Dextranase from a deep-sea vent of the South China Sea

Abstract

This invention discloses a dextranase derived from an ancient hydrothermal vent area in the South China Sea, belonging to the field of genetic engineering technology. The acid-base- and thermostable dextranase FQZMS7044, screened from this ancient hydrothermal vent area, exhibits a high specific activity of 20,500 U / mg and strong catalytic ability. This enzyme demonstrates excellent thermostability: its activity remains stable after prolonged incubation at 60°C; after treatment at 80°C for 60 minutes, the enzyme activity retention rate is >60%; and after storage at 45°C and under light for 8 weeks, the enzyme activity retention rate is >85%. It also exhibits a wide pH tolerance range, with an enzyme activity retention rate >80% after treatment at 60°C for 60 minutes within a pH range of 4.0–10.0. It shows significant effectiveness in removing dental plaque biofilm, far exceeding the effect of 0.12% chlorhexidine. Furthermore, this enzyme exhibits good compatibility with commonly used additives in toothpaste and mouthwash, with enzyme activity retention rates generally >80% after co-incubation for 168 hours, ensuring its commercial application.

Description

Technical Field

[0001] This invention relates to a dextranase from an ancient hydrothermal vent area in the South China Sea, belonging to the field of genetic engineering technology. Background Technology

[0002] Oral health is an essential component of overall health, and the formation and accumulation of dental plaque is a core contributing factor to various oral diseases such as tooth decay, gingivitis, and periodontitis. Dental plaque is a bacterial biofilm attached to the tooth surface. It is formed by extracellular polysaccharides synthesized and secreted by cariogenic bacteria in the oral cavity (especially *Streptococcus mutans*) in the presence of sucrose, which act as a structural framework to adhere and aggregate bacteria, salivary proteins, food debris, and other substances. Dextran is the main component of the extracellular polysaccharide matrix of dental plaque, formed by glucose residues linked by α-1,6-glycosidic bonds. Some molecules also contain branched structures such as α-1,3, α-1,4, or α-1,2. Dextran possesses high adhesiveness and film-forming properties, enabling it to tightly adhere cariogenic bacteria to the tooth surface, forming a stable three-dimensional network structure that protects the bacteria from the effects of oral mechanical cleaning and the salivary defense system. Mature dental plaque biofilms exhibit high structural stability and resistance to erosion, making them difficult to remove effectively with ordinary brushing and rinsing. Therefore, effectively degrading dextran and breaking down the biofilm structure of dental plaque are key ways to prevent oral diseases and maintain oral health.

[0003] Dextranase (EC 3.2.1.11) is a class of enzymes that specifically catalyze the hydrolysis of α-1,6-glycosidic bonds in dextran. It precisely cleaves the polysaccharide scaffold of dental plaque biofilm, disrupting the three-dimensional network structure of the biofilm. This loosens the plaque structure, reduces its adhesion, and makes it easier for oral mechanical cleaning and saliva rinsing to remove. Simultaneously, it inhibits the maturation and thickening of new plaque, suppressing the adhesion and reproduction of cariogenic bacteria at the source. In the field of oral care, dextranase has been increasingly used in toothpaste, mouthwash, and oral gels, becoming a core functional ingredient in next-generation bio-based oral care products. Compared to traditional chemical cleaning ingredients, dextranase has significant advantages such as strong targeting, minimal interference with the oral microecology, and high safety.

[0004] However, existing dextranases are mainly derived from terrestrial microorganisms (such as Penicillium, Fusarium, and Arthrobacter) and some marine environmental microorganisms. Although some enzymes have been reported to have dextran degradation activity, their performance is still insufficient to meet the requirements of dental plaque polysaccharide removal applications, mainly due to the following problems: First, insufficient thermal stability. Dental cleaning products may experience temperature fluctuations during production (such as ingredient mixing, homogenization, and sterilization), storage, transportation, and use, which can easily lead to conformational changes, resulting in reduced or even inactivated enzyme activity, affecting the product's functional stability and shelf life. To improve this issue, existing technologies often employ auxiliary methods such as enzyme immobilization and microencapsulation (e.g., sodium alginate microspheres, liposomes, or chitosan microcapsules) to enhance enzyme stability. However, this increases process costs and complexity and may affect the enzyme's release efficiency in the oral cavity, limiting its large-scale application in dental cleaning products.

[0005] Second, the ability to degrade dextran in dental plaque biofilms is limited. Dextran in dental plaque exhibits a complex three-dimensional biofilm structure with poor substrate accessibility, a wide molecular weight distribution, and diverse branched structures. While some existing dextranases can hydrolyze soluble dextran standards (such as dextran T70 and T2000), their ability to degrade insoluble, multi-branched, or biofilm-state dextran is limited, making it difficult to effectively disrupt the spatial structure of mature dental plaque. Furthermore, some enzymes have low specific activities, requiring higher dosages to achieve the desired effect, increasing product costs.

[0006] Third, insufficient pH and temperature adaptability. The pH of the oral environment is typically between 6.5 and 7.5, and the temperature is approximately 36.5 to 37.5°C. Existing dextranases often deviate from this optimal pH range, or their activity is significantly reduced under neutral pH conditions, affecting their efficiency in the oral cavity. Ideally, dextranases for oral care should maintain high activity within the pH range of 5.5 to 8.0 to adapt to the dynamic changes in the oral environment.

[0007] Fourth, insufficient formula compatibility and stability. Dental cleaning products contain a variety of functional ingredients, including abrasives (such as calcium carbonate and silica), humectants (such as glycerin and sorbitol), surfactants (such as sodium lauryl sulfate), preservatives, fluoride, thickeners, and fragrances. These ingredients may disrupt the spatial structure of enzymes, reduce water activity, adsorb enzyme molecules, or inhibit their functional activity. Ideally, dextranase should maintain stable enzyme activity in the presence of these complex ingredients and remain functionally stable over a shelf life of 2-3 years.

[0008] To address the problems of poor thermostability, weak degradation ability against complex biofilms, and insufficient adaptability to the oral environment associated with existing dextranases used in dental plaque polysaccharide removal, a novel dextranase is urgently needed that possesses the following characteristics: 1. Excellent thermostability, capable of withstanding temperature fluctuations during the production of dental cleaning products and maintaining stable enzyme activity during product storage and use, without relying on additional immobilization or embedding treatments; 2. Good degradation ability of dextran in dental plaque biofilms, effectively acting on insoluble, multi-branched, high-molecular-weight dextran to disrupt the three-dimensional network structure of the biofilm; 3. pH adaptability to the oral physiological environment, with an optimal pH close to the oral physiological range and maintaining high activity over a wide pH range; 4. Good formulation compatibility and stability, able to withstand the influence of various components in the complex formulation system of dental cleaning products. Summary of the Invention

[0009] In view of the above-mentioned prior art, the present invention provides a thermostable dextranase obtained from an ancient hydrothermal vent area in the South China Sea, the amino acid sequence of which is shown in SEQ ID NO.1.

[0010] The present invention also provides a gene encoding the dextranase.

[0011] In one embodiment, the nucleotide sequence of the gene is shown in SEQ ID NO.6.

[0012] The present invention also provides a recombinant expression vector containing the said gene.

[0013] In one embodiment, the expression vector includes, but is not limited to, the pET-28a plasmid.

[0014] The present invention also provides a host cell expressing the dextranase.

[0015] In one embodiment, the host cell is *Escherichia coli* (E. coli). Escherichia coli The preferred strain is Escherichia coli BL21(DE3).

[0016] In one embodiment, the dextranase is expressed in Escherichia coli BL21(DE3) using pET-28a as the expression vector.

[0017] The present invention also provides a method for preparing dextranase, comprising culturing the recombinant host cell under conditions suitable for expressing the dextranase, and recovering the dextranase from the culture.

[0018] In one embodiment, the method includes using IPTG at a final concentration of 0.3-0.5 mM as an inducer to induce expression at 28-30°C.

[0019] The present invention also provides compositions containing the dextranase, comprising the dextranase, and carriers or excipients acceptable in the food, pharmaceutical or oral care fields.

[0020] In one embodiment, the composition is an oral care product, including but not limited to toothpaste, mouthwash, tooth powder, oral spray, or chewing gum.

[0021] In one embodiment, the composition further comprises one or more of the following: an abrasive, a humectant, a surfactant, a preservative, a fluoride, and a thickener.

[0022] The present invention also provides a method for degrading dextran or dextran-containing biofilms, the method comprising contacting a substrate or biofilm containing dextran with an effective amount of the dextranase or the composition.

[0023] In one embodiment, the biofilm is composed of Streptococcus mutans (Streptococcus mutans). Streptococcus mutans Dental plaque biofilm formed.

[0024] Beneficial effects: (1) The present invention screened and obtained acid-base heat-stable dextran enzyme FQZMS7044 from the ancient hydrothermal vent area of ​​the South China Sea. The amino acid sequence is shown in SEQ ID NO.1. The specific activity of this enzyme is as high as 20500 U / mg. The enzyme can achieve a high efficiency catalytic effect with a very low concentration of enzyme, which is crucial for reducing the raw material cost of industrial production and end products.

[0025] (2) The dextranase provided by this invention exhibits excellent thermal stability and a wide pH tolerance range. Its activity remains essentially stable after prolonged incubation at 60°C; even after incubation at 80°C for 60 minutes, the enzyme activity retention rate remains above 60%; and after being placed at 45°C under light for up to 8 weeks, the enzyme activity retention rate still exceeds 85%. Regarding pH tolerance, after treatment at 60°C in buffer solutions with pH ranges of 4.0-10.0 for 60 minutes, the enzyme activity retention rate exceeds 80%. This broad temperature and pH adaptability allows it to remain stable and less prone to inactivation in various demanding processing techniques (such as processes involving high temperatures or acidic / alkaline environments), providing unparalleled stability and storage advantages compared to similar competing products on the market.

[0026] (3) The dextranase provided by this invention is extremely effective in removing dental plaque biofilm. After 24 hours of treatment with the enzyme of this invention at a concentration of 800 U / L, the degradation rate of mature Streptococcus mutans biofilm reached 98.73%, and the EPS content decreased by 91.58%, almost achieving complete removal of the biofilm. This effect not only far exceeds that of other enzymes at the same concentration, but also outperforms the clinical standard anti-plaque agent 0.12% chlorhexidine (with a degradation rate of only 69.72% after 24 hours) at all time points. This effect confirms the great medical and daily chemical application value of this invention in caries prevention and oral plaque control.

[0027] (4) The dextranase provided by the present invention exhibits excellent compatibility with commonly used additives in oral care products. After being co-incubated with common toothpaste and mouthwash components such as abrasives, humectants, preservatives, fluorides, and thickeners at commercially available concentrations for 168 hours, its enzyme activity retention rate is generally above 80%, which provides a guarantee for its stable application in commercial products. Attached Figure Description

[0028] Figure 1 SDS-PAGE analysis of dextranase; where M: marker; 1: FQZMS7044 cells; 2: FQZMS7044 supernatant; 3: WP8845 cells; 4: WP8845 supernatant; 5: MGY94070 cells; 6: MGY94070 supernatant; 7: WP4350 cells; 8: WP4350 supernatant; 9: FQZMS16967 cells; 10: FQZMS16967 cells; BL21(DE3) control: BL21(DE3) wild-type strain.

[0029] Figure 2 Three-dimensional structural model of dextranase FQZMS7044.

[0030] Figure 3 The optimal reaction temperature for dextranase FQZMS7044 is [value missing].

[0031] Figure 4 The thermostability of dextranase FQZMS7044.

[0032] Figure 5 For comparison of thermal stability; where A: dextranase FQZMS7044; B: competitor dextranase.

[0033] Figure 6 The optimal reaction pH for dextranase FQZMS7044.

[0034] Figure 7 pH stability of dextranase FQZMS7044.

[0035] Figure 8 The stability of dextranase FQZMS7044 enzyme activity. Detailed Implementation

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. The specific embodiments described below further illustrate the present invention.

[0037] The culture medium used in the examples: LB medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH natural (approximately 7.0). For solid medium, add 20 g / L agar to this mixture. 0.1 M PBS pH 6.5 buffer: Pour approximately 800 mL of deionized water into a beaker. Weigh 12 g of anhydrous NaH2PO4 and 14.2 g of anhydrous Na2HPO4 and add them to the beaker, stirring until completely dissolved. Adjust the pH to 6.50: Transfer the solution to a 1 L volumetric flask, dilute to the mark with deionized water, and mix well.

[0038] 40 mM PBS pH 6.5 buffer: Add 400 mL of 0.1 M PBS 6.5 buffer to 500 mL of deionized water, transfer the solution to a 1 L volumetric flask, and dilute to the mark with deionized water. Mix well.

[0039] 20 mM PBS pH 6.5 buffer: Add 200 mL of 0.1 M PBS 6.5 buffer to 500 mL of deionized water, transfer the solution to a 1 L volumetric flask, and dilute to the mark with deionized water. Mix well.

[0040] Example 1: Screening and homology comparison of dextranase In the microbial gene resource library obtained from the ancient hydrothermal vent area of ​​the South China Sea, screening was conducted based on highly conserved motifs of dextranase donor-substrate binding, and the thermostability of dextranase was predicted. The amino acid sequences of the dextranases FQZMS7044, WP8845, MGY94070, WP4350, and FQZMS16967 were finally screened and are shown in SEQ ID NO.1 to SEQ ID NO.5, respectively.

[0041] Example 2: Expression and purification of dextranase Shanghai Diwin Biotechnology Co., Ltd. synthesized plasmids containing the gene sequences shown in SEQ ID NO. 6 to SEQ ID NO. 10 (encoding the enzymes shown in SEQ ID NO. 1 to SEQ ID NO. 5, respectively) linked to pET-28a. The obtained plasmids were transformed into wild-type Escherichia coli BL21(DE3), and the resulting strains were transferred to LB liquid medium containing 100 μg / mL kanamycin and cultured at 37 ℃ and 200 rpm for 8 h. The inoculum was then transferred to 200 mL of LB liquid medium at a 1% (v / v) inoculation rate, and kanamycin was added to a final concentration of 100 μg / mL. The culture was incubated at 37°C and 200 rpm for 3 h until the OD600nm value reached 0.6-0.8. Subsequently, IPTG was added to a final concentration of 0.4 mM as an inducer, and the culture was continued at 30°C and 200 rpm for 8 h. The cells were collected by centrifugation at 5000 rpm and 4°C for 10 min, and the precipitate was resuspended in 10 mM Tris-HCl (pH 10.0) to a final concentration of 20 OD / mL to obtain a recombinant *E. coli* cell suspension. This suspension was then sonicated at 30% power on an ice-water bath with a 10 s pause followed by a 5 s pause for 30 min. The suspension was then centrifuged at 12000 rpm and 4°C for 30 min to obtain the supernatant. The supernatant was filtered through a 0.22 μm filter membrane to obtain the crude enzyme solution prepared for each strain. The purification method was as follows: Ni was previously equilibrated with 100 mL of 50 mM Tris-HCl at pH 7.4. 2+ -NTASepharose (Hilden, Germany) loaded the crude enzyme supernatant (5 mL) onto an IMAC column (0.8 cm² × 10 cm, 1.5 mL / min). After washing with buffer 2 containing 0.3 M NaCl and 20 mM imidazole, the protein was eluted with buffer (50 mM Tris-HCl pH 7.4, 0.3 M NaCl, and 500 mM imidazole), and the collected eluent was the pure enzyme. The results were analyzed by SDS-PAGE (…). Figure 1 The results showed that the supernatant of cell lysates expressing FQZMS7044 or WP8845 contained bands, while the supernatant of other strains did not, which may be due to the formation of inclusion bodies.

[0042] Example 3: Enzyme activity detection method Standard curve: Measure 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mL of glucose standard solution (1 mg / mL) into 15 mL test tubes, respectively. Add distilled water to bring the volume to 1.0 mL, shake well, and accurately add 2 mL of dinitrosalicylic acid (DNS, purchased from Shanghai Yuanye Biotechnology Co., Ltd.). Heat in a boiling water bath for 2 min, cool, and then add water to bring the volume to the 15 mL mark. Measure the absorbance at 540 nm and plot the standard curve based on the glucose concentration and the corresponding absorbance value.

[0043] The dextranase activity unit (U) is defined as the amount of enzyme that catalyzes the hydrolysis of dextran to produce 1 μmol of reducing sugar (in glucose equivalent) per minute under specific conditions (pH 6.5, 37℃).

[0044] Enzyme activity assay method: Add 150 μL of substrate 60 g / L dextran T70 + 40 mM PBS 6.5 buffer to the test tubes of the experimental group and the control group. Experimental group: Add 50 μL of dextranase enzyme solution prepared in Example 2, mix well, and react at the set temperature for 15 min; after the reaction is completed, add 200 μL of DNS reagent to terminate the reaction.

[0045] Control group: No enzyme solution was added, and the mixture was placed at the same temperature for 15 min; then 200 μL of DNS reagent was added to terminate the reaction, and then 50 μL of the enzyme solution prepared in Example 2 was added and mixed well.

[0046] Subsequently, the experimental and control group samples were simultaneously subjected to color development treatment, and the OD value (540 nm) was measured to calculate the dextranase activity.

[0047] Table 1 Enzyme activity detection results

[0048] The enzyme activity results are shown in Table 1. FQZMS7044 showed the highest enzyme activity at 93,000 U / L, followed by WP8845 at 1,140 U / L. The other three bacteria likely contained inclusion bodies, making enzyme activity undetectable. FQZMS7044 was purified using a nickel column and then directly lyophilized; the specific activity obtained was 20,500 U / mg.

[0049] The structural prediction results of FQZMS7044 are as follows: Figure 2As shown, 88-395aa is predicted to be the GH66 domain (shown in blue), with D235E301 being the core catalytic site (shown in pink). It contains two cysteines: C83 and C202 (shown in yellow), but they are not spatially adjacent and cannot form an SS bond. There are up to 17 pairs of salt bridges (shown in orange), mainly distributed on the protein surface, connecting α-helices, loops, and domains. These salt bridge pairs effectively reduce the protein's thermal motion and maintain structural stability through Coulomb forces between positive and negative charges. The synergistic effect of multiple salt bridges significantly improves the protein's heat resistance.

[0050] Example 4: Dextranase Optimal Temperature Test The dextranase FQZMS7044 prepared in Example 2 was subjected to enzymatic reaction and enzyme activity detection at 20℃, 30℃, 37℃, 40℃, 50℃, 60℃, 70℃, 80℃, 90℃ and 100℃ in 20mM PBS (pH 6.5) buffer, respectively, according to the method in Example 3. The results are as follows Figure 3 As shown, the relative enzyme activity is 100% at 60℃, and decreases with increasing or decreasing temperature, indicating that the optimal reaction temperature for dextranase FQZMS7044 is approximately 60℃. Furthermore, under oral environmental conditions of 30–37℃, the dextranase activity retention rate can reach 80%.

[0051] Example 5: Temperature stability test of dextranase Temperature stability determination: Dextranase FQZMS7044 was incubated in 20mM PBS (pH 6.5) buffer at 37℃, 60℃, and 80℃ for 5 min, 10 min, 20 min, 30 min, and 60 min, respectively, before enzymatic reaction. Untreated enzyme solution was used as a control. Enzyme activity was detected according to the method in Example 3.

[0052] The results are as follows Figure 4 As shown, the dextranase FQZMS7044 maintained stable activity after incubation at 37℃ and 60℃ for 60 min, and its activity remained above 60% after incubation at 80℃ for 60 min, indicating that the dextranase FQZMS7044 has strong thermostability.

[0053] Example 6: Dextranase Storage Stability Test Storage stability determination: Dextranase FQZMS7044 and the competing dextranase (Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd.) were incubated in 20mM PBS buffer at 60°C for 5 days. Enzymatic reactions were performed on days 0, 1, 2, and 5, with untreated enzyme solution as a control. Enzyme activity was detected according to the method in Example 3.

[0054] The results of the thermal stability comparison are as follows Figure 5 As shown, the dextranase FQZMS7044 maintained stable enzyme activity after being placed at 60℃ for 5 days, while the competing dextranase showed almost undetectable enzyme activity after being placed at 60℃ for 3 days, indicating that the dextranase FQZMS7044 has strong thermostability.

[0055] Example 7: Optimal pH Test To determine the optimal pH for the enzyme, 20 mmol / L buffer solutions with pH values ​​ranging from 3.0 to 11.0 were prepared using different buffer systems. Specifically, citrate-sodium citrate buffer was used for pH 3.0–5.0, phosphate buffer for pH 6.0–8.0, and glycine-sodium hydroxide buffer for pH 9.0–11.0. Dextranase FQZMS7044 enzyme solution was added to each of these buffer solutions, and the dextranase activity was measured under the same reaction conditions at 60°C.

[0056] The results showed that the optimal reaction pH for dextranase FQZMS7044 was 6.0. Figure 6 ).

[0057] Example 8: pH stability test pH stability determination: Dextranase FQZMS7044 was subjected to enzymatic reaction after being tolerated for 60 min in buffer systems (prepared according to Example 7) at 60℃ and pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0. Untreated enzyme solution was used as a control. Enzyme activity was detected according to the method in Example 3.

[0058] The results showed that buffer solutions ranging from pH 4.0 to pH 10.0 retained more than 80% of their activity after treatment at 60°C for 60 min, while buffer solutions at pH 3.0 and pH 11.0 retained more than 70% of their activity after treatment at 60°C for 60 min. Figure 7 This indicates that dextranase FQZMS7044 has good pH stability.

[0059] Example 9: Verification of the thermostability of dextranase Dextranase FQZMS7044 was placed in stability chambers at different temperature environments (5℃, 25℃, 45℃, and light exposure), and tested after 1, 2, 4, and 8 weeks. After removal, the samples were allowed to return to room temperature before enzyme activity was detected according to Example 3. The light stability test conditions were as follows: light source: LED lamp, power: 12 W, light intensity: 760 lux, color temperature: 3800 K, distance between light source and sample: 50 cm, temperature: 45℃.

[0060] Table 2 Stability of dextranase activity

[0061] The stability results of dextranase FQZMS7044 are shown in Table 2 and Figure 8 As shown, after being placed at 5℃, 25℃, 45℃ and under light conditions for 8 weeks, the activity was maintained at more than 70%, indicating that dextranase FQZMS7044 has good thermal stability.

[0062] Example 10: Degradation test of mature biofilm of Streptococcus mutans by dextranase Sample: Dextranase FQZMS7044; Strains: Streptococcus mutans CGMCC 1.2499, purchased from the Institute of Microbiology, Chinese Academy of Sciences; Reagents: pH 7.2 sterile PBS buffer, 0.1% crystal violet staining solution, 33% (v / v) glacial acetic acid, phenol-sulfuric acid total sugar assay kit, 2.5% glutaraldehyde fixative, gradient ethanol (30%-100%), 0.12% chlorhexidine solution (positive control, clinical standard antimicrobial plaque agent).

[0063] Culture medium: BHI broth (Beijing Luqiao, CM206), BHI broth containing 10 g / L sucrose.

[0064] Preparation of mature biofilms from Streptococcus mutans: Take a suspension of Streptococcus mutans in the logarithmic growth phase and adjust the concentration to 1×10⁻⁶ with BHI broth containing 10 g / L sucrose. 6 CFU / mL. Add 200 μL of bacterial suspension (for quantitative detection) to each well of a 96-well polystyrene plate. Place a sterile round coverslip (φ14 mm) in each well of a 24-well plate and add 1 mL of bacterial suspension (for SEM observation). Incubate anaerobically at 36℃±1℃ for 48 h to form a dense, mature biofilm. Discard the culture medium and gently wash three times with sterile PBS to remove any unattached airborne bacteria.

[0065] Grouping and processing: Set up 6 groups, with 6 parallel holes in each group: (1) Blank control group: only 200 μL PBS (without biofilm) was added; (2) Negative control group: 200 μL PBS (with biofilm, no enzyme treatment) was added. (3) Positive control group: Add 200 μL of 0.12% chlorhexidine solution; (4) Low concentration enzyme group: Add 200 μL of dextranase solution with a concentration of 50 U / L; (5) Medium concentration enzyme group: Add 200 μL of dextranase solution with a concentration of 200 U / L; (6) High concentration enzyme group: Add 200 μL of dextranase solution with a concentration of 800 U / L; After treatment at 36℃±1℃ for 1h, 3h, 6h and 24h respectively, all groups were discarded and gently rinsed 3 times with PBS.

[0066] Testing indicators: (1) Quantification of total biofilm using crystal violet staining: Add 200 μL of 0.1% crystal violet to each well and stain at room temperature for 15 min. Wash with PBS until colorless, dissolve by shaking in 33% glacial acetic acid for 10 min, and measure the OD value at 595 nm using a microplate reader. Formula for calculating biofilm degradation rate: Degradation rate (%) = (OD595 of negative control group - OD595 of experimental group) / OD595 of negative control group × 100%; (2) Determination of extracellular polysaccharide (EPS) content: Collect biofilms after 24h treatment, sonicate (200W, 3s working / 5s intermittent, 30 times), centrifuge at 12000r / min at 4℃ for 20min, collect the supernatant, determine the total sugar content at 490nm using the phenol-sulfuric acid method, and calculate the EPS concentration using the glucose standard curve.

[0067] The biofilm results for each group at different treatment times are shown in Table 3. (1) Concentration dependence: The degradation effect of marine dextranase FQZMS7044 on mature biofilms increased significantly with increasing enzyme concentration; the degradation effect of the low concentration group (50 U / L) was weaker than that of the positive control group; the degradation rate of the biofilm in the 200 U / L group reached 80.10% after 1 hour of treatment, which was far higher than the 34.92% of the positive control group at the same time; the degradation rate of the high concentration group (800 U / L) reached 98.73% after 24 hours, achieving almost complete degradation of mature biofilms.

[0068] (2) Time dependence: The degradation efficiency of the enzyme FQZMS7044 of the present invention at medium and high concentrations continued to increase with the extension of the reaction time; the degradation rate of the 800 U / L group reached 92.76% after 3 h of reaction, tended to stabilize after 6 h, and completely removed the biofilm after 24 h.

[0069] Compared with the positive control: the degradation rate of 0.12% chlorhexidine, which is commonly used in clinical practice, is only 69.72% after 24 hours; the degradation effect of the 200 U / L and 800 U / L concentration groups of this invention is better than that of chlorhexidine at all time points.

[0070] Table 3. Biomembrane OD at different treatment times for each group 595 Value and degradation rate (n=6)

[0071] The degradation effect of extracellular polysaccharides (EPS) is shown in Table 4. Dextranase FQZMS7044 specifically hydrolyzes dextran-type extracellular polysaccharides (Table 4), the core matrix of dental plaque. The EPS reduction rate in the 800 U / L group reached 91.58%, effectively disrupting the polysaccharide backbone of the biofilm. The EPS content was significantly positively correlated with the total biofilm content (r=0.942, P<0.01), confirming that dextranase FQZMS7044 achieves plaque removal through targeted polysaccharide degradation.

[0072] Table 4. EPS content of biofilm in each group after 24 hours of treatment (n=3)

[0073] Example 11: Tolerance test of dextranase to commonly used additives in oral care products Take 1 mL of crude dextran enzyme solution with an initial enzyme activity of 200 U / L, and add 9 mL of pH 6.0 phosphate buffer containing different concentrations of commonly used oral additives (humectants, surfactants, preservatives, fluoride, thickeners), and water-insoluble abrasives (light calcium carbonate and silica). The specific steps are as follows: First, accurately add the target concentration of abrasive to 9 mL of pH 6.0 buffer, vortex to mix and prepare a homogeneous suspension, then add 1 mL of enzyme solution (to make the final enzyme concentration 200 U / L), and incubate at a constant temperature with shaking throughout (simulating the actual toothpaste stirring environment, 37℃, 150~180 rpm). At the same time, a blank control group without additives is set up. All samples are incubated at 37℃, and samples are taken at 0 h and 168 h, respectively. Centrifuge at 12000 r / min for 10 min and collect the supernatant. The residual enzyme activity is determined by the DNS method of Example 3. The formula for calculating the enzyme activity retention rate is: Enzyme activity retention rate (%) = Remaining enzyme activity of sample group after 168h / Enzyme activity of sample group after 0h × 100%.

[0074] Table 5. Retention rate of dextranase activity after 168 h of treatment with different additives (n=3)

[0075] The results are shown in Table 5. Within the concentration range of commonly used additives in oral products, after incubation for 168 hours, the enzyme activity retention rate of the dextranase of the present invention was above 80% in most groups, while the enzyme activity retention rate of the blank control group was 92.35%, indicating that the enzyme itself has good thermal stability and storage stability.

[0076] Moisturizers (glycerin, sorbitol) have no negative impact on enzyme activity; on the contrary, they slightly increase it with increasing concentration (the enzyme activity retention rate of the 30% glycerin group reached 95.78%). This is because polyols can stabilize the spatial conformation of enzymes and prevent them from denaturing and becoming inactive.

[0077] The abrasives, preservatives, fluorides, and thickeners have little effect on enzyme activity at commonly used concentrations. The enzyme activity retention rate in the highest concentration group is still above 82%, which fully meets the efficacy requirements within the product's shelf life.

[0078] Surfactants (SDS) have a certain impact on enzyme activity, but in toothpaste and mouthwash, the enzyme activity retention rate can still reach 78.91%-85.63% when the concentration is in the range of 0.5%-1.0%. When the concentration exceeds 2.0%, the enzyme activity decreases significantly. It is recommended that the SDS concentration in the formulation be controlled below 1.0%.

[0079] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Dextranase, characterized in that, The amino acid sequence is shown in SEQ ID NO.

1.

2. The gene encoding the dextranase of claim 1.

3. A recombinant expression vector containing the gene of claim 2.

4. Host cells expressing the dextranase of claim 1.

5. Recombinant Escherichia coli, characterized in that, Using Escherichia coli BL21(DE3) as the host and pET-28a as the expression vector, the dextranase described in claim 1 is expressed.

6. A method for preparing dextranase, characterized in that, The recombinant Escherichia coli of claim 5 is cultured under conditions suitable for expressing the dextranase, and the dextranase is recovered from the culture.

7. A composition containing the dextranase of claim 1, characterized in that, The composition comprises the dextranase, and a carrier or excipient acceptable in the food, pharmaceutical, or oral care fields.

8. The composition according to claim 7, characterized in that, The composition also contains one or more of the following: abrasive, humectant, surfactant, preservative, fluoride, and thickener.

9. The composition according to claim 7, characterized in that, The composition is an oral care product, including: toothpaste, mouthwash, tooth powder, oral spray or chewing gum.

10. A method for degrading dextran or biofilms containing dextran, characterized in that, The method comprises contacting a substrate or biofilm containing dextran with an effective amount of the dextranase of claim 1 or any of the compositions of claims 7-8.

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