**1. Introduction**

Dextranases (α-1,6-D-glucan-6-glucanohydrolase; EC 3.2.1.11) hydrolyze dextran to oligosaccharides at the α-1,6 glucosidic bond, and are widely used in medical, dental, and sugar industries. In clinical applications, specific clinical dextran produced by dextranase can be used as a blood substitute in emergencies [1–3]. In the sugar industry, dextranase has been used to resolve the poor clarification and throughput that dextran can cause in sugarcane juice [4–7]. It is worth noting that this enzyme can be used with commercial dextran to directly synthetize isomaltose and isomaltooligosaccharides which exhibit prebiotic effects [8–10]. A previous report proposed that dextranase may be capable of treating dental plaques [2,3,11]. For this reason, the use of dextranase to treat dental caries has attracted a great deal of attention, particularly with respect to the degradation of dextran in dental plaques. Bacterial cells form biofilms as a protective barrier from external conditions, serving as a mechanism for improving survival and dispersion [12,13]. *Streptococcus mutans* is the main cause of dental decay in human teeth and key modulator of the development of cariogenic biofilms [14,15]. Accumulation of this cariogenic bacterium within the biofilm may lead to the onset of periodontal inflammation. Thus, dislodging the biofilm is the main therapy for periodontal inflammation.

An alkaline dextranase may be suitable for the treatment of dental caries because alkaline tooth-rinse products are expected to be more amenable to enamel than acidic products [16]. In addition, dextranase works efficiently at temperatures of about 37 ◦C and may also contribute to the degradation of human dental plaques. However, the most common source of dextranase, fungi, produce many acidic and megathermal dextranases, which catalyze at pH values ranging from pH 5.0 to 6.5 and temperatures above 50 ◦C, and are unstable under alkaline conditions. Dextranases are seldom capable of catalysis under both alkaline and moderate-temperature conditions [1]; however, enzymes from marine microorganisms may be an exception [17–19].

In this study, a dextranase produced by a marine bacterium *Catenovulum* sp. DP03 (CGMCC No. 7386) was sequenced and selected for extracellular experiments [20]. This study demonstrated a method for purifying, characterizing and hydrolyzing products of dextranase from the marine strain DP03 and analyzed its effects on *S. mutans* biofilm. The results provide insights into additional applications for this enzyme.

#### **2. Results**

#### *2.1. Purification of Dextranase*

A summary of the efficient extraction protocol used to purify *Catenovulum* sp. DP03 dextranase (hereafter called Cadex), which included ultrafiltration, ethanol precipitation, ammonium sulfate precipitation, and ion exchange chromatography (Table 1). The activity of crude dextranase was 77.9 U/mg, and the activity of Cadex sequentially purified by ultrafiltration, ethanol precipitation, and ammonium sulfate precipitation was 163.5 U/mg, 223.3 U/mg and 341.6 U/mg, respectively. When ion exchange chromatography was used for purification, Cadex was purified 29.6-fold with a specific activity of 2309 U/mg protein and a yield of 16.9%. About 5.4% of enzymatic activity was lost and specific enzyme activity was increased by 48.7% after ultrafiltration. Ethanol precipitation and ammonium sulfate precipitation were used to remove polysaccharides and proteins. Ion exchange chromatography was performed to optimize the purification. Ten fractions containing proteins were eluted from Q-sepharose column, among which only one fraction showed dextranase activity. A Q-sepharose column with gradient elution of 45% 800 mM NaCl is an efficient method for Cadex purification. Finally, a homogenetic dextranase was purified. As shown in Figure 1, Cadex appeared as a single band of an estimated molecular weight of 75 kDa. The isoelectric point (pI) was 5.0.


**Table 1.** Purification of Cadex.

**Figure 1.** Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of Cadex. Lane Maker: protein marker. Lane 1: purified Cadex.

#### *2.2. Characterization of Dextranase*

2.2.1. Effects of pH and Temperature on Dextranase Activity

The optimum pH for maximum Cadex activity was 8.0 within the range of pH 7.0–9.0 (Figure 2a). The effects of temperature on dextranase activity are shown in Figure 2b. The optimal temperature for dextranase activity was 40 ◦C within the range of 25–50 ◦C, showing peak relative activity no less than 87%. However, dextranase activity decreased up to 60 ◦C. Cadex also showed 33.8% relative activity at 0 ◦C.

**Figure 2.** *Cont*.

**Figure 2.** (**a**) Optimum pH and stability curves of Cadex. For each pH, activity was assayed at 40 ◦C and considered relative activity (ȣ). The pH stability curve (-) represents residual activity after pre-incubation for 1 h at 25 ◦C. (**b**) Effects of temperature on the activity. (**c**) Effects of temperature on thermal stability of Cadex. Thermal stability: -30 ◦C, ȣ 40 ◦C,Ÿ50 ◦C.

#### 2.2.2. Enzymatic Stability

Figure 2a shows that Cadex retained more than 90% activity within a pH range of 5–11 (100% activity at pH 7.0). Purified Cadex showed thermal stability at 30 ◦C (pH 7.5) for 5 h. Under these conditions, almost 100% activity was retained. At 40 ◦C, 40% of activity was lost after 5 h of exposure. Above 50 ◦C, activity rapidly declined to undetectable levels at 1 h (Figure 2c).

#### 2.2.3. Effects of Metal Ions and Reagents on Dextranase Activity

Alkaline proteases from microorganism require a divalent cation such as Ca2+, Mg2+, and Mn2+ or a combination of these cations, for maximum activity [19]. The effects of various metal ions on Cadex were investigated and the results are shown in Table 2. Enzyme activity was slightly increased upon exposure to 1 mM and 5 mM MgCl2 or SrCl2, respectively. This is consistent with previous observations [21,22]. However, enzyme activity was inhibited by exposure to 1 mM CuCl2, FeCl3, ZnCl2, CdCl2, NiCl2, or CoCl2, and even more so at concentrations of 5 mM, consistent with the results

from another report [23], although these data conflict with the results reported by Birol et al. [24]. No apparent effects on dextranase activity were observed after exposure to BaCl2, NH4Cl, CaCl2, KCl, or LiCl. However, the cations Co2+ and Ca2+ had the opposite effects on dextranase from *Chaetomium erraticum* and *Arthrobacter oxydans* KQ11 [3,10]. The effects of several types of chemical treatments for dental caries on the activity of Cadex are shown in Table 3. The data indicate that 5% ethanol, 0.1% carboxybenzene, sodium fluoride and xylitol had no apparent effects on the Cadex activity.


**Table 2.** Effects of metal ions on Cadex activity.



2.2.4. Substrate Specificity and the Hydrolysis Products of Dextranase

Dextranase activity in the catalyzed hydrolysis of carbohydrates with different glucosidic linkages was determined and used as an indicator of the substrate specificity of Cadex (Table 4). Dextranase showed high specificity towards dextrans containing the α-1,6 glucosidic bond. The poor hydrolysis of soluble starch might indicate that Cadex can only cleave α-1,6 glycosidic bond. Soluble starch was formed using only a few α-1,6 glucosidic and α-1,4 glucosidic linkages [25]. No activity was detected in pullulan, which was mainly formed by α-1,4 glucosidic linkages. The same results were observed with chitin and cellulose, which are formed by β-1,4 glucosidic linkages. First, isomalto-triose (G3), isomalto-tetraose (G4), isomalto–pentaose (G5) and higher molecular weight maltooligosaccharides were found to be the products after a 3 h reaction. The hydrolysis products after a 24 h reaction were glucose (G1), G3, G4, and G5, and G6 was an extra sugar product when the reaction time was extended to 72 h, as shown by thin-layer chromatography (TLC) (Figure 3).

Second, high-performance liquid chromatography (HPLC) indicated that isomaltooligomers were the main products released by Cadex regardless of reaction time (Figure 4). Each hydrolyzed product was quantified by Empower GPC software (Table 5). G1, G2, G3, G4, G5, G6, and G7 were products of glucose, maltose, isomalto-triose, isomalto-tetraose, isomalto-pentaose, isomalto-hexaose, and isomalto-heptaose, respectively. The results indicated that a small amount of glucose was detected, which is similar to most fungal dextranases. They maintained a dynamic equilibrium, which did not increase with longer reaction times. Moreover, the amounts of maltose, isomalto-triose and

isomalto-tetraose slightly declined, and an increase in time from 15 min to 5 h did not increase the yield. However, isomalto-hexaose and isomalto-heptaose accumulated more quickly than the other sugars as the time increased from 15 min to 5 h. At the same time, the yield of these intermediate high molecular weight oligomers (15–20%) was significantly higher than that of glucose (about 2%). This was similar to other reports in which macromolecule isomaltooligomers accumulated in the first period (6 h), and the other sugars were hydrolyzed to produce other smaller sugars [26]. The isolation of small amounts of glucose and some intermediate high molecular weight oligomers seemed to be random rather than through the stepwise hydrolysis of polysaccharide, making Cadex an endo-type dextranase.


**Table 4.** Substrate specificity of Cadex.

**Table 5.** Content of sugar (%) in hydrolysates after enzymatic hydrolysis of dextran by Cadex.


**Figure 3.** Thin-layer chromatogram of the products from Cadex. Symbols: G1 to G7 a series of authentic sugar standards of glucose, maltose, isomalto-triose, isomalto-tetraose, isomalto-pentaose, isomalto-hexaose, and isomalto-heptaose, respectively. M is the standard marker, S1 to S3 show the 3 h, 24 h, and 72 h reaction times, respectively.

**Figure 4.** *Cont*.

**Figure 4.** The 3% dextran T70 was treated at 40 ◦C and pH 8.0 for different periods with the products measured by HPLC: (**a**) the results for standards (G1 to G7 a series of authentic sugar standards of glucose, maltose, isomalto-triose, isomalto-tetraose, isomalto-pentaose, isomalto-hexaose, and isomalto-heptaose, respectively); and (**b**–**f**) the results for 3% dextran T70 treated for 15 min, 30 min, 1 h, 3 h, and 5 h with Cadex.

Sugars were identified and measured by HPLC. The hydrolysis condition yielded 3% dextran T70 at 40 ◦C and pH 8.0.
