Investigation of bacteriolytic enzyme effect to oral streptococci clinical isolation

溶菌酶对口腔链球菌临床分离效果的探讨

• 批准号: 17592140
• 负责人: KOZAI Katsuyuki
• 金额: $ 2.18万
• 依托单位: HIROSHIMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17592140/
• 关键词: Mutans Streptococci; Dental Caries; Bacteriolytic Enzymes; Oral Streptococci; 口腔レンサ球菌(属); Streptococcus mutans; Streptococcus sobrinus; S.mutans; S.sobrlnus

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.

口腔链球菌，特别是变形链球菌和远缘链球菌，是引起龋齿的主要病原体。去年，我们鉴定了Aml，参与变形链球菌的肽聚糖水解酶，并经历了其对变形链球菌和S. sobrinus的特异性裂解活性。本研究建立了重组蛋白Aml的大规模生产和纯化方法。其次，对影响酶解活性的因素进行了研究，找到了酶解活性的最佳条件。最后，我们研究了Aml对变形链球菌和远缘链球菌的裂解活性。1）重组蛋白Aml的大量生产和纯化方法首先，我们对Aml进行了蛋白质序列鉴定，并获得了Aml的DNA信息。其次，我们利用这些信息构建了重组蛋白，并建立了大量生产方法和纯化方法。2）Aml的性质研究了影响Aml裂解活性的因素，发现Ca^<2+>和Na^+对Aml裂解活性有促进作用。此外，triton-X还增强了裂解活性。最后确定了最佳裂解条件并配制了裂解缓冲液。(0.1M 3）Aml对变形链球菌和远缘链球菌临床分离株的裂解活性研究对儿童临床分离的变形链球菌和远缘链球菌进行了分离，得到39株变形链球菌和9株远缘链球菌。然后，我们在相同的条件下研究了Aml对这些分离物的裂解活性：加入10 μ g/ml Aml并孵育约90分钟。结果表明，该菌株对所有菌株均有较强的溶菌活性，但溶菌活性与致龋活性无相关性。

项目成果

Identification and molecular characterization of an N-acetylmuraminidase, Aml, involyed in Streptococcus mutans cell separation

变形链球菌细胞分离中涉及的 N-乙酰胞壁胺酶 Aml 的鉴定和分子表征

• 发表时间: 2006
• 期刊: Microbiology and Immunology 50・9
• 作者: Yoshimura G;Komatsuzawa H;Hayashi I;Fujiwara T;Yamada S;Nakano Y;Tomita Y;Kozai K;Sugai M
• 通讯作者: Sugai M

Identification and molecular characterization of an N-acetylmuraminidase, Aml, involved in Streptococcus mutans cell separation

参与变形链球菌细胞分离的 N-乙酰胞壁胺酶 Aml 的鉴定和分子表征

• 发表时间: 2006
• 期刊: Microbiology and Immunology 50・9
• 作者: Yoshimura G;Komatsuzawa H;Hayashi I;Fujiwara T;Yamada S;Nakano Y;Tomita Y;Kozai K;Sugai M.
• 通讯作者: Sugai M.