Development of mutacin MT6223 from Streptococcus sobrinus as an anti-caries agent

开发来自 Sobrinus 链球菌的 mutacin MT6223 作为抗龋齿剂

• 批准号: 06557099
• 负责人: HAMADA Shigeyuki
• 金额: $ 5.95万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06557099/
• 关键词: mutacin; Streptococcus mutans; dental caries; Streptococcus sobrinas; bacteriocin; う蝕

Streptococcus sobrinus MT6223 extracellularly produces mutacin, a bactericidal agent. We have attempted to purify and characterize this bacteriocin.1.Dialyzed TTY (dTTY) broth was better than BHI or Eagle medium for the growth of Streptococcus sobrinus MT6223. Production of mutacin MT6223 was also the highest among the three medium when cultured in dTTY.2.After culture supernatant from dTTY-grown S.sobrinus was precipitated with ammonium sulfate, the resultant precipitant was applied onto several column chromatography. Recovery of mutacin using Sepharose column was more efficient than Bio-Gel or Sephadex.3.Mutacin was purified using Sepharose CL-6B,Develosil ODS-HG-5, and Resource RPC column chromatography. It was found that the bacteriocin is heat and acid-stable, and that mutacin MT6223 is composed of peptide. Since mutacin in 1 M urea solution exerted killing bactericidal activity against S.mutans MT8148, the bacteriocin proved to exhibit the anti-microbial activity as a monomeric form.4.FAB/MS and MALDI-TOF MS analyzes revealed that mutacin possesses a molecular mass of 2,716. Amino acid analysis determined that the N-terminal sequence is Ala-Val-X-and composition of mutacin is rich with Gly and Ala.5.We have isolated a variant of S.sobrinus that can produce mutacin by fourfold higher compared with parent strain MT6223. Purification of mutacin from this high producer is now in progress using the above methods established in this project.

sobrinus链球菌MT6223在细胞外产生诱变素，一种杀菌剂。我们已经尝试提纯这种细菌素并对其进行表征。透析后的TTY培养液对sobrinus Streptococcus MT6223的生长效果优于BHI和Eagle培养基。在dtty培养基中培养时，诱变霉素MT6223的产量也最高。用硫酸铵沉淀dtty培养的sobrinus的培养上清，得到的沉淀剂应用于几柱层析。Sepharose色谱柱比Bio-Gel或sephadex回收率高。采用Sepharose CL-6B、developsil ODS-HG-5和Resource RPC柱层析纯化Mutacin。结果表明，细菌素具有热稳定性和耐酸性，变异素MT6223是由多肽组成。由于诱变素在1 M尿素溶液中对S.mutans MT8148具有杀灭活性，证明该细菌素以单体形式表现出抑菌活性。FAB/MS和MALDI-TOF MS分析显示，mutacin的分子质量为2716。氨基酸分析确定其n端序列为ala - val - x，突变素的组成中含有丰富的Gly和Ala.5。我们已经分离出一种变异的s.s sobrinus，它产生的突变素比亲本菌株MT6223高4倍。利用本项目建立的上述方法，目前正在从这种高产植物中提纯诱变素。

项目成果

T.Ooshima et al.: "Detection of caries-inducing microorganisms in hyposalivated rats without infection of mutans streptococci" Microbiology and Immunology. 38(1). 39-45 (1994)

T.Ooshima 等人：“在没有变形链球菌感染的唾液分泌不足的大鼠中检测龋齿诱导微生物”微生物学和免疫学。

T. Ooshima: "Detection of caries-inducing microorganisms in hyposalivated rats without infection of mutans streptococci" Microbiology and Immunology. 38・1. 39-45 (1994)

T. Ooshima：“在没有变形链球菌感染的唾液分泌不足的大鼠中检测龋齿诱导微生物”微生物学和免疫学38・1（1994）。

S.Hamada et al.: "Peroxidase-catalyzed generation of catechin oligomers that inhibit glucosyltransferases from Streptococcus sobrinus." FEMS Microbiology Letters. 143. 35-40 (1996)

S.Hamada 等人：“过氧化物酶催化生成儿茶素低聚物，可抑制 Sobrinus 链球菌的葡萄糖基转移酶。”

T. Ooshima: "Reduction of dental plaque deposition in human by oolong tea extract" Caries Research. 28. 146-149 (1994)

T. Ooshima：“乌龙茶提取物减少人体牙菌斑沉积”，龋齿研究。

T. Fujiwara: "Deletion and reintroduction of glucosyltransferase genes of streptococcus mutans and role of their gene product in sucrose dependent cellular adherence" Microbial Pathogenesis. 20. 225-233 (1996)

T. Fujiwara：“变形链球菌葡萄糖基转移酶基因的删除和重新引入及其基因产物在蔗糖依赖性细胞粘附中的作用”微生物发病机制。