초록
Streptococcus mutans has been implicated as primary causative agents of dental caries by insoluble glucan (IG) in human and experimental animals. An attempt was made to search for the ${\alpha}$-1,3 glucanase that degrades IG produced by S. mutans. ${\alpha}$-1,3 glucanase was detected in the culture supernatant of microorganisms, which are isolated from soils on agar medium containing IG as a sole carbon source. This Streptomyces sp. hydrolysed IG produced by immobilized S. mutans and was named as Y9373. This enzyme required ${\alpha}$-1,3 glucan (IG) as an inducer. The optimum conditions for enzyme production were studied. The enzyme was purified by 30~70% $(NH_4)_2SO_4$ precipitation, anion exchange chroma tography on DEAE-cellulose and gel filtration on Sepadex G-75. The purified enzyme has a specific activity of 7840.0 U/mg protein giving 32.1-fold purification and final yield of 0.53%. The molecular weight was estimated to be about 22.5 kDa by SDS-PAGE. The optimum pH and temperature for enzyme reaction were 6.5 and 37$^{\circ}C$, respectively and the enzyme was relatively stable at the temperature below 60$^{\circ}C$. The activity of purified enzyme was enhanced by adding $Co^{2+},\;Mn^{2+}\;and\;Mg^{2+}$ into the medium, whereas inhibited by adding $Hg^{2+},\;Zn^{2+}$ and SDS. The $K_m\;and\;V_{max}$ value of ${\alpha}$-1,3 glucanase for IG were estimated to be 2.50 mM and 0.0431 mM/min, respectively. The thin layer chromatographic analysis of hydrolysates from IG with ${\alpha}$-1,3 glucanase showed that glucose was the main product of reaction. This enzyme activity was about 14 times higher than marketing dextranase as preventive agent against artificial dental caries by S. mutans in TH medium including 5% sucrose after 30 minutes.