Improvements of Antimicrobial Activity of Lysozyme by Protein Engineering

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The effect of the length of hydrophobic peptide (H3 ; Phe-Val-Pro, H5 ; Phe-Phe-Val-Ala-Pro, H7 ; Phe-Phe-Val-Ala-Ile-Pro) at the C-terminus of lysozyme on the bactericidial action against Escherichia coli was investgated by using the yeast harboring the expression vector inserted the cDNA of the hydrophobic fusion lysozyme. Although these hydrophobic fusion lysozyms were little secreted in the culture medium of yeast harboring the expression plasmid, they were highly experssed in the medium when the substitution of Gly with Asn at the position 49 was combined to the fusion lysozyme (H3/G49N-Lz, H5/G49N-Lz, H7/G49N-Lz) to insert the signal sequence (Asn-X-Ser/Thr) of N-linked glycosylation. Interestingly, the amount of secretion of glycosylated fusion lysozyme was much smaller than that of G49N-Lz. These results suggest that the glycosylated fusion lysozyme become unstable and then is subjected to proteolysis due to quality control in endoplasmic reticulum, because the folding dose not work smoothly. On the other hand, the folding of nonglycosylated hydrophobic fusion lysozyme (H3,5,7/G49N-Lz) works so well in ER when their secretion increase considerably. The hydrophobic fusion lysozyme retained 75-80% lytic activity of wild type-Lz. The antimicrobial activity increased in propotion to the length of hydrophobic peptide in fusion lysozyme, although H5/G49N-Lz and H7/G49N-Lz showed almost the same antimicrobial activity. These results suggested that when the hydrophobic residues form the beta-sheet conformation, the length of pentapeptide is enough to penetrate into the inner membrane through the outer LPS membrane. The denaturated fusion lysozyme without lytic activity did not show any killing action against E.coli, suggesting the importance of catalytic domein.