific gravity and overrun of the foams between PP- and DH-EWP (P < 0.05), the specific gravity and overrun of the foams from PP-EWP were smaller and higher, respectively, than that of the foams from N-EWP. The drainage volume was smaller in the foams from PP-EWP than in those from N- and DH-EWP. These results demonstrated that phosphorylation of EWP by dry-heating in the presence of pyrophosphate improved the foaming properties, and that it was more effective for the foam stability than for the foam formation.