Until recently research on the genetics, metabolism and membrane structure of Streptococcus mutans has been hampered by the resistance of this bacterium to enzymatic hydrolysis (3). The difficulty, however, was partially overcome by the development of several techniques involving addition of high concentrations of salt (4), high pH (14), or detergent to cause lysis of lysozyme-treated cells (2). On the other hand, the search for enzymes capable of hydrolyzing cell walls or intact cells of S. mutans indicated that the L-ll enzyme of a Flavobacterium species (9), the M-l enzyme of Streptomyces globisporus (16) and the H-402 enzyme from Streptomyces griseus (17) could cause lysis. Only a few of these enzymes enabled a few investigators to succeed in the formation of protop lasts or osmotically fragile bodies from S. mutans (13, 15) opening opportunities for many types of studies. The present paper describes a new enzyme preparation, achromopeptidase (TBL-l, Wako Pure Chemical Industries, Osaka, Japan) capable of hydrolyzing intact cells as well as cell walls of S. mutans, its serotype-dependent sensitivity to the enzyme and successful formation of osmotically fragile bodies from this bacterium.Strains of Streptococcus mutans used were HSI (serotype a), FAI (serotype b), Ingbritt (serotype c), OMZ 176 (serotype d), LM 7 (serotype e), SE 17 (serotype f), and OMZ 65 (serotype g). All strains were gifts from Dr. Shigeyuki Hamada (National Institute of Health of Japan, Tokyo). Each strain of these bacteria was cultivated in brain heart infusion broth (BHI, Difco) at 37 C for 15 hr. Bacterial cells were harvested and washed three times by centrifugation in a 0.01 M Tris-HCl buffer solution (pH 8.0) containing 0.01 M NaCl. The cells were resuspended in the buffer solution to give an optical density (O.D.) reading of about 0.5 at 660 nm in a Bausch and Lomb spectrophotometer (Shimadzu Seisakusho, Ltd., Kyoto, Japan). The reaction was started by incubating the cell suspension at 37 C in a total volume of 5.0 ml with achromopeptidase at a concentration of 100 to 1,600 ftg per ml. At intervals, a decrease in turbidity was followed by measurement of O.D. at the same wavelength. To obtain purified cell walls of each serotype, cells were treated for 12 min in a glass bottle with glass beads (0.1 mm in diameter) in a B. Braun disintegrator (Melsungen, West Germany). The cell wall fraction was purified by differential centrifugation of the treated cells in a solution of 1.0 M NaCl containing 1.0% Triton X-lOO. The fraction was further subjected to repeated 957
