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
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
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DOI：
10.1128/aem.02332-07
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发表时间：
2008-05-01
影响因子：
4.4
通讯作者：
Nakashima, Syozi
中科院分区：
文献类型：
--
作者：
Shirnotsutira, Isao;Kigawa, Hiromitsu;Nakashima, Syozi
从类芽孢杆菌属（Paenibacillus sp.）菌株RM 1中分离到一种新型的突变酶（称为突变酶RM 1）。纯化的酶特异性水解α-1，3-葡聚糖（变聚糖），并有效地降解由变形链球菌形成的生物膜，变形链球菌是龋齿发展的主要病原体，即使在短暂的孵育后。该蛋白质的基因的核苷酸序列含有3，873-bp的开放阅读框，编码1，291个氨基酸，计算分子量为135 kDa。该蛋白包含两个主要结构域，N-末端结构域（277个残基）和C-末端结构域（937个残基），由脯氨酸和苏氨酸重复组成的特征序列分开。在大肠杆菌中表达的每个结构域的重组蛋白的表征表明，N-末端结构域具有强的突变体结合活性，但没有突变酶活性，而C-末端结构域负责突变酶活性，但具有突变体结合活性显着低于完整蛋白。重要的是，在完整蛋白质中观察到的生物膜降解活性并不表现为单独的结构域或与另一个结构域组合。因此，这些结果表明，含有N-末端突变体结合结构域的突变酶RM 1的结构完整性是生物膜降解活性所必需的。
A novel type of mutanase (termed mutanase RM1) was isolated from Paenibacillus sp. strain RM1. The purified enzyme specifically hydrolyzed alpha-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.