4.7.2. Effects of Cadex on Biofilm Formation

Base on the biofilm mass assay, MBIC of *Streptococcus mutans* ATCC 25175 (American Type Culture Collection (ATCC), Manassas, VA, USA) was measured. The effects of Cadex and a homogenetic purity dextranase from *Penicillium* (SA D8144; Sigma) on *S. mutans* biofilm formation were investigated using SEM at MBIC90. *S. mutans* was pre-inoculated in BHI medium without sucrose at 37 ◦C for 15 h. Then, 1 mL of this precultured solution was inoculated into fresh BHI medium with 1% sucrose (20 mL in 100 mL Erlenmeyer-type flask). Sterile glass coverslips were placed in the BHI medium. The media were co-cultured with *S. mutans* and incubated with Cadex at 37 ◦C for 3 h, 9 h, and 18 h. An identical assay with an equal volume of cell-free deionized water served as the blank control. All coverslips were collected for fixation, and were dehydrated and dried according to the procedure described by Tao et al. [52]. The coverslips were sputter-coated with gold (JFC-1600, JEOL, Tokyo, Japan) and viewed by SEM (JSM-6390LA; JEOL).

#### **5. Conclusions**

Cadex from the marine bacterium *Catenovulum* sp. was purified to 29.6-fold homogeneity. It showed a specific activity of 2309 U/mg protein and a molecular weight of 75 kDa. Its optimum pH and temperature were 8.0 and 40 ◦C, respectively. The enzyme was stable at temperatures below 30 ◦C and pH values within 5–11. Some mental ions and chemicals might activate Cadex, but others might inhibit it or leave it unaffected. Cadex was identified as a typical endo-dextranase. The main hydrolysis products were isomaltoogligosaccharides which may be included as a prebiotic supplement to promote the growth and proliferation of intestinal flora. Cadex inhibited biofilm formation by *S. mutans*. Thus, this alkaline- and cold-adapted dextranase from the marine bacterium *Catenovulum* sp. appears to be efficacious under both mesophilic and alkalophilic conditions, thus is a potential candidate for development into a novel marine oral biofilm removal drug.

**Acknowledgments:** This work was supported by the National Natural Science Foundation of China (Grant No: 31471719), the Key Research and Development Program of Jiangsu [Social Development] (Grant No: BE2016702), and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

**Author Contributions:** Wei Ren, Ruanhong Cai, Yaowei Fang, Mingsheng Lyu, and Shujun Wang designed the experiments. Wei Ren, Ruanhong Cai, and Wanli Yan performed the experiments. Wei Ren, Wanli Yan, Ruanhong Cai, Yaowei Fang, and Mingsheng Lyu analyzed the data. Shujun Wang supervised the study and reviewed the manuscript. Wei Ren and Ruanhong Cai wrote the manuscript. All authors have read and approved the final manuscript.

**Conflicts of Interest:** The authors have no conflicts of interest to declare.
