*2.3. Effects of Cadex on Biofilm*

Table 6 shows the comparison of biofilm inhibitory rates between *Penicillium* dextranase and Cadex under various concentrations. It can be clearly seen that both *Penicillium* dextranase and Cadex impeded the biofilm formation. The minimum biofilm inhibitory concentration for 90% inhibition (MBIC90) was calculated when 40 U/mL *Penicillium* dextranase and 30 U/mL Cadex were added to the media after which the effects of the dextranases on *S. mutans* biofilm formation were analyzed. Cadex was more efficient than *Penicillium* dextranase in inhibiting *S. mutans* biofilm formation. Figure 5 shows the bacterial morphology and biofilm, as observed by scanning electron microscopy (SEM.). In the blank control group, *S. mutans* grew well and the biofilm developed smoothly with prolonged time. At 18 h, a thick biofilm with dense cells were seen, with no obvious structural breakdown. In contrast, biofilm did not form easily when *S. mutans* was cultured in brain heart infusion (BHI) medium by adding the MBIC90 of Cadex or *Penicillium* dextranase. Dextranase impeded biofilm formation and reduced the number of *S. mutans* cells that adhered to the glass coverslips. Cadex had inhibitory effects on *S. mutans* biofilm formation. A previous report proposed that crude *Catenovulum* dextranase can prevent *S. mutans* from forming biofilms, however, it used crude dextranase and was a preliminary assessment [20].

**Table 6.** Biofilm inhibitory rates with different concentrations of dextranase.


<sup>a</sup> The biofilm inhibitory rate was calculated at an absorbance of 595 (A595) of the crystal violet stained biofilm without dextranase subtracted from A595 of biofilm with dextranase, and divided by A595 of biofilm without dextranase multiplied by 100%.

**Figure 5.** *Cont*.

**Group a-18 h Group b-18 h Group c-18 h** 

**Figure 5.** Electron microscopy of *S. mutans* biofilm formed on glass coverslips in the presence and absence of dextranase at different periods: (**Group a**) blank control, note the equal volume of cell-free pure water was added to replace dextranase; (**Group b**) biofilm subjected to 40 U/mL *Penicillium dextranase*; and (**Group c**) biofilm subjected to 35 U/mL Cadex.

#### **3. Discussion**

A psychrotolerant dextranase-producing bacterium named *Catenovulum* sp. DP03 was previously studied [20]. However, to the best of our knowledge, this is the first report of the purification and characterization of dextranase from *Catenovulum.* Purification of crude dextranase by ammonium sulfate fractionation and Sepharose 6B chromatography, which resulted in a 6.69-fold increase in specific activity and an 11.27% recovery, was previously reported [23]. This system of the aforementioned procedure may be used to produce homogenetic dextranase. The process can easily be scaled up and is cost-effective. The molecular weight of Cadex was about 75 kDa, which resembled that of dextranase from *Sporotrix schencki* (79 kDa) [27]. Bacteria producing dextranases generally have molecular weights ranging from 60 to 114 kDa [28–30]. The smallest dextranase (23 kDa) is from *Lipomyces starkeyi* [24], and the largest (175 kDa) is from *Streptococcus sobrinus* [31].

Endo-type Cadex showed high specificity towards dextrans containing α-1,6 glucosidic bonds. Moreover, the main hydrolysis products of Cadex were isomaltooligomers [30,32,33]. Dextranase from *Chaetomium* [34], *Aspergillus* [35], *Penicillium* [36], and *Fusarium* [37] synthesize comparatively low amounts of glucose and higher amounts of isomaltooligosaccharides. Isomaltooligosaccharides can promote the growth and proliferation of *Bifidobacteria* and *Lactobacillus* [1,38]. Numerous isomaltooligosaccharides are prebiotics, which are produced endodextranases and have garnered much commercial interest [33].

The optimum pH for Cadex activity tends to be alkaline, and recently reported alkalophilic cases were *Streptomyces* sp. NK458 and *Bacillus subtilis* NRC-B233b, which had maximum activities at pH 9.0 and pH 9.2 [7,39]. Evidence is accumulating that alkali generation plays a major role in pH homeostasis which may modulate the initiation and progression of dental caries [40]. Therefore, alkalophilic Cadex may be suitable for the development of novel marine agents for the treatment of this condition [16]. Cadex had catalytic efficiency at 0 ◦C, similar characteristics to other cold-adapted enzymes: for example, a cold-adapted ι-carrageenase showed 36.5% relative activity at 10 ◦C [41] and a cold-adapted β-glucosidase retained more than 60% of its activity at temperatures ranging from 15 ◦C to 35 ◦C [42]. Cold-adapted enzymes have optimal catalyst temperatures near 30 ◦C and remain efficient at 0 ◦C. Cadex can be classified as a cold-adapted enzyme according to the system developed by Margesin and Schinner [43]. The excellent pH stability of Cadex distinguishes it from other dextranases, which are generally unstable across a broad pH range [1,7,22,27]. It would be easier to hydrolyze dextran than dextranases in acid/alkaline catalysis conditions. We speculate that Cadex may be suitable for widespread use. We have classified Cadex as a cold-adapted dextranase, which may explain its lower thermo-stability than terrestrial dextranases [1]. Nevertheless, in our early studies, crude Cadex showed greater thermostability than purified dextranase, as it was stable at 45 ◦C, and its half-life was 10 h (data not shown).

The present study proposes that purified *Catenovulum* dextranase, namely Cadex, is an alkalophilic and cold-adapted dextranase that is considered to be a novel marine dextranase of dealing with biofilms. The failure of biofilm formation is attributable to the failure of extracellular polysaccharides to form efficiently, possibly due to cleavage of the α-1,6 glucosidic linkages in the biofilm that occurs in the presence of Cadex. The oral *Streptococcus* biofilm is formed by α-(1,3)-glucan and α-(1,6)-glucan, in which the α-1,6 glucosidic linkages are degradable by dextranase while the α-1,3 glucosidic linkages can be cleaved by mutanase [44]. *Penicillium* dextranase is often used as the standard dextranase in studies of this enzyme such as studies showing dextran removal during sugar manufacturing [7]. Cadex was a favorable biofilm inhibitor that surpassed the inhibitory ability of *Penicillium* dextranase at the same concentration. In addition, common teeth rinsing products, such as carboxybenzene, ethanol, sodium fluoride, and xylitol, had no negative effects on Cadex activity. Marine organisms are regarded as a prolific resource of novel bioactive metabolites, including a vast array of macrolides, cyclic peptides, pigments, polyketides, terpenes, steroids and alkaloids [45]. At the same time, marine enzymes are important bioactive metabolites which characterized by high salt tolerance, hyperthermostability, and low ideal temperature tolerance. These beneficial properties make Cadex an attractive candidate for development as a novel reagent for dental plaque treatment [46,47].

#### **4. Materials and Methods**

#### *4.1. Chemicals*

Q-Sepharose FF, dextran (T20, T40, T70, and T500), and PhastGel IEF 3-9 were obtained from GE Healthcare (Uppsala, Sweden). A prestained protein PAGE ruler was obtained from Fermentas (Waltham, MA, USA). An oligosaccharide kit, an IEF protein mix 3.6–9.3, bovine serum albumin, crystal violet, (CV), and Coomassie brilliant blue R250 and G250 were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were purchased from Sinopharm Chemical Reagent Corporation (Shanghai, China) and were of the highest analytical grade.

#### *4.2. Crude Dextranase Production*

Extracellular dextranase production was performed in medium containing 5 g/L yeast extract, 5 g/L peptone, 10 g/L dextran T20, and 5 g/L NaCl. The pH was adjusted to about 8.0 before autoclaving. Then, the production medium was inoculated with *Catenovulum* sp. DP03. After fermenting at 30 ◦C for 28 h, a cell-free culture broth was obtained by centrifugation for 20 min at 12,000× *g* and 4 ◦C.
