LYTIC RESPONSE OF LACTOCOCCUS-LACTIS SUBSP LACTIS-484 TO MURALYTIC ENZYMES

Conditions for production and regeneration of Lactococcus lactis subsp lactis 484 protoplasts are described. Protoplasts of the test culture were obtained by treatment with mutanolysin or lysozyme in Tris-HCl-MgCl2-sucrose (THMS) buffer (pH 8.0). Efficient cell wall digestion was observed in the cells obtained from 4-h-old cultures. Addition of mutanolysin resulted in complete cell wall digestion, generating a much higher yield of true protoplasts compared to that after lysozyme treatment as evidenced from N-acetyl hexosamine release and scanning electron microscopic studies. Regeneration of the protoplasts was accomplished with a hypertonic medium that contained sucrose (20%), gelatin (2.5%), and bovine serum albumin (0.5%). Protoplast production and their subsequent regeneration were achieved at the rate of 99% and 10-15%, respectively, on using lysozyme (200-mu-g ml-1 for 60 min or 300-mu-g ml-1 for 30 min) or mutanolysin (50-mu-g ml-1 for 20 min or 100-mu-g ml-1 for 10 min).