Purification and Characterization of Cell Wall Hydrolase from AlkalophilicBacillus mutanolyticus YU5215

Streptococcus mutans has the capacity of inducingdental caries. Thus, to develop a novel way of preventing dentalcaries, a cell wall hydrolase-producing strain was isolated andits characteristics were investigated. Among 200 alkalophilicstrains isolated from soil, 8 strains exhibited lytic activitiesagainst Streptococcus mutans. However, strain YU5215 withthe highest cell wall hydrolase activity was selected forfurther study. Strain YU5215 was identified as a novel strainof Bacillus based on analyzing its 16S rDNA sequenceand Bergey's Manual of Systematic Bacteriology, and thusdesignated as Bacillus mutanolyticus YU5215. The optimalconditions for the production of the cell wall hydrolase fromBacillus mutanolyticus YU5215 consisted of glucose (0.8%),yeast extract (1.2%), polypeptone (0.5%), K2HPO4 (0.1%),MgSO4·7H2O (0.02%), and Na2CO3 (1.0%) at pH 10.0.Bacillus mutanolyticus YU5215 was cultured at 30oC for72 h to produce the cell wall hydrolase, which was thenpurified by acetone precipitation and CM-agarose columnchromatography. The molecular weight of the lytic enzymewas determined as 22,700 Da by SDS-PAGE. When the cellwall peptidoglycan of Streptococcus mutans was digestedwith the lytic enzyme, no increase in the reducing sugars wasobserved, while the free amino acids increased, indicatingthat the lytic enzyme had an endopeptidase-like property. Theamino terminus of the cell wall peptidoglycan digested by thelytic enzyme was determined as a glutamic acid, while thelytic site of the lytic enzyme in the Streptococcus mutanspeptidoglycan was identified as the peptide linkage of L-Alaand D-Glu. KCI Citation Count: 4