Production of genetically modified lysozymes having extreme heat stability and antimicrobial activity against gram negative bacteria in yeast and in plant.

Hen egg white lysozyme was genetically modified to have extreme heat stability and strong antimicrobial activity against Gram negative bacteria and the modified lysozymes were secreted in yeast and tobacco. Complementary DNA encoding lysozyme was subjected to site-directed mutagenesis to have the Asn-X-Thr(Ser) sequence that is the signal for asparagine-linked glycosylation at the positions 49. The glycosyl lysozyme enhanced heat stability was expressed in the yeast carrying the modified lysozyme cDNA. The expression amount of glycosyl lysozyme was about 10 mg/l of yeast culture medium. Using the same yeast expression system, the lysozyme enhanced antimicrobial action by inserting hydrophobic penta-peptide at the C-terminus were secreted in a small amount (less than 100 micrograms/l in the yeast culture medium). These cDNA constructs of modified lysozymes were engineered into tabacco through Agrobacterium-mediated transformation in order to construct antimicrobial plant. The expression of lysozymes was confirmed by the reverse transcriptional PCR, SDS-PAGE analysis and lytic activity of transformants of tobacco. The transformant having the highest lytic activity expressed about 40 micrograms of lysozyme per g of leaf tissue.