Bacillus sp.SQ8, method for producing dextranase by bacillus sp.SQ8 and application of bacillus sp.SQ8

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to bacillus sp. SQ8, a method for producing dextranase by using the bacillus sp. SQ8 and application of the bacillus sp. SQ8. The strain is strong in growth adaptability and high in enzyme production efficiency. The dextranase produced by the strain SQ8 can be used for preparing high-polymerization-degree isomaltooligosaccharides, in hydrolysate of the strain SQ8, the ratio of tetrasaccharide is 10.8%, the ratio of pentasaccharide is 19.7%, the ratio of hexasaccharide is 42.1%, the ratio of heptasaccharide is 22.7%, the ratio of high-polymerization-degree isomaltooligosaccharides is 95.3%, and the dextranase produced by the strain SQ8 can be used for preparing high-polymerization-degree isomaltooligosaccharides and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a Bacillus sp. SQ8 and a method and application of dextranase produced by the Bacillus sp. SQ8. BACKGROUND

[0002] The ocean has extremely rich microbial resources. Unlike terrestrial environments, microorganisms growing in marine environments usually have salt tolerance, alkali tolerance, and low-temperature tolerance. With the increasing attention to marine resources, more and more novel biological enzymes have been discovered. Due to the unique environment of the ocean, these enzymes have unique properties in structure and function, which broaden the sources and applications of biological enzyme preparations.

[0003] Dextran is a polysaccharide in which glucose is connected by alpha-1, 6 glycosidic bonds to form a main chain. Dextranase (EC 3.2.1.11) is a glycoside hydrolase that specifically hydrolyzes alpha-1, 6 glycosidic bonds in dextran. The enzymatic product is a low polysaccharide and an isomaltulose oligosaccharide. Dextranase has been applied in medicine, sugar production, food, health care and other fields. In the food industry, dextranase hydrolyzes high molecular dextran to prepare functional isomaltulose and produce prebiotics; dextranase and dextran with different degrees of hydrolysis are both food additives.

[0004] Oligosaccharides have been widely concerned and applied in nutrition, health care, disease prevention and treatment, etc. due to their effects of regulating blood sugar, regulating human immunity, reducing constipation, and enhancing intestinal probiotic flora. It is found that the effect of oligosaccharides is closely related to their degree of polymerization. High degree of polymerization (degree of polymerization greater than 4) oligosaccharides not only can promote the secretion of insulin, but also can significantly promote the growth of intestinal probiotics. In the existing isomaltulose oligosaccharide products, the main components are di- and tri-saccharides, and their effects are far lower than those of high degree of polymerization oligosaccharides. Therefore, increasing the proportion of high degree of polymerization oligosaccharides in oligosaccharides can improve the quality and biological activity of oligosaccharides.

[0005] The dextranase produced by the strain SQ8 of the present application can be used to prepare high degree of polymerization isomaltulose oligosaccharides. In the hydrolysis product, the proportion of tetrasaccharides is 10.8%, the proportion of pentasaccharides is 19.7%, the proportion of hexasaccharides is 42.1%, the proportion of heptasaccharides is 22.7%, and the proportion of high degree of polymerization isomaltulose oligosaccharides is 95.3%. Isomaltulose oligosaccharides have biological activity, can significantly promote the growth of probiotics, improve the balance of intestinal flora, enhance the body's immunity, have antioxidant, hypolipidemic and constipation prevention functions, and are widely used in the fields of medicine and functional food. The above results show that the strain and its dextranase have significant advantages in preparing high degree of polymerization oligosaccharides. SUMMARY

[0006] The present application aims at the defects of the prior art, and provides a Bacillus sp. SQ8 and a method and application of producing dextranase thereof, so as to solve the problems in the background art.

[0007] To achieve the above object, the present application provides the following technical scheme: a Bacillus sp. SQ8, which is preserved in the China General Microbiological Culture Collection Center on January 20, 2025, with a preservation number of CGMCC No. 33459 and a preservation unit address of No. 3, Institute of Microbiology, Chinese Academy of Sciences, 1 Xibeinanli, Chaoyang District, Beijing, China.

[0008] A method for expanding culture of the Bacillus sp. SQ8, comprising the following steps:

[0009] Inoculate the strain SQ8 slant seed into 2216E culture medium, with pH 8.0, 35℃, 180r / min, liquid loading amount of 20%, and culture for 12h to obtain a seed liquid;

[0010] Inoculate the seed liquid into a growth culture medium with an inoculation amount of 2%, pH 8.0, 35℃, 180r / min, and culture for 14h;

[0011] The growth culture medium is prepared by using 1g / L yeast, 5g / L beef extract powder, and pure water, with pH 8.0, high-temperature 121℃ sterilization for 20min.

[0012] A method for producing dextranase by the Bacillus sp. SQ8, comprising the following steps:

[0013] S1: inoculate the strain SQ8 into a growth culture medium, with liquid loading amount of 20%, 180r / min, 35℃, and culture for 12h to obtain a bacterial suspension;

[0014] S2: inoculate the bacterial suspension into an enzyme production culture medium with an inoculation amount of 2%, liquid loading amount of 40%, 180r / min, 30℃, and culture for 60h to obtain a fermentation liquid;

[0015] S3: centrifuge the fermentation liquid at 4℃, 8000r / min for 10min to obtain a supernatant as a crude enzyme liquid, filter the crude enzyme liquid by using a 0.22μm filter membrane to remove bacteria, and centrifuge the filtered liquid at 4℃, 4500r / min for 10min by using a 30kDa ultrafiltration tube to concentrate and purify, to obtain an enzyme liquid.

[0016] Preferably, the growth culture medium in step S1 is prepared by using 1g / L yeast, 5g / L beef extract powder, and pure water, with pH 8.0, high-temperature 121℃ sterilization for 20min.

[0017] Preferably, the enzyme-producing culture medium in step S2 is prepared by using 1 g / L of potato starch, 5 g / L of beef extract powder, 2 g / L of dextran T500, and pure water, and sterilized at 121 DEG C for 20 min at pH 9.0.

[0018] The application provides an application of the dextranase produced by the Bacillus sp. SQ8 in hydrolysis of dextran T500 to prepare isomaltulose.

[0019] A method for hydrolyzing dextran T500 by using the dextranase produced by the Bacillus sp. SQ8, comprising the following steps: mixing the enzyme solution and the substrate at a ratio of 1:3, reacting at 35 DEG C for 12 h, boiling in boiling water for 5 min, centrifuging at 8000 r / min for 5 min, filtering the supernatant by using a 0.22 mu m filter membrane, drying the filtrate, and obtaining isomaltulose as the product, wherein the content of the oligosaccharide with a polymerization degree greater than or equal to 4 is greater than 95%.

[0020] Compared with the prior art, the application has the following beneficial effects:

[0021] Screening of enzyme-producing microorganisms from marine samples is one of important ways for mining novel enzymes, and the application provides a novel Bacillus sp. SQ8 capable of producing dextranase, which enriches marine microbial strains capable of producing dextranase. The strain has the following characteristics: the growth temperature range is 15-40 DEG C, and the initial growth pH range is 5.0-10.0. The optimal growth temperature of the strain SQ8 is 35 DEG C, and the optimal initial growth pH is 8.0. The optimal action temperature of the dextranase produced by the strain SQ8 is 40 DEG C. The dextranase produced by the strain SQ8 of the application can be used to prepare high-polymerization isomaltulose, and in the hydrolysis product, the content of tetrasaccharide is 10.8%, the content of pentasaccharide is 19.7%, the content of hexasaccharide is 42.1%, the content of heptasaccharide is 22.7%, and the content of high-polymerization isomaltulose is 95.3%. The above results show that the dextranase produced by the strain can be used to prepare isomaltulose with high-polymerization oligosaccharide content. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 A transparent circle diagram formed on a screening plate of the strain SQ8 of the application;

[0023] Figure 2 A gram staining diagram of the strain SQ8 of the application;

[0024] Figure 3 A scanning electron microscope diagram of the strain SQ8 of the application;

[0025] Figure 4 A 16S rDNA system evolution tree diagram of the strain SQ8 of the application;

[0026] Figure 5Figure for the effect of time on the growth of strain SQ8 according to the present application;

[0027] Figure 6 Figure for the effect of temperature on the growth of strain SQ8 according to the present application;

[0028] Figure 7 Figure for the effect of initial pH of the medium on the growth of strain SQ8 according to the present application;

[0029] Figure 8 Figure for the effect of NaCl concentration of the medium on the growth of strain SQ8 according to the present application;

[0030] Figure 9 Figure for the effect of carbon source on the growth of strain SQ8 according to the present application;

[0031] Figure 10 Figure for the effect of nitrogen source on the growth of strain SQ8 according to the present application;

[0032] Figure 11 Figure for the effect of time on the enzyme production of strain SQ8 according to the present application;

[0033] Figure 12 Figure for the effect of temperature on the enzyme production of strain SQ8 according to the present application;

[0034] Figure 13 Figure for the effect of initial pH of the medium on the enzyme production of strain SQ8 according to the present application;

[0035] Figure 14 Figure for the effect of NaCl concentration of the medium on the enzyme production of strain SQ8 according to the present application;

[0036] Figure 15 Figure for the effect of carbon source on the enzyme production of strain SQ8 according to the present application;

[0037] Figure 16 Figure for the effect of nitrogen source on the enzyme production of strain SQ8 according to the present application;

[0038] Figure 17 Figure for the effect of different molecular weight inducers on the enzyme production of strain SQ8 according to the present application;

[0039] Figure 18 Figure for the effect of T500 concentration on the enzyme production of strain SQ8 according to the present application;

[0040] Figure 19 Figure for the effect of the temperature of action of the enzyme on the enzyme activity according to the present application;

[0041] Figure 20 Figure for the thermal stability of the enzyme according to the present application;

[0042] Figure 21 Figure for the effect of the pH of action of the enzyme on the enzyme activity according to the present application;

[0043] Figure 22 pH stability graph of the enzyme of the present application;

[0044] Figure 23 hydrolysis T50012 h high performance liquid chromatogram of the dextranase of the present application; DETAILED DESCRIPTION

[0045] The preferred embodiments of the present application are described in detail below with reference to the accompanying drawings, so that the advantages and features of the present application can be more easily understood by those skilled in the art, and the scope of protection of the present application can be more clearly defined.

[0046] A Bacillus sp. SQ8, which is preserved in the China General Microbiological Culture Collection Center on January 20, 2025, has a preservation number of CGMCC No. 33459 and a preservation unit address of No. 3, Yikuangli, Beichenxili, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0047] The colony of the bacteria is milky white, viscous, has an irregular edge, a smooth surface, and is a gram-positive bacillus; 16S rDNA of the strain SQ8 is analyzed, combined with morphological characteristics, the strain SQ8 is identified as Bacillus sp.; the optimal growth conditions of the strain SQ8 are as follows: yeast, 1 g / L; beef extract powder, 5 g / L; pH 8.0; and 35℃ culture.

[0048] A method for producing dextranase by Bacillus sp. SQ8, the specific steps are as follows:

[0049] S1: inoculate the strain SQ8 into a growth culture medium, with a liquid loading amount of 20%, 180 r / min, 35℃ culture for 12 h, to obtain a bacterial suspension;

[0050] S2: inoculate the bacterial suspension into an enzyme production culture medium with an inoculation amount of 2%, a liquid loading amount of 40%, 180 r / min, 30℃ culture for 60 h to obtain a fermentation broth;

[0051] S3: centrifuge the fermentation broth at 4℃, 8000 r / min for 10 min to take the supernatant as a crude enzyme liquid, filter the crude enzyme liquid using a 0.22 μm filter membrane to remove bacteria, and centrifuge the filtered liquid at 4℃, 4500 r / min for 10 min using a 30 kDa ultrafiltration tube to concentrate and purify, to obtain an enzyme liquid.

[0052] Application of the dextranase produced by Bacillus sp. SQ8, the dextranase hydrolyzes dextran T500, and the product is analyzed by high performance liquid chromatography.

[0053] The specific steps are as follows:

[0054] Mix the enzyme solution with the substrate 1:3, react for 12h at 35℃, boil in boiling water for 5min, centrifuge at 8000r / min for 5min, take the supernatant, filter with a 0.22μm filter membrane, and store at 4℃. Prepare for subsequent high-performance liquid analysis. The chromatographic column is a waters sugar-pak1 (6.5*300mm), the column temperature is 75℃, the mobile phase is deionized water, and the flow rate is 0.4mL / min.

[0055] The identification method of the Bacillus sp. SQ8 is as follows: morphological observation is performed on the strain, the strain is divided into three zones on a plate, the color, size, shape and size of the transparent circle of the colony are observed, and photographs and records are taken; an appropriate amount of bacterial liquid is spread on a glass slide, fixed, and then subjected to primary staining, medium staining, decolorization and restaining with Gram staining solution, and observed under a microscope. Then, 16S rDNA sequence analysis and molecular biology identification are performed, the gene of the strain is extracted by using a kit, and universal 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTACGACTT-3') are selected for PCR amplification. The PCR amplification system is 25μL (1μL of DNA template, 12.5μL of PCR Mix, 1μL of 27F, 1μL of 1492R, 9.5μL of dd H2O), and the amplification conditions are as follows: denaturation at 94℃ for 4min; denaturation at 94℃ for 30s, annealing at 55℃ for 30s, extension at 72℃ for 2min, 35 cycles; final extension at 72℃ for 10min, and preservation at 4℃. The PCR product is sent to Shanghai Shengong Biotechnology for sequencing. The sequencing result is submitted to the national center of biotechnology information (NCBI) database for comparison, and a phylogenetic tree is constructed by using MEGA7.0 software through the neighbor-joining method.

[0056] The features of the present application include that the marine strain Bacillus sp. SQ8 is a Bacillus isolated from a marine sample (36°05'N, 120°28'E) in the sea area of Qingdao, Shandong, China by using the transparent circle method, and the strain was preserved in the China General Microbiological Culture Collection Center on January 20, 2025, with the preservation number: CGMCC No. 33459, and the address of the preservation unit: No. 3, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The present application provides strain culture and the use of the strain to produce dextranase, and a method for preparing high-polymerization-degree isomalto-oligosaccharide by hydrolyzing dextran T500 with the enzyme.

[0057] 1. Morphological characteristics of the strain SQ8 of the present application;

[0058] 1.1. Colony morphological characteristics:

[0059] The dextranase activity produced by the strain SQ8 is the highest, the colony of the strain is milky white, viscous, the edge is rodent, the surface is flat and the center is slightly convex, Figure 1 ), it is a gram-positive bacillus, Figure 2 ), and the electron microscope morphology is as shown in Figure 3 ).

[0060] 1.2, molecular biology identification of the strain SQ8;

[0061] Molecular biology identification, the kit extracts the gene of the strain, the universal primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTACGACTT-3') of the prokaryotic microorganism are selected to carry out PCR amplification. The PCR amplification system is 25 uL (DNA template 1 uL, PCR Mix 12.5 uL, 27F 1 uL, 1492R 1 uL, dd H2O 9.5 uL), and the amplification condition is as follows: 94 DEG C denaturation for 4 min; 94 DEG C denaturation for 30 s, 55 DEG C annealing for 30 s, 72 DEG C extension for 2 min, 35 cycles; 72 DEG C terminal extension for 10 min, 4 DEG C preservation. The PCR product is sent to Shanghai Shenguo Biological Sequencing. The sequencing result is submitted to the national center of biotechnology information (NCBI) database comparison, the phylogenetic tree is constructed by the neighbor-joining method (neighbor-joining) through MEGA7.0 software. Through detection, the 16S rDNA base length is 1458 bp. The sequence is analyzed by BLAST on NCBI, the homology of the 16S rDNA of the strain SQ8 and Bacillus subtilis is 100%, the strain SQ8 is identified as Bacillus sp. according to the comprehensive morphological characteristics, the NJ method in MEGA7.0 software is used to construct a phylogenetic tree, and the result is shown in Figure 4 .

[0062] 2, the growth characteristics of the strain SQ8 of the application;

[0063] The strain SQ8 provided by the application is studied, and the growth conditions of the strain are obtained.

[0064] 2.1, preparation of seed liquid

[0065] The strain SQ8 slope seed is inoculated into 2216E medium, pH 8.0, 35 DEG C, 180 r / min, liquid volume 20%, and cultured for 12 h.

[0066] 2.2, effect of time on growth of the strain SQ8;

[0067] The seed liquid was inoculated in 2216E medium at 2% inoculation amount, pH 8.0, and cultured at 35℃, 180r / min. The OD value was measured every 2h. 600nm The strain SQ8 grew most actively at 0-14h, in the logarithmic phase, and the growth rate slowed down after 14h, entering the stationary phase. Figure 5

[0068] 2.3, Effect of temperature on the growth of strain SQ8;

[0069] The seed liquid was inoculated in 2216E medium at 2% inoculation amount, pH 8.0, and cultured at different temperatures at 180r / min for 12h. The strain SQ8 could grow at 15-40℃, and the optimum growth temperature was 35℃. Figure 6

[0070] 2.4, Effect of initial pH value of the medium on the growth of strain SQ8;

[0071] The seed liquid was inoculated in 2216E medium at different pH values at 2% inoculation amount, and cultured at 35℃, 180r / min for 12h. The optimum pH range of the strain SQ8 was 7-9, and the growth was best at pH 8. Figure 7

[0072] 2.5, Effect of NaCl concentration of the medium on the growth of strain SQ8;

[0073] The seed liquid was inoculated in 2216E medium with different NaCl concentrations at 2% inoculation amount, pH 8.0, and cultured at 35℃, 180r / min for 12h. The strain SQ8 could grow at 0-8% NaCl concentration, and the growth effect was best without NaCl, which was beneficial to pollution control in industrial fermentation. Figure 8

[0074] 2.6, Effect of carbon and nitrogen sources on the growth of strain SQ8;

[0075] Maltose, sucrose, lactose, dextrin, yeast, soluble starch, potato starch, cassava starch, and corn starch were used to replace yeast as carbon sources, and fish meal peptone, ammonium sulfate, soybean peptone, ammonium chloride, beef infusion powder, and urea were used to replace fish meal peptone as nitrogen sources. The absorbance value (OD600 nm value) of the bacterial suspension was measured after 12h of culture at 35℃, 180r / min, and the optimum carbon and nitrogen sources were selected. The effects of the above factors on the growth of strain SQ8 were studied. The strain SQ8 had good adaptability to most carbon sources, and the best carbon source was yeast, and the best nitrogen source was beef infusion powder. Figure 9 Figure 10 ​​​​​​

[0076] 3. The strain SQ8 dextranase production conditions optimization;

[0077] 3.1, fermentation time on the strain SQ8 enzyme production effect;

[0078] The seed liquid is inoculated in the enzyme production medium with 2% inoculation amount, pH 8.0, 35℃, 180r / min, and the enzyme activity is determined every interval time. When the fermentation time is 60h, the enzyme activity is the highest Figure 11 )。

[0079] 3.2, fermentation temperature on the strain SQ8 enzyme production effect;

[0080] The seed liquid is inoculated in the enzyme production medium with 2% inoculation amount, pH 8.0, 20-40℃, 180r / min for 60h. The optimum enzyme production temperature of the strain is 30℃ Figure 12 )。

[0081] 3.3, initial pH value of the culture medium on the strain SQ8 enzyme production effect;

[0082] The seed liquid is inoculated in the enzyme production medium with 2% inoculation amount, 30℃, 180r / min for 60h. The optimum enzyme production pH of the strain is 9.0 Figure 13 )。

[0083] 3.4, NaCl concentration of the culture medium on the strain SQ8 enzyme production effect;

[0084] The seed liquid is inoculated in the enzyme production medium with 2% inoculation amount, pH 9.0, 30℃, 180r / min for 60h. The optimum NaCl concentration of the strain enzyme production is 0% Figure 14 )。

[0085] 3.5, carbon and nitrogen source on the strain SQ8 enzyme production effect;

[0086] The basic medium is the enzyme production medium, the formula is 0.1% yeast, 0.5% fish powder peptone, 1% T20, pH 9.0, liquid volume 40%, respectively, maltose, sucrose, lactose, dextrin, yeast, soluble starch, potato starch, cassava starch, corn starch instead of yeast as carbon source in the original medium, 2% inoculation amount, 30℃, 180r / min, culture for 60h, sample determination of enzyme activity, select the optimum carbon source. Again, fish powder peptone, ammonium sulfate, soybean peptone, ammonium chloride, beef infusion powder, urea instead of fish powder peptone as nitrogen source in the original medium, to study the above factors on the strain enzyme production. Among them, the best carbon source for the strain SQ8 enzyme production is potato starch Figure 15 ), the best nitrogen source is beef infusion powder Figure 16 ).

[0087] 3.6. Effects of different molecular weight inducers on enzyme production of strain SQ8;

[0088] 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 9.0, 30°C, and 180 r / min. The optimal inducer for the strain was dextran T500 ( Figure 17 ).

[0089] 3.7. Effect of dextran T500 concentration on enzyme production of strain SQ8;

[0090] The seed solution was inoculated with 2% inoculum into enzyme production medium with T500 concentration of 0-1.6%, and cultured at pH 9.0, 30℃, and 180r / min for 60h. The optimal T500 concentration for enzyme production of the strain was 0.2% ( Figure 18 ).

[0091] 4. Study on the enzymatic properties of dextranase produced by strain SQ8 of the present invention;

[0092] 4.1. Enzyme activity determination;

[0093] The enzyme activity was determined by the DNS method. 150 μL of substrate (75 μL of Tris-HCl buffer 7.5 + 75 μL of 6% dextran T500) 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 value was 0. 540nm 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 other operations remained unchanged. The dextranase activity was calculated according to the following formula:

[0094]

[0095] (a is the slope of the DNS curve; b is the intercept of the curve)

[0096] 4.2 Effect of temperature on dextranase activity and thermal stability;

[0097] Depend on Figure 19 It can be seen that the optimal temperature for the enzyme is 40°C. It maintains high catalytic activity in the temperature range of 30-45°C, and its relative enzyme activity is not less than 80%. After being kept at 30°C for 5 hours, the residual activity level remains at almost 100%. After being kept at 40°C for 5 hours, it still retains more than 80% of the enzyme activity. Figure 20 ).

[0098] 4.3 Effect of pH on dextranase activity and stability;

[0099] The enzyme maintains a high activity level in the pH range of 5.0-9.0, and its optimum pH is 7.5 ( Figure 21 ),Depend on Figure 21 It can be seen that after 1 hour in a pH 5.0-9.0 buffer at 30℃, the enzyme activity still remains above 60% ( Figure 22 ).

[0100] 4.4 Effects of metal ions on enzyme activity;

[0101] The effects of metal ions on dextranase activity are shown in Table 1. As shown in Table 1, Ca 2+ 、Ba 2+ Can increase enzyme activity, Cu 2+ , Fe 3+ , Mn 2+ ,Co 2+ , Zn 2+ , K + , Na + , Mg 2+ , Ni 2+ Inhibit enzyme activity, 1mmol / L Mn 2+ It has little effect on enzyme activity.

[0102] Table 1: Effects of metal ions on dextranase activity

[0103]

[0104]

[0105] 4.5. Effects of organic solvents on enzyme activity;

[0106] The organic solvents used all had varying degrees of inhibitory effect on the enzyme activity. Ethyl acetate, glycerol, Tween 20, and petroleum ether had weaker inhibitory effects on enzyme activity, reducing enzyme activity to about 90%, while n-butanol had the strongest inhibitory effect on enzyme activity, only about 33%.

[0107] Table 2: Effects of organic solvents on dextranase activity

[0108]

[0109] 4.6. Enzyme substrate specificity;

[0110] As shown in Table 3, the optimal substrate of the enzyme is dextran T500. The enzyme can specifically hydrolyze dextran of different molecular weights composed of α-1, 6 glycosidic bonds, and still has 7.05% catalytic activity on soluble starch composed of α-1, 4 and α-1, 6 glycosidic bonds, and has no catalytic activity on pullulan composed of α-1, 4 and α-1, 6 glycosidic bonds and sucrose composed of β-1, 2 glycosidic bonds.

[0111] Table 3: Substrate specificity of dextranase

[0112]

[0113] 5. Application of the dextranase of strain SQ8;

[0114] 5.1 High performance liquid chromatography analysis of the product of dextranase of strain SQ8 hydrolyzing dextran T500

[0115] The enzyme solution was mixed with the substrate at a ratio of 3:1, and reacted at 35°C for 12h, boiled in boiling water for 5min, centrifuged at 8000r / min for 5min, and the supernatant was filtered with a 0.22μm filter membrane, and stored at 4°C. It was prepared for subsequent high performance liquid chromatography analysis. The chromatographic column was waters sugar-pak1 (6.5x300mm), the column temperature was 75°C, the mobile phase was deionized water, and the flow rate was 0.4mL / min. Figure 23 ).

[0116] The above examples only express the embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as limiting the scope of the patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of protection of the present application.

Claims

1. A Bacillus sp. SQ8, which was deposited at the General Microbiology Center of the China Culture Collection Administration on January 20, 2025, with the deposit number: CGMCC No. 33459. The depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The method for expanding the culture of Bacillus sp. SQ8 according to claim 1, comprising the following steps: Strain SQ8 slant seeds were inoculated into 2216E medium at pH 8.0, 35°C, 180 rpm, and 20% liquid volume for 12 h to obtain seed solution. The seed solution was inoculated into the growth medium at a 2% inoculum size, pH 8.0, 35°C, and 180 rpm for 14 h; The growth medium is prepared by using 1g / L yeast, 5g / L beef extract powder, and pure water, with a pH of 8.0, and is sterilized at a high temperature of 121°C for 20 minutes.

3. The method for producing dextranase by Bacillus sp. SQ8 according to claim 1, comprising the following steps: S1: Inoculate strain SQ8 into growth medium with a liquid volume of 20%, culture at 180 rpm and 35°C for 12 h to obtain a bacterial suspension; S2: The bacterial suspension was inoculated into the enzyme production medium with an inoculum volume of 2% and a liquid volume of 40%, and cultured at 180 rpm and 30°C for 60 h to obtain the fermentation liquid; S3: The fermentation broth was centrifuged at 4°C and 8000 rpm for 10 min to obtain the supernatant as the crude enzyme solution. The crude enzyme solution was sterilized by filtration using a 0.22 μm filter membrane. The filtrate was concentrated and purified by centrifugation using a 30 kDa ultrafiltration tube at 4°C and 4500 rpm for 10 min to obtain the enzyme solution.

4. The method for producing dextranase according to claim 3, wherein In step S1, the growth medium is prepared with 1 g / L yeast, 5 g / L beef extract powder, and pure water, with a pH of 8.0, and is sterilized at a high temperature of 121° C. for 20 min.

5. The method for producing dextranase according to claim 3, wherein In step S2, the enzyme production culture medium is prepared with 1 g / L potato starch, 5 g / L beef extract powder, 2 g / L dextran T500, and pure water, with a pH of 9.0 and sterilized at a high temperature of 121° C. for 20 min.

6. Use of the dextranase produced by Bacillus sp. SQ8 according to claim 1 in hydrolyzing dextran T500 to prepare isomalto-oligosaccharides.

7. A method for hydrolyzing dextran T500 using the dextranase produced by Bacillus sp. SQ8 according to claim 1, comprising the following steps: mixing the enzyme solution and the substrate in a ratio of 1:3, reacting at 35°C for 12 hours, boiling in boiling water for 5 minutes, centrifuging at 8000 rpm for 5 minutes, filtering the supernatant through a 0.22 μm filter membrane, and drying the filtrate. The product is isomaltooligosaccharide, wherein the content of oligosaccharides with a degree of polymerization greater than or equal to 4 is greater than 95%.

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