A microorganism producing mutanase was isolated from soil on RL, agar containing blue mutan as sole carbon source and identified as Microbispora rosea on the basis of the cultural, morphological and physiological observations. Mutanase production was enhanced when Microbispora rosea was cultured in the media containing 0.01% glucose, 10mM KCl, 0.1mM MgCl_2, 16mM CaCl_2 and 0.2% mutan.
The enzyme was purified by (NH_4)_2SO_4 precipitation, anion exchange column chromatography on Mono Q following DEAE cellulose column chromatography and gel filtration on superose 12 column. The molecular weight of the purified enzyme was 48,000Da by SDS-PAGE and the amino-terminal sequences were Ser-Pro-Gly-Asn-Ser-Pro-Gly-Ala. The optimum pH and temperature for enzyme activity were 5.5 and 40℃, respectively. The enzyme was stable up to 50℃ on heat treatment at pH 5.5 for 30 min. The enzyme activity was inhibited by the presence of Zn^++, Fe^++ at 10mM. Tween 20 increased enzyme activity in contrast with the effect of SDS and Triton X-100.
In vitro, the purified mutanase had strong inhibitory effect on the formation of artificial plaque while the reduction of preformed glucan was weak. The mutanase from Microbispora rosea had an effect on the formation of glucan as well as reduction of glucan produced by other Streptococcus mutans.
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