Investigation of bacteriolytic enzyme effect to oral streptococci clinical isolation

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Oral Streptococci, especially S.mutans and S.sobrinus, are major pathogens causing dental caries. Last year, we identified Aml, the peptidoglycan hydrolase involved in S.mutans, and experienced its specific lytic activity against S.mutans and S.sobrinus. This activity showed some possibility of suppressing material against mutans streptococci.This year, we established mass-producing and refining method of recombinant protein Aml. Next, we searched for the influencing factors and found the best condition of lytic activity. And last, we investigated Aml lytic activity against S.mutans and S.sobrinus clinical isolates.1) Mass-producing and refining method of recombinant protein AmlAt first, we identified the protein sequence and got the DNA information about Aml. Next, we made up the recombinant protein from these informations, and we established mass-producing method and refining method. At last, we made 1mg Aml recombinant protein from the llitter cell culture.2) Characterization of AmlWe investigated the influencing factor of Aml lytic activity, and we found Ca^<2+> and Na^+ enhanced the activity. Moreover, triton-X also enhanced the lytic activity. Finally, we identified the best condition of lytic activity and made up the lysis buffer. (0.1M phosphate buffer, 0.1M NaCl, 0.1mM CaCl_2, 0.1% triton-X)3) Investigation of Aml lytic activity against S.mutans and S.sobrinus clinical isolatesWe separated the clinical isolation of S.mutans and S.sobrinus from children, and got the 39 S.mutans stocks and 9 S.sobrinus stocks. Next, we investigated the Aml lytic activity to these isolates in the same condition: added 10ug/ml Aml and incubated it for about 90 minutes. As a result, we found the strong lytic activity to all isolates, but there was no correlation between lytic activity and caries activity.