Purification and properties of LIQ 4, an antibacterial substance produced by Streptococcus faecalis var. liquefaciens K 4

Summary: An antibacterial substance (LIQ 4) produced by Streptococcus faecalis var. liquefaciens K 4 was isolated in extracellular and cell associated form. It markedly inhibited the growth of Gram-positive bacteria, whereas only a few Gram-negative bacteria were susceptible. LIQ 4 was purified by hydrophobic chromatography (Servachrome XAD-2; octyl-Sepharose CL-4B; Sephadex LH 60) and Sephacryl S-200. TLC yielded a fluorescent spot as the active component and several inactive ninhydrin-positive substances. These contaminating peptides strongly adsorbed to LIQ 4 and could only be removed by repeated reversed phase HPLC. Furthermore, HPLC separated LIQ 4 into seven closely related substances. All showed strong fluorescence under UV light, stained yellow with ninhydrin, and contained aspartate and lysine after acid hydrolysis. The molecular weight was estimated by Amicon ultrafiltration to be less than 2000. LIQ 4 was stable at 80°C (30 min) and pH 2, but considerably inactivated above pH 8. It was apparently affected by proteolytic enzymes, but the activity could be fully restored upon heating.