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

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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.