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

Abstract

A novel type of mutanase (termed mutanase RM1) was isolated from Paenibacillus sp. strain 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 brief 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 135 kDa. The protein contains two major domains, the N-terminal domain (277 residues) and the C-terminal domain (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 had mutan-binding activity significantly lower than that of the intact protein. Importantly, the biofilm-degrading activity observed with the intact protein was not exhibited by either domain alone or in combination with the 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.

Domain Structure and Function of α-1,3-Glucanase fromBacillus circulansKA-304, an Enzyme Essential for Degrading Basidiomycete Cell Walls

“…15) A biochemical study of mutanase RM indicated that the N-terminal region, corresponding to DS1 and CB6, exhibited -1,3-glucan binding activity and enhanced degrading activity as to biofilms from Streptococcus mutans, 16) but no functions of individual domains, DS1 and CB6, have been investigated in it. In this study, we determined in detail the role of each N-terminal domain, containing DS1, CB6, DS2, and UCD, of Alg-KA by characterizing the domain deletion enzymes, and confirmed their relevance to cell-wall lysis.…”

“…Deleting DS1 caused a decrease in binding activity that was nearly lost on deletion of DS1 and CB6, suggesting that at least DS1 and CB6 are necessary for the strong adsorption of this enzyme on -1,3-glucan. Shimotsuura et al 16) reported that the binding activity of mutanase RM1 from Paenibacillus sp. RM1 remarkably decreased after deletion of the N-terminal region containing DS1 and CB6, which have high amino acid sequence similarities to DS1 and CB6 of Agl-KA.…”

“…Shimotsuura et al 16) have reported that mutanase RM1 contains two major functional regions, an Nterminal region (277 residues) and a C-terminal region (937 residues), separated by TP. They also reported that the N-terminal region of mutanase RM1 exhibited -1,3-glucan-binding activity and that this region was indispensable in degrading the biofilms formed by Streptoy To whom correspondence should be addressed.…”

Cloning and Expression of the Mutanase Gene of Paenibacillus humicus from Fermented Food

“…strain RM1 and Bacillus circulans strain KA-304, were isolated previously. 7,8) Other mutanases were characterized, and some examined for dental caries preventive efficacy. 9) Those samples, however, were obtained from the soil.…”

“…The forward primer 5 -GCGTTCCCGCAATGGATTCAAG-3 and reverse primer 5 -GGAATTGTCACCGTATTGCC-3 were designed based on the conserved region of mutanase genes. 4,6,7) The compositions of PCR were the same as described above, except for the primers. Thermocycling consisted of one cycle at 94 • C for 10 min, followed by 30 cycles at 94 • C for 30 sec, 50 • C for 30 sec, and 72 • C for 1 min.…”