Biochemical and Molecular Characterization of a Novel Type of Mutanase from Paenibacillus sp. RM1: Identification of Its Mutan-binding Domain Essential for Degradation of Biofilms of Streptococcus mutans

Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan, Oral Care Research Laboratories, Lion Corporation, 3-7, Honjyo 1-chome, Sumida-ku, Tokyo, 130-8644, Japan, Biological Science Research Laboratories, Lion Corporation, 100 Tajima, Odawara, Kanagawa, 256-0811, Japan, C3-Jian, Los Angeles, CA, USA

^* To whom correspondence should be addressed. Email: isaosimo{at}lion.co.jp.

	Abstract

A novel type of mutanase (termed mutanase RM1) was isolated from Paenibacillus sp. RM1. The purified enzyme specifically hydrolyzed -1,3-glucan (mutan) and effectively degraded biofilms formed by Streptococcus mutans, a major etiologic agent in the progression of dental caries, even following short-time incubation. The nucleotide sequence of the gene for this protein contains a 3,873-bp open reading frame encoding 1,291 amino acids with a calculated molecular mass of 132 kDa. The protein contains two major domains, N-terminal (277 residues) and C-terminal (937 residues), separated by a characteristic sequence composed of proline and threonine repeats. The characterization of the recombinant proteins for each domain which were expressed in Escherichia coli, demonstrated that the N-terminal domain had strong mutan-binding activity but no mutanase activity, whereas the C-terminal domain was responsible for mutanase activity but its mutan-binding activity was significantly lower than the intact protein. Importantly, the biofilm-degrading activity observed with the intact protein was not exhibited by either domain alone or in combination with each other. Therefore, these results indicate that the structural integrity of mutanase RM1 containing the N-terminal mutan-binding domain is required for the biofilm-degrading activity.