Marine bacterium Bacillus sp.SQ3, method for producing dextranase by using marine bacterium Bacillus sp.SQ3 and application of marine bacterium Bacillus sp.SQ3

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses a marine bacterium Bacillus sp.SQ3 and a method for producing dextranase and application thereof, the strain is preserved in China General Microbiological Culture Collection Center (CGMCC) on January 20, 2025, the preservation number is CGMCC No.33458, the preservation unit address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.33458. Chinese Academy of Sciences Institute of Microbiology. The dextranase produced by the strain SQ3 has the advantage of preparing high-polymerization-degree isomaltooligosaccharides, in hydrolysate of the dextranase, the ratio of tetrasaccharide is 9.9%, the ratio of pentasaccharide is 15.5%, the ratio of hexasaccharide is 32.7%, the ratio of heptasaccharide is 33.9%, the total ratio reaches 92%, and the strain and the enzyme produced by the strain have the remarkable advantage of being applied to preparing the high-polymerization-degree oligosaccharides.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to a marine bacterium Bacillus sp. SQ3 and a method and application thereof for producing dextranase. Background Art

[0002] The ocean's ecological environment exhibits minimal temperature fluctuations and boasts a rich biodiversity. Microorganisms within the ocean are well adapted to its temperature, pH, and salinity. Marine microorganisms primarily inhabit a neutral to alkaline environment, and the enzymes they secrete exhibit low optimal reaction temperatures and alkaline pH levels. Fungi, yeasts, and bacteria have all been found to produce dextranase. Dextranase produced by marine bacteria is also known to be salt- and alkali-tolerant, and has a low operating temperature.

[0003] Dextran is a poly-D-glucose with a specific structure, its backbone connected by α-1,6 glycosidic bonds. Dextranase (EC3.2.1.11) is an enzyme that specifically hydrolyzes the α-1,6 anhydroglucose bonds in dextran, producing oligosaccharides and isomalto-oligosaccharides (IMOs). Dextranase is primarily used in the pharmaceutical, food, healthcare, sugar production, and biotechnology industries. Dextranase can hydrolyze high-molecular-weight dextran to produce functional isomalto-oligosaccharides. Dextran with varying degrees of hydrolysis can be used as a food additive to improve food softness.

[0004] IMO is a non-digestible oligosaccharide with a low glycemic index, which helps maintain blood sugar homeostasis. It also has the potential to lower cholesterol levels, promote intestinal probiotics, and alleviate intestinal inflammation, making it widely used in the food and health sector. Studies have shown that the effects of oligosaccharides are closely related to their degree of polymerization (DP). High-DP oligosaccharides (DP greater than 4) can significantly promote the growth of intestinal probiotics such as bifidobacteria and lactic acid bacteria, regulating the balance of intestinal flora. They can also enhance intestinal barrier function and immunity, improving resistance to infection and inflammation. Furthermore, high-DP oligosaccharides help improve metabolism and have certain hypoglycemic and hypolipidemic effects. In contrast, low-DP oligosaccharides are easily digested and absorbed by the body and have lower biological activity. Testing has found that commercially available prebiotic oligosaccharide products are primarily composed of low-DP components, which to some extent weakens their effectiveness in promoting probiotic growth. Therefore, increasing the proportion of high-DP oligosaccharides in products is important for enhancing the function of prebiotics. However, current oligosaccharide production and processing technologies lack key enzymes for the efficient production of high-DP oligosaccharides. To this end, a marine bacterium Bacillus sp. SQ3 and a method and application for producing dextranase thereof are provided. Summary of the Invention

[0005] The purpose of the present invention is to address the defects of the prior art and provide a marine bacterium Bacillus sp. SQ3 and a method and application for producing dextranase thereof to solve the problems raised by the above background technology.

[0006] To achieve the above-mentioned purpose, the present invention provides the following technical solution: a marine bacterium Bacillus sp. SQ3, which was deposited in the General Microbiology Center of the China Culture Collection Administration on January 20, 2025, with the deposit number: CGMCC No. 33458, and the deposit unit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] A method for producing dextranase using the marine bacterium Bacillus sp. SQ3 as described above, comprising the following specific steps:

[0008] Step 1: Store the strain SQ3 slant in a 4°C refrigerator; inoculate into growth medium at 10% liquid volume, incubate at 180 rpm and 40°C for 12 h to obtain a bacterial suspension;

[0009] Step 2: The bacterial suspension was inoculated into the enzyme production medium with an inoculum volume of 2% and a liquid volume of 10%, and cultured at 180 rpm and 30°C for 60 h to obtain the fermentation liquid;

[0010] Step 3: The fermentation broth was centrifuged at 8000 rpm at 4°C for 10 min. The supernatant was sterilized by filtration using a 0.22 μm filter membrane. The filtrate was centrifuged at 4500 rpm at 4°C for 10 min using a 30 kDa ultrafiltration tube. The portion with a molecular weight greater than 30 kDa was the enzyme solution.

[0011] As a preferred technical solution of the present invention, the growth medium in step 1 is prepared by: 1g / L potato starch, 5g / L soy peptone, and pure water, pH 9.0, and high temperature sterilization at 121°C for 20 minutes.

[0012] As a preferred technical solution of the present invention, the enzyme-producing culture medium in step 2 is: 1 g / L cassava starch, 5 g / L fish meal peptone, 4 g / L dextran T20, pH 7.5, and is sterilized at high temperature of 121°C for 20 minutes.

[0013] An application of the dextranase produced by the method described above, wherein the enzyme solution is used to hydrolyze dextran T20 to produce isomaltooligosaccharides;

[0014] The specific steps are as follows: the enzyme solution and the substrate are mixed in a ratio of 1:3, reacted at 35°C for 12 hours, boiled in boiling water for 5 minutes, centrifuged at 8000 rpm for 5 minutes, the supernatant is filtered with a 0.22 μm filter membrane, and the filtrate is dried to obtain isomaltooligosaccharide, wherein the content of oligosaccharides with a degree of polymerization greater than 4 is greater than 90%.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention provides a new dextranase-producing Bacillus sp. SQ3, which enriches the varieties of marine bacteria that produce dextranase. The strain has the following characteristics: a growth temperature range of 20-45°C, an initial growth pH range of 5.0-11.0, an optimum growth temperature of 40°C for the strain SQ3, and an optimum initial growth pH of 9.0; the dextranase produced by the strain SQ3 has an optimum action temperature of 40°C, which is relatively low.

[0017] The dextranase produced by the strain SQ3 of the present invention has the advantage of preparing high-polymerization oligosaccharides. Among the hydrolyzed products of the enzyme, tetrasaccharides account for 9.9%, pentasaccharides account for 15.5%, hexasaccharides account for 32.7%, and heptasaccharides account for 33.9%, with the total proportion of high-polymerization oligosaccharides reaching 92%. This bacterium and its enzyme have significant advantages in the preparation of high-polymerization oligosaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a transparent circle formed on the screening plate of the strain SQ3 of the present invention;

[0019] Figure 2 This is the Gram staining image of the strain SQ3 of the present invention;

[0020] Figure 3 This is a scanning electron micrograph of the strain SQ3 of the present invention;

[0021] Figure 4 This is a phylogenetic tree diagram of the rDNA phylogeny of the strain SQ316S of the present invention;

[0022] Figure 5 This is a graph showing the effect of time on the growth of strain SQ3 of the present invention;

[0023] Figure 6 This is a graph showing the effect of temperature on the growth of strain SQ3 of the present invention;

[0024] Figure 7 This is a graph showing the effect of the initial pH value of the culture medium of the present invention on the growth of strain SQ3;

[0025] Figure 8 This figure shows the effect of NaCl concentration in the culture medium of the present invention on the growth of strain SQ3;

[0026] Figure 9 This is a graph showing the effect of the carbon source of the present invention on the growth of strain SQ3;

[0027] Figure 10 This is a graph showing the effect of nitrogen sources on the growth of strain SQ3 of the present invention;

[0028] Figure 11 This is a graph showing the effect of time on enzyme production of strain SQ3 of the present invention;

[0029] Figure 12 This is a diagram showing the effect of temperature on enzyme production by strain SQ3 of the present invention;

[0030] Figure 13 This is a graph showing the effect of the initial pH of the culture medium on the enzyme production of the strain SQ3;

[0031] Figure 14 This figure shows the effect of NaCl concentration in the culture medium of the present invention on enzyme production by strain SQ3;

[0032] Figure 15 This is a graph showing the effect of the carbon source of the present invention on the enzyme production of strain SQ3;

[0033] Figure 16 This is a diagram showing the effect of nitrogen sources on enzyme production by strain SQ3 of the present invention;

[0034] Figure 17 This is a graph showing the effects of different molecular weight inducers of the present invention on enzyme production by strain SQ3;

[0035] Figure 18 This is a graph showing the effect of T20 concentration on enzyme production by strain SQ3 of the present invention;

[0036] Figure 19 Graph showing the effect of the action temperature of the enzyme of the present invention on the enzyme activity;

[0037] Figure 20 is the thermostability diagram of the enzyme of the present invention;

[0038] Figure 21 This is a diagram showing the effect of pH on enzyme activity of the enzyme of the present invention;

[0039] Figure 22 is the pH stability diagram of the enzyme of the present invention;

[0040] Figure 23 Graph showing the HPLC test results of dextran T20 hydrolyzed by dextranase of the present invention;

[0041] (A): HPLC chromatogram of sugar standard; (B): HPLC chromatogram of dextran hydrolysis T2012h by strain SQ3 dextranase. DETAILED DESCRIPTION

[0042] The preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more precise definition of the protection scope of the present invention.

[0043] A marine bacterium Bacillus sp.SQ3, which was deposited at the General Microbiology Center of China Culture Collection of Microorganisms on January 20, 2025, with the deposit number: CGMCC No.33458. The depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0044] The colonies of this bacterium are milky white and sticky, with jagged edges, a flat surface and a slightly raised center, indicating that it is a Gram-positive bacillus. Phylogenetic tree analysis of the 16S rDNA of strain SQ3 and comprehensive morphological characteristics identified strain SQ3 as a Bacillus sp. The optimal growth medium for strain SQ3 is 1 g / L potato starch, 5 g / L soy peptone, pH 9.0, and a culture temperature of 40°C.

[0045] A method for producing dextranase using marine bacteria Bacillus sp. SQ3, comprising the following steps:

[0046] S1: strain SQ3, obtained from a slant screened from marine mud and stored in a refrigerator at 4°C, was inoculated into growth medium (1 g / L potato starch, 5 g / L soy peptone, pH 9.0, sterilized at 121°C for 20 min before use) with a 10% liquid volume, 180 rpm, and 40°C for 12 h to obtain a bacterial suspension.

[0047] S2: The bacterial suspension was inoculated into enzyme production medium (1 g / L cassava starch, 5 g / L fish meal peptone, 4 g / L dextran T20, pH 7.5, sterilized at 121°C for 20 min before use) with an inoculum size of 2% and a liquid volume of 10%. The culture was performed at 180 rpm and 30°C for 60 h to obtain a fermentation broth.

[0048] S3: The fermentation broth was centrifuged at 4°C and 8000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane for sterilization. The filtrate was purified by ultrafiltration using a 30 kDa ultrafiltration tube at 4°C and 4500 rpm for 10 min to obtain the enzyme solution.

[0049] The invention discloses an application of dextranase produced by marine bacteria Bacillus sp.SQ3 in the preparation of prebiotics. The product of dextran T20 hydrolyzed by dextranase is subjected to high performance liquid chromatography analysis.

[0050] The specific steps are as follows:

[0051] Mix the enzyme solution and substrate in a 1:3 ratio, react at 35°C for 12 hours, boil in boiling water for 5 minutes, and centrifuge at 8000 rpm for 5 minutes. Filter the supernatant through a 0.22 μm filter and store at 4°C for subsequent HPLC analysis. The column used was a Waters Sugar-PAK1 (6.5 × 300 mm) column, with deionized water as the mobile phase, at a flow rate of 0.4 mL / min and a column temperature of 75°C.

[0052] Example: The features of the present invention include that the marine strain Bacillus sp. SQ3 is a marine bacterium isolated from the sea mud in Qingdao, Shandong, China. The strain was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms on January 20, 2025, with a deposit number of CGMCC No. 33458. The depository address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The present invention provides a strain and a method for producing dextran using the strain, as well as a product obtained by hydrolyzing dextran T20 using the dextranase produced by the strain, wherein the product contains 90% oligosaccharides with a high degree of polymerization.

[0053] 1. Morphological characteristics of strain SQ3 of the present invention

[0054] 1.1 Colony morphology characteristics

[0055] Dextranase produced by strain SQ3, the colony is milky white, sticky, with jagged edges, a flat surface and a slightly raised center ( Figure 1 ), a Gram-positive bacillus ( Figure 2 ), electron microscopic morphology such as ( Figure 3 ).

[0056] 1.2 Molecular Biological Identification of Strain SQ3

[0057] Genes from strain SQ3 were extracted using a kit, and PCR amplification was performed using universal prokaryotic primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTACGACTT-3'). The PCR amplification system consisted of 25 μL of DNA template (1 μL, PCRMix 12.5 μL, 27F 1 μL, 1492R 1 μL, and 9.5 μL of ddH2O). The amplification conditions were as follows: denaturation at 94°C for 4 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 2 min, followed by a final extension at 72°C for 10 min, and storage at 4°C. The PCR product was sequenced at Shanghai Sangon Biotechnology Co., Ltd. Sequencing results were compared with the National Center of Biotechnology Information (NCBI) database, and a phylogenetic tree was constructed using the neighbor-joining method using MEGA 7.0 software. The 16S rDNA base length was 1444bp. BLAST analysis of the sequence on NCBI showed that the 16S rDNA of strain SQ3 was 98% homologous to that of Bacillus subtilis. Based on the morphological characteristics, strain SQ3 was identified as Bacillus sp. The phylogenetic tree was constructed using the NJ method in MEGA7.0 software. The results are shown in the figure. Figure 4 .

[0058] 2. Growth characteristics of strain SQ3 of the present invention

[0059] The growth characteristics of the strain SQ3 provided by the present invention are studied, and the growth conditions of the strain are obtained.

[0060] 2.1. Preparation of seed solution

[0061] The strain SQ3 was inoculated into 2216E medium at pH 8.0, 35°C, 180 rpm, and a liquid volume of 20%, and cultured for 12 h.

[0062] 2.2 Effect of time on the growth of strain SQ3

[0063] The seed liquid was inoculated into 2216E medium at a 2% inoculum, pH 8.0, 35°C, 180 r / min, and samples were taken every 2 hours to measure OD 600nm Strain SQ3 grew most actively from 0 to 18 hours and was in the logarithmic phase. After 18 hours, the growth rate slowed down and entered the stable growth phase ( Figure 5 ).

[0064] 2.3 Effect of temperature on the growth of strain SQ3

[0065] The seed solution was inoculated into 2216E medium at a pH of 8.0 at a 2% inoculum rate and cultured at 180 rpm for 12 h at different temperatures. Strain SQ3 can grow at temperatures between 20 and 45°C, with an optimum growth temperature of 40°C ( Figure 6 ).

[0066] 2.4 Effect of the initial pH value of the culture medium on the growth of strain SQ3

[0067] The seed solution was inoculated into 2216E medium with different pH values at a 2% inoculum and cultured at 40°C and 180 rpm for 12 h. The optimal pH range for strain SQ3 was 8-10, with the best growth at pH 9. Figure 7 ).

[0068] 2.5 Effect of NaCl concentration in the culture medium on the growth of strain SQ3

[0069] The seed solution was inoculated with 2% inoculum into 2216E medium with different NaCl concentrations, pH 9.0, 40℃, 180r / min for 12h. Strain SQ3 can grow at 0-8% NaCl concentration, and the best growth effect is achieved under the condition of no NaCl ( Figure 8 ).

[0070] 2.6 Effects of carbon and nitrogen sources on the growth of strain SQ3

[0071] Maltose, sucrose, lactose, dextrin, yeast, soluble starch, potato starch, cassava starch, and corn starch were used as carbon sources instead of yeast; the culture was carried out at 40°C and 180 r / min for 12 h, and the absorbance of the bacterial suspension (OD600 nm value) was measured. The optimal carbon source was selected, and fish meal peptone, ammonium sulfate, soy peptone, ammonium chloride, beef extract powder, and urea were used as nitrogen sources instead of fish meal peptone. The effect of nitrogen source on the growth of strain SQ3 was studied. Strain SQ3 grew well under various carbon source conditions, among which potato starch ( Figure 9 ), the best nitrogen source is soy peptone ( Figure 10 ).

[0072] 3. Dextranase production conditions of strain SQ3 of the present invention

[0073] 3.1 Effect of fermentation time on enzyme production of strain SQ3

[0074] The seed liquid was inoculated into the enzyme production medium at a 2% inoculum, pH 8.0, 35℃, 180r / min, and the enzyme activity was measured at intervals. The enzyme activity was the highest when the fermentation time was 60h ( Figure 11 ).

[0075] 3.2 Effect of fermentation temperature on enzyme production of strain SQ3

[0076] The seed liquid was inoculated into the enzyme production medium at a 2% inoculum, pH 8.0, and cultured at 20-40°C and 180 rpm for 60 h. The optimal enzyme production temperature of the strain is 30°C ( Figure 12 ).

[0077] 3.3 Effect of the initial pH value of the culture medium on enzyme production by strain SQ3

[0078] The seed solution was inoculated into enzyme production medium with different pH values at a 2% inoculum and cultured at 30°C and 180 rpm for 60 h. The optimal pH for enzyme production of the strain was 7.5 ( Figure 13 ).

[0079] 3.4 Effect of NaCl concentration in the culture medium on enzyme production by strain SQ3

[0080] The seed solution was inoculated into enzyme production medium with different NaCl concentrations at a 2% inoculum and cultured for 60 h at pH 7.5, 30°C, and 180 rpm. The optimal NaCl concentration for enzyme production was 0% ( Figure 14 ).

[0081] 3.5 Effects of carbon and nitrogen sources on enzyme production by strain SQ3

[0082] The basal culture medium is an enzyme production medium with a formula of 0.1% yeast, 0.5% fish meal peptone, 1% dextran T20, pH 9.0, and a liquid volume of 40%. Maltose, sucrose, lactose, dextrin, yeast, soluble starch, potato starch, cassava starch, and corn starch are used as carbon sources instead of yeast. The inoculation amount is 2%, the temperature is 30°C, and the r / min is 180. The enzyme activity is measured after sampling. The optimal carbon source is selected, and fish meal peptone, ammonium sulfate, soy peptone, ammonium chloride, beef extract powder, and urea are used as nitrogen sources to replace fish meal peptone. The effects of the above factors on the enzyme production of the strain are studied. Among them, the best carbon source for enzyme production of strain SQ3 is cassava starch ( Figure 15 ), the best nitrogen source is fish meal peptone ( Figure 16 ).

[0083] 3.6 Effects of different molecular weight inducers on enzyme production of strain SQ3

[0084] The same amount of dextran T40, T70, and T500 were used as inducers instead of T20, and the seed solution was inoculated into the enzyme production medium at a 2% inoculum, and cultured for 60 hours at pH 7.5, 30°C, and 180 r / min. The optimal inducer for the strain was dextran T20 ( Figure 17 ).

[0085] 3.7 Effect of Dextran T20 Concentration on Enzyme Production of Strain SQ3

[0086] The seed solution was inoculated with 2% inoculum into enzyme production medium with T20 concentration of 0-1.6%, and cultured at pH 7.5, 30℃, and 180r / min for 60h. The optimal dextran T20 concentration for enzyme production of the strain was 0.4% ( Figure 18 ).

[0087] 4. Enzymatic properties of dextranase produced by strain SQ3 of the present invention

[0088] 4.1. Enzyme activity determination

[0089] The enzyme activity was determined by the DNS method. 150 μL of substrate (75 μL Tris-HCl buffer 8.0 + 75 μL 6% dextran T20) and 50 μL of enzyme solution were placed in a water bath at 40°C for 20 min. 200 μL of DNS was added and the mixture was boiled in boiling water for 5 min. 3 mL of distilled water was added to the mixture and the OD was determined. 540 nm Under the same conditions, the absorbance of the sample was measured and the enzyme activity was calculated. In the control group, after adding the substrate, DNS was added first and then the enzyme solution. The rest of the operation was the same. The dextranase activity was calculated according to the following formula:

[0090]

[0091] (a is the slope of the DNS curve; b is the intercept of the curve)

[0092] 4.2 Effect of Temperature on Dextranase Activity and Enzyme Thermal Stability

[0093] The optimum temperature of the enzyme is 40°C, and it maintains high catalytic activity in the temperature range of 35-40°C, with a relative enzyme activity of not less than 80% ( Figure 19 After being kept at 30℃ for 5 hours, the enzyme activity lost nearly 60%, and after being kept at 40℃ for 5 hours, the enzyme activity lost nearly 80%. ( Figure 20 ).

[0094] 4.3 Effect of pH on dextranase activity and stability

[0095] The enzyme maintains a high activity level in the pH range of 6.0-9.0, and its optimum pH is 8.0 ( Figure 21 ), after being in a pH 6.0-9.0 buffer at 30℃ for 1 hour, the enzyme activity was still more than 60% ( Figure 22 ).

[0096] 4.4 Effects of Metal Ions on Enzyme Activity

[0097] The effects of metal ions on dextranase activity are shown in Table 1. 2+ Can increase enzyme activity, Cu2+ , Fe 3+ , Mn 2+ ,Co 2+ , Zn 2+ , K + , Na + , Mg 2+ , Ni 2+ Inhibit enzyme activity, 1mmol / L 5mmol / L of Ca 2+ It promotes enzyme activity.

[0098] Table 1: Effects of metal ions on dextranase activity

[0099]

[0100]

[0101] 4.5 Effects of organic solvents on enzyme activity

[0102] Hexane and Tween 20 have a certain promoting effect on the enzyme, while ethyl acetate and petroleum ether have little effect on it. The other organic solvents have a certain inhibitory effect on it.

[0103] Table 2: Effects of organic solvents on dextranase activity

[0104] organic solvents Relative enzyme activity (%) comparison 100.0±6.73 ethanol 70.48±0.45 Ethylene glycol 66.03±6.22 Methanol 75.51±5.50 Ethyl acetate 100.53±3.33 glycerin 70.77±8.97 Acetonitrile 59.33±3.31 n-Propanol 56.34±7.01 n-Butanol 29.66±8.80 n-hexane 115.72±1.75 Twain 20 115.50±7.04 Twain 80 58.09±7.90 Petroleum ether 102.82±7.97

[0105] 4.6 Enzyme Substrate Specificity

[0106] As shown in Table 3, the optimal substrate for this enzyme is dextran T20. This enzyme can specifically hydrolyze dextran of varying molecular weights composed of α-1,6 glycosidic bonds, and still has 10.98% catalytic activity against soluble starch composed of both α-1,4 and α-1,6 glycosidic bonds. It is unable to hydrolyze pullulan composed of both α-1,4 and α-1,6 glycosidic bonds, nor sucrose composed of β-1,2 glycosidic bonds.

[0107] Table 3: Substrate specificity of dextranase

[0108]

[0109]

[0110] 5. Application of dextranase from strain SQ3

[0111] 5.1 HPLC analysis of dextran T20 hydrolyzed by dextranase from strain SQ3

[0112] The enzyme solution and substrate were mixed in a ratio of 1:3, reacted at 35°C for 12 hours, boiled in boiling water for 5 minutes, centrifuged at 8000 rpm for 5 minutes, and the supernatant was filtered through a 0.22 μm filter membrane and stored at 4°C for subsequent HPLC analysis. The chromatographic column was Waters Sugar-PAK1 (6.5×300 mm), the mobile phase was deionized water, the flow rate was 0.4 mL / min, and the column temperature was 75°C ( Figure 23 ), and the proportion of oligosaccharides in the product was calculated based on the peak area.

[0113] The above embodiments merely illustrate the implementation methods of the present invention. Although the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that a person skilled in the art may make various modifications and improvements without departing from the scope of the present invention, and these modifications and improvements fall within the scope of protection of the present invention.

Claims

1. A marine bacterium, Bacillus sp. SQ3, characterized by: This strain was deposited in the General Microbiology Center of China Culture Collection of Microorganisms on January 20, 2025, with the deposit number: CGMCC No.33458. The depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. A method for producing dextranase by the marine bacterium Bacillus sp. SQ3 according to claim 1, characterized in that: The specific steps are as follows: Step 1: Store the strain SQ3 slant in a 4°C refrigerator; inoculate into growth medium at 10% liquid volume, incubate at 180 rpm and 40°C for 12 h to obtain a bacterial suspension; Step 2: The bacterial suspension was inoculated into the enzyme production medium with an inoculum volume of 2% and a liquid volume of 10%, and cultured at 180 rpm and 30°C for 60 h to obtain the fermentation liquid; Step 3: The fermentation broth was centrifuged at 8000 rpm at 4°C for 10 min. The supernatant was sterilized by filtration using a 0.22 μm filter membrane. The filtrate was centrifuged at 4500 rpm at 4°C for 10 min using a 30 kDa ultrafiltration tube. The portion with a molecular weight greater than 30 kDa was the enzyme solution.

3. The method for producing dextranase by the marine bacterium Bacillus sp. SQ3 according to claim 2, characterized in that: The growth medium in step 1 is prepared by: 1 g / L potato starch, 5 g / L soy peptone, and pure water, pH 9.0, and sterilized at high temperature of 121° C. for 20 min.

4. The method for producing dextranase by the marine bacterium Bacillus sp. SQ3 according to claim 3, characterized in that: The enzyme production culture medium in step 2 is: 1 g / L cassava starch, 5 g / L fish meal peptone, 4 g / L dextran T20, pH 7.5, and is sterilized at high temperature of 121° C. for 20 min.

5. Use of the dextranase produced by the method according to any one of claims 2 to 4, characterized in that: Dextran T20 was hydrolyzed with enzyme solution to produce isomaltooligosaccharide; The specific steps are as follows: the enzyme solution and the substrate are mixed in a ratio of 1:3, reacted at 35°C for 12 hours, boiled in boiling water for 5 minutes, centrifuged at 8000 rpm for 5 minutes, the supernatant is filtered with a 0.22 μm filter membrane, and the filtrate is dried to obtain isomaltooligosaccharide, wherein the content of oligosaccharides with a degree of polymerization greater than 4 is greater than 90%.

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