Recombinant Bacillus subtilis BLCY-013 and its application in preparing mannose from glucose

Abstract

The present invention provides recombinant Bacillus subtilis BLCY-013 and its application in preparing mannose with glucose as raw material, belonging to the field of biotechnology. The recombinant Bacillus subtilis BLCY-013 of the present invention was stored in the General Microbiological Center of China Microbiological Culture Collection Administration Committee on November 25, 2024, with an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the strain collection number is CGMCC No. 32760; the present invention uses glucose as a raw material, and prepares mannose by recombinant Bacillus subtilis enzyme reaction, does not use chemical reagents, has strong reaction specificity, and is significantly reduced in raw material cost compared with the traditional method of preparing with fructose as a substrate; the unreacted glucose in the preparation process is recycled, and the raw material utilization rate is high; evaporation crystallization is adopted, and the prepared mannose is mannose without crystal water by controlling the crystallization parameters, which has good fluidity, weak moisture absorption, and is not easy to agglomerate.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to recombinant Bacillus subtilis BLCY-013 and application thereof in preparing mannose using glucose as a raw material. Background Art

[0002] Mannose is a hexose sugar and an isomer of glucose. Mannose has the functions of regulating immunity, resisting bacterial and viral infection, promoting glycoprotein synthesis in the body, and anti-cancer. Therefore, mannose is widely used in biology and medicine. Mannose can not only be used as a raw material for synthesizing certain pharmaceutical chemical raw materials and important carbohydrate drug precursors, but also can be used as a sweetener for food and beverages. In recent years, the demand for mannose in functional foods, feed additives and pharmaceutical fields has increased significantly, and it has huge economic development value.

[0003] At present, the bioconversion method has great application potential. This method uses isomerase catalysis to realize the conversion of monosaccharides to mannose. For example, using glucose as a substrate, cellobiose 2-epimerase is used to catalyze the conversion of glucose into mannose, with a conversion rate of 15%. Using D-fructose as a substrate, Liu Zuyi and others from the School of Food Science and Engineering of Jiangnan University reported in the Journal of Bioengineering that the recombinant Bacillus subtilis whole cell efficiently catalyzed the synthesis of D-mannose with D-fructose as a substrate. The conversion rate of whole cell catalyzed efficient synthesis of D-mannose reached 27.75%, which is the highest yield of D-mannose production reported so far by food-grade strains. Japanese patent JP01 / 06384 discloses a method of using high-purity fructose liquid as a raw material, using isomerase for epimerization, obtaining a mixed solution of mannose and fructose, and then purifying and crystallizing in water to obtain 96% crystalline mannose. In this preparation process, due to the difficulty in separating mannose and fructose, it is difficult to achieve a purity of crystalline D-mannose above 99% under the condition of a yield of more than 20%. Moreover, in industrial production, more than 85% of high fructose syrup is produced from glucose isomerization, resulting in the purchase cost of fructose being higher than that of glucose. Therefore, the preparation process of this patent is not only costly but also difficult to separate the finished product, resulting in low yield and purity of the product, and is not suitable for industrial production.

[0004] Therefore, there is an urgent need for a strain and method for directly preparing mannose using glucose as a substrate and achieving a higher conversion rate. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a recombinant Bacillus subtilis BLCY-013 and its application in preparing mannose using glucose as a raw material. The present invention uses glucose as a raw material and prepares mannose through a biological enzyme reaction. Compared with the traditional chemical method, no chemical reagents are used, and the reaction specificity is strong. Compared with the traditional preparation method using fructose as a substrate, the raw material cost is significantly reduced; the enzyme preparation obtained by fermentation can use glucose as a substrate, with a conversion rate of 25-29%, which greatly reduces the production cost.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a recombinant Bacillus subtilis ( Bacillus subtilis ) BLCY-013, characterized in that the recombinant Bacillus subtilis BLCY-013 was preserved on November 25, 2024 at the General Microbiological Center of China Culture Collection Administration, with the address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the culture collection number is CGMCC No.32760.

[0008] Recombinant Bacillus subtilis ( Bacillus subtilis BLCY-013, the strain is from the National Engineering Research Center for Grain Fermentation and Food Biomanufacturing of Jiangnan University, and was obtained through genetic modification. Its gene source is Streptomyces ( Streptomyces aerocolorigenes ).

[0009] In a second aspect, the present invention provides the use of the above-mentioned recombinant Bacillus subtilis in preparing mannose using glucose as raw material.

[0010] The invention produces mannose-2-epimerase by fermentation of recombinant Bacillus subtilis, and mannose-2-epimerase can convert glucose into mannose, and then obtains anhydrous mannose product through the steps of refining, purification, crystallization, drying and the like.

[0011] In a third aspect, the present invention provides a method for preparing mannose using glucose as a raw material, comprising the following steps:

[0012] S1. The above recombinant Bacillus subtilis ( Bacillus subtilis ) BLCY-013 is sequentially subjected to primary seed culture, secondary seed culture, and fermentation culture to obtain a crude solution of mannose 2-epimerase;

[0013] S2. adding mannose 2-epimerase crude solution to the glucose solution to prepare mannose crude solution through conversion;

[0014] S3. The crude mannose liquid is sequentially subjected to first refining, chromatographic separation, second refining, evaporative crystallization, centrifugation, and drying to obtain mannose.

[0015] The present invention uses the above-mentioned recombinant Bacillus subtilis ( Bacillus subtilis ) BLCY-013 was subjected to primary seed culture, secondary seed culture and fermentation culture in sequence to obtain a crude solution of mannose 2-isomerase.

[0016] The specific method of primary seed cultivation in S1 of the present invention is:

[0017] The recombinant Bacillus subtilis was streaked on an LB plate and incubated upside down at 33-37°C for 18-23 h;

[0018] Then, a single colony was picked and inoculated into an LB shake flask, and then cultured at 170-220 rpm and 33-37°C for 8-12 h to obtain a primary seed solution;

[0019] The liquid volume of the LB shake flask is 1 / 10 to 1 / 5 of the container volume;

[0020] The specific method of secondary seed cultivation is:

[0021] The first-level seed solution was inoculated into a seed tank at an inoculation rate of 0.5% to 1%, and then cultured at a speed of 150 to 250 rpm and 33 to 37°C. 600 When the temperature reaches 8-12, stop the culture and obtain the secondary seed solution;

[0022] The specific method of fermentation culture is:

[0023] The secondary seed liquid is inoculated into the fermentation medium at an inoculation rate of 5-15%, and then fermented at 35-37° C., pH 6.8-7.2, and a dissolved oxygen rate of 25-35%;

[0024] When dissolved oxygen rebound occurs during fermentation, feed addition is started to control the glucose concentration to 5-8 g / L.

[0025] During the fermentation process, the OD 600 When the temperature reaches 45-55, lower the temperature to 25-28°C and add ITPG at a final concentration of 0.2-0.4 mM for induction;

[0026] When the fermentation liquid OD 600 When the count reached 100-120, ITPG was added for the second time with a final concentration of 0.2-0.4 mM for induction;

[0027] When the fermentation liquid OD 600 When the concentration reaches 170-190, the fermentation culture is stopped to obtain the crude mannose 2-isomerase solution.

[0028] Furthermore, the fermentation medium is: 2.5-4 g / L corn steep liquor powder, 6-10 g / L glucose, 10.5-13.5 g / L potassium dihydrogen phosphate, 1.0-1.5 g / L magnesium sulfate heptahydrate, 3.0-5.0 g / L diammonium hydrogen phosphate, 1.2-1.8 g / L citric acid monohydrate, and the balance water, pH 7.0-7.2.

[0029] The supplementary feed includes: glucose 400~500g / L, corn dry pulp powder 8~10g / L, and magnesium sulfate heptahydrate 16~20g / L.

[0030] The invention adds mannose 2-epimerase crude liquid into glucose solution, and prepares mannose crude liquid through conversion.

[0031] In the present invention, the parameters in S2 are as follows:

[0032] The concentration of glucose solution is 550~600g / L;

[0033] The amount of enzyme added is 0.7~1.0g / L;

[0034] The conversion reaction temperature is 40~45℃;

[0035] The conversion reaction pH is 6.0~7.0;

[0036] The conversion reaction time is 22~26h;

[0037] The purity of the obtained mannose crude liquid is 25-29%.

[0038] The invention sequentially performs first refining, chromatographic separation, second refining, evaporation crystallization, centrifugation and drying on the crude mannose liquid to obtain mannose.

[0039] In the present invention, the first refined liquid is separated by continuous simulated mobile chromatography to obtain mannose liquid and glucose liquid, and the glucose liquid is returned to the first refining step for recycling; the continuous simulated mobile chromatography separation material-water ratio is 1:3.5~1:4.0, and the processing capacity is 0.025~0.028T.DS / h / m 3 Resin, yield 80~83%;

[0040] The purity of the mannose solution obtained by chromatographic separation is above 98%.

[0041] In the present invention, the specific method of the second refining in S3 is:

[0042] The mannose solution is sequentially subjected to decolorization, ion exchange, and concentration to obtain a second refined solution;

[0043] The second refined liquid has a solid content of 75-78%.

[0044] In the present invention, the decolorization process is: decolorization is performed using a granular activated carbon column, wherein the material temperature is 55-65°C, and then filtered through an Ama filter, wherein the material temperature is 50-60°C; the working pressure of the Ama filter is 0.2-0.4Mpa;

[0045] The ion exchange process is as follows: the decolorized material is sequentially passed through a cation exchange column, an anion exchange column, a cation exchange column, an anion exchange column, and a cation exchange column; wherein the material temperature is 35-45° C., and the flow rate is 2-4 BV / h;

[0046] Furthermore, the separated glucose solution is fed back to the first refining step for recycling.

[0047] The concentration process is: using a six-effect evaporator to concentrate the material after ion exchange, wherein the concentration temperature is 65~75°C and the pressure is -0.06~-0.1Mpa.

[0048] In the present invention, the specific method of the evaporative crystallization is:

[0049] The second refined liquid was concentrated to increase the fixed matter content to 78-81%, the temperature was increased to 55-60°C, 0.1-0.5‰ of dry basis weight of 180-250 mesh mannose was added as seed crystals, and after 60-120 minutes, continuous feeding was started. During the feeding process, the temperature was controlled at 57-60°C, the fixed matter content was 79-81%, and the crystallization liquid was obtained after 16-20 hours;

[0050] The feeding liquid used in the feeding is a mannose solution with a solid content of 45%-50%.

[0051] The preparation method of the feed solution is the same as that of the second refined solution, except that the solid content is controlled to be 45-50%.

[0052] In the present invention, the specific method of centrifugation and drying is: centrifuging and drying the crystallization liquid to obtain mannose crystals.

[0053] Furthermore, centrifugation is performed using a vertical scraper centrifuge with a rotation speed of 600-900 rpm, a centrifugation time of 30-40 min, and a washing water volume of 1-5% of the volume of the feed liquid.

[0054] Furthermore, the drying is carried out by fluidized bed drying, with an air inlet temperature of 58-65°C, a cooling temperature of 25-30°C, and a drying time of 20-30 minutes.

[0055] In the present invention, the purity of mannose crystals is above 99.5%, and the water content is less than 0.5%.

[0056] Beneficial technical effects:

[0057] 1. The present invention uses glucose as a raw material and prepares mannose through a biological enzyme reaction. Compared with the traditional chemical method, no chemical reagents are used, the reaction specificity is strong, and compared with the traditional preparation method using fructose as a substrate, the raw material cost is significantly reduced.

[0058] 2. The unreacted glucose in the preparation process of the present invention is recycled, has no by-products, and has a high raw material utilization rate.

[0059] 3. The present invention adopts evaporation crystallization, and by controlling the crystallization parameters, the prepared mannose is mannose without crystal water, which has good fluidity, weak moisture absorption and is not easy to agglomerate.

[0060] 4. The enzyme preparation obtained by fermentation in the present application can use glucose as a substrate to prepare mannose, with a conversion rate of 25-29%, greatly reducing production costs.

[0061] Description of biological deposit:

[0062] The recombinant Bacillus subtilis ( Bacillus subtilis ) BLCY-013 was deposited on November 25, 2024 at the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the culture collection number is CGMCC No.32760. DETAILED DESCRIPTION

[0063] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0064] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0065] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0066] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0067] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0068] The "room temperature" and "normal temperature" mentioned in the present invention are all calculated as 25±2°C unless otherwise specified.

[0069] Recombinant Bacillus subtilis ( Bacillus subtilis BLCY-013, the strain is from the National Engineering Research Center for Grain Fermentation and Food Biomanufacturing of Jiangnan University, and was obtained through genetic modification. Its gene source is Streptomyces ( Streptomyces aerocolorigenes ).

[0070] Unless otherwise specified, the equipment or raw materials used in the following examples can be commercially available.

[0071] Example 1

[0072] 1. Preparation of mannose 2-epimerase crude solution:

[0073] (1) Streak the recombinant Bacillus subtilis on an LB plate and incubate at 33°C for 19 h.

[0074] (2) Primary seed culture: Pick a single colony from the culture in step (1) and inoculate it into a 500 mL LB shake flask, where the liquid culture medium is 1 / 10 of the container volume, and culture at 170 rpm for 8 h at a temperature of 33°C;

[0075] (3) Secondary seed culture: After the primary seed solution in step (2) is bottled, it is inoculated into a 1000L seed tank for culture, wherein the inoculation amount is 0.5%, the culture temperature is 33°C, and the rotation speed is 150 rpm. When the OD of the seed solution reaches 8, the culture is stopped;

[0076] (4) Fermentation culture: The fermentation broth described in step (3) was inoculated into the fermentation medium, with an inoculum of 5%, cultured at 35°C, pH 6.9, and a dissolved oxygen rate of 25. When dissolved oxygen rebounded, feed addition was started, and the glucose concentration was controlled at 5 g / L. When the OD of the fermentation broth reached 5 g / L, the fermentation medium was inoculated with 5% of the inoculum and cultured at 35°C, pH 6.9, and a dissolved oxygen rate of 25. 600 When the temperature reached 45, the temperature was lowered to 25°C, and ITPG was added to induce the fermentation broth at a final concentration of 0.2 mM. 600 When the OD value of the fermentation liquid reached 100, ITPG was added for the second time with a final concentration of 0.2 mM. 600When the temperature reaches 170°C, the fermentation culture is stopped to obtain the crude mannose 2-epimerase solution.

[0077] The fermentation medium is as follows: 2.5 g / L corn steep liquor powder, 6 g / L glucose, 10.5 g / L potassium dihydrogen phosphate, 1.0 g / L magnesium sulfate heptahydrate, 3.0 g / L diammonium hydrogen phosphate, 1.2 g / L citric acid monohydrate, and the balance is water, pH 7.0.

[0078] The supplementary feed includes: 400 g / L glucose, 8 g / L corn pulp powder, and 16 g / L magnesium sulfate heptahydrate.

[0079] 2. Enzyme conversion reaction: Add mannose 2-isomerase crude solution to glucose solution to convert and prepare mannose crude solution.

[0080] The concentration of glucose solution was 600 g / L, the amount of enzyme added was 0.7 g / L, the reaction temperature was 45°C, the pH was 6.0, and the reaction time was 23 h. After the reaction, the purity of mannose was 25.8%.

[0081] 3. First refining: The crude mannose solution described in step 2 is subjected to first refining by decolorization, separation and concentration.

[0082] The decolorization is carried out by using granular activated carbon column, the material temperature is 55°C, and then it is filtered by Ama filter, the material temperature is 50°C, and the working pressure of Ama filter is 0.2Mpa.

[0083] The ion exchange process is to pass the decolorized material through a cation exchange column, anion exchange column, cation exchange column, anion exchange column, and cation exchange column in sequence, with a material temperature of 35° C. and a flow rate of 2BV / h.

[0084] The concentration process uses a six-effect evaporator to concentrate the material after separation, the concentration temperature is 65°C, the pressure is -0.06Mpa, and the solid content of the material is increased to 55% in one concentration to obtain the first refined liquid.

[0085] 4. Chromatographic separation: The first refined liquid in step 3 is separated by continuous simulated mobile chromatography to obtain mannose solution and glucose solution, and the glucose solution is returned to the first refining step for recycling.

[0086] The continuous simulated mobile chromatography separation material-water ratio is 1:3.5, and the processing capacity is 0.025T.DS / h / m 3 Resin, yield 82%. Obtained high-purity mannose solution with a purity of 98.4%.

[0087] 5. Second refining: The high-purity mannose solution described in step 4 is subjected to second refining by decolorization, separation and concentration.

[0088] The decolorization is carried out by using granular activated carbon column, the material temperature is 55°C, and then it is filtered by Ama filter, the material temperature is 50°C, and the working pressure of Ama filter is 0.2Mpa.

[0089] The ion exchange process is to pass the decolorized material through a cation exchange column, anion exchange column, cation exchange column, anion exchange column, and cation exchange column in sequence, with a material temperature of 35° C. and a flow rate of 2BV / h.

[0090] The concentration process uses a six-effect evaporator to concentrate the material after separation, with a concentration temperature of 65°C and a pressure of -0.06Mpa, increasing the solid content of the material to 75% to obtain the second refined liquid;

[0091] 6. Evaporation and crystallization: the second refined liquid described in step 5 is subjected to evaporation and crystallization operation.

[0092] The concentration of the second refined liquid was further concentrated to increase the fixed matter content to 78%, the temperature was increased to 55°C, and 200-mesh mannose at 0.02% by dry weight was added as seed crystals. After 60 minutes, continuous feeding began, and mannose with a solid content of 45% was used as the crystallization feeding liquid. During the feeding process, the temperature was controlled at 57°C, the fixed matter content was 80%, and the feeding was completed after 18 hours. The preparation method of the feeding liquid used for the feeding was the same as that of the second refined liquid, except that the solid content was controlled at 45%.

[0093] 7. Centrifugation and drying: The crystals and sugar solution obtained in step 6 are centrifuged and dried to obtain mannose crystals.

[0094] The centrifugation was carried out using a vertical scraper centrifuge with a rotation speed of 600 rpm and a centrifugal time of 30 min. The amount of water used for washing was 2% of the volume of the feed liquid.

[0095] The drying was carried out in a fluidized bed with an air inlet temperature of 58°C, a cooling temperature of 25°C and a drying time of 20 minutes.

[0096] The purity of the obtained mannose crystals was 99.4% and the water content was 0.26%.

[0097] Example 2

[0098] 1. Preparation of mannose 2-epimerase crude solution:

[0099] (1) Streak the recombinant Bacillus subtilis on an LB plate and incubate at 37°C for 23 h.

[0100] (2) Primary seed culture: Pick a single colony from the culture in step (1) and inoculate it into a 500 mL LB shake flask, where the liquid culture medium is 1 / 5 of the container volume, and culture at 1220 rpm for 12 h at a temperature of 37°C;

[0101] (3) Secondary seed culture: After the primary seed solution in step (2) is bottled, it is inoculated into a 1000L seed tank for culture, wherein the inoculation amount is 1%, the culture temperature is 37°C, and the rotation speed is 250 rpm. When the OD of the seed solution reaches 12, the culture is stopped;

[0102] (4) Fermentation culture: The fermentation broth described in step (3) was inoculated into the fermentation medium with an inoculum of 15%, cultured at 37°C, pH 7.2, and a dissolved oxygen rate of 35%. When dissolved oxygen rebounded, feed addition was started, and the glucose concentration was controlled at 8 g / L. When the OD of the fermentation broth reached 1.5, ... and the OD of the fermentation broth reached 1.5. 600 When the fermentation broth reached 55, the temperature was lowered to 28°C, and ITPG was added at a final concentration of 0.4 mM for induction. 600 When the OD value of the fermentation liquid reached 120, ITPG was added for the second time with a final concentration of 0.4 mM. 600 When the temperature reaches 190°C, the fermentation culture is stopped to obtain the crude mannose 2-epimerase solution.

[0103] The fermentation medium is as follows: 4 g / L corn steep liquor powder, 10 g / L glucose, 13.5 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate heptahydrate, 5.0 g / L diammonium hydrogen phosphate, 1.8 g / L citric acid monohydrate, and the balance is water, pH 7.2.

[0104] The supplementary feed includes: 500 g / L glucose, 10 g / L corn pulp powder, and 20 g / L magnesium sulfate heptahydrate.

[0105] 2. Enzyme conversion reaction: Add mannose 2-isomerase crude solution to glucose solution to convert and prepare mannose crude solution.

[0106] The concentration of glucose solution was 550 g / L, the amount of enzyme added was 1.0 g / L, the reaction temperature was 40°C, the pH was 7.0, and the reaction time was 26 h. After the reaction, the purity of mannose was 29%.

[0107] 3. First refining: The crude mannose solution in step 2 is first refined by decolorization, separation and concentration. The decolorization is carried out by a granular activated carbon column, the material temperature is 65°C, and then filtered by an Ama filter, the material temperature is 60°C, and the working pressure of the Ama filter is 0.4Mpa.

[0108] The ion exchange process is to pass the decolorized material through a cation exchange column, anion exchange column, cation exchange column, anion exchange column, and cation exchange column in sequence, with a material temperature of 45° C. and a flow rate of 4BV / h.

[0109] The concentration process uses a six-effect evaporator to concentrate the material after separation. The concentration temperature is 75°C and the pressure is -0.1Mpa. One concentration increases the solid content of the material to 60%.

[0110] 4. Chromatographic separation: The first refined liquid in step 3 is separated by continuous simulated mobile chromatography to obtain mannose solution and glucose solution, and the glucose solution is returned to the first refining step for recycling.

[0111] The continuous simulated mobile chromatography separation material-water ratio is 1:4.0, and the processing capacity is 0.028T.DS / h / m 3 Resin, yield 83%. A high-purity mannose solution with a purity of 98.9% was obtained.

[0112] 5. Second refining: The high-purity mannose solution described in step 4 is subjected to second refining by decolorization, separation and concentration.

[0113] The decolorization is carried out by using granular activated carbon column, the material temperature is 65°C, and then it is filtered by Ama filter, the material temperature is 60°C, and the working pressure of Ama filter is 0.4Mpa.

[0114] The ion exchange process is to pass the decolorized material through a cation exchange column, anion exchange column, cation exchange column, anion exchange column, and cation exchange column in sequence, with a material temperature of 45° C. and a flow rate of 4BV / h.

[0115] The concentration process uses a six-effect evaporator to concentrate the material after separation, with a concentration temperature of 75°C and a pressure of -0.1Mpa, increasing the solid content of the material to 78% to obtain the second refined liquid.

[0116] 6. Evaporation and crystallization: the second refined liquid described in step 5 is subjected to evaporation and crystallization operation.

[0117] The concentration of the second refined liquid was further concentrated to increase the fixed matter content to 81%, the temperature was increased to 60°C, and 250-mesh mannose at a dry weight of 0.50 millionths was added as a seed crystal. After 120 minutes, continuous feeding began, and mannose with a solid content of 50% was used as the crystallization feeding liquid. During the feeding process, the temperature was controlled at 60°C, the fixed matter content was 81%, and the feeding was completed after 20 hours. The preparation method of the feeding liquid used for the feeding was the same as that of the second refined liquid, except that the solid content was controlled to 50%.

[0118] 7. Centrifugation and drying: The crystals and sugar solution obtained in step 6 are centrifuged and dried to obtain mannose crystals.

[0119] The centrifugation was carried out using a vertical scraper centrifuge with a rotation speed of 900 rpm, a centrifugal time of 40 min, and a washing water volume of 5% of the volume of the feed liquid.

[0120] The drying was carried out in a fluidized bed with an air inlet temperature of 65°C, a cooling temperature of 30°C and a drying time of 30 minutes.

[0121] The purity of the obtained mannose crystals was 99.7% and the water content was 0.21%.

[0122] Comparative Example 1:

[0123] Comparative Example Mannose was prepared according to the preparation method of Example 1, except that in step 1, the fermentation medium was: 2.5 g / L corn syrup powder, 6 g / L glucose, 8 g / L potassium dihydrogen phosphate, 1.0 g / L magnesium sulfate heptahydrate, 3.0 g / L diammonium hydrogen phosphate, 1.2 g / L citric acid monohydrate, and the balance was water, pH 7.0.

[0124] Under the conditions of this comparative example, the enzyme conversion rate was 18.2%, the purity of mannose in the mannose solution after chromatographic separation was 87.8%, the purity of the mannose finished product crystals was 93.7%, and the water content was 0.30%.

[0125] Comparative Example 2:

[0126] In this comparative example, mannose was prepared according to the preparation method of Example 1, except that, in step 1, the fermentation medium was: 2.5 g / L corn steep liquor powder, 6 g / L glucose, 10.5 g / L potassium dihydrogen phosphate, 1.0 g / L magnesium sulfate heptahydrate, 2 g / L diammonium hydrogen phosphate, 1.2 g / L citric acid monohydrate, and the balance was water, with a pH of 7.0.

[0127] Under the conditions of this comparative example, the enzyme conversion rate is 15.4%, the purity of mannose in the mannose solution after chromatographic separation is 85.3%, the purity of the mannose finished product crystals is 91.4%, and the water content is 0.36%.

[0128] Comparative Example 3:

[0129] In this comparative example, mannose was prepared according to the preparation method of Example 1, except that, in step 1, the feed was: 500 g / L glucose, 10 g / L corn slurry powder, and 15 g / L magnesium sulfate heptahydrate.

[0130] Under the conditions of this comparative example, the enzyme conversion rate was 21.4%, the purity of mannose in the mannose solution after chromatographic separation was 89.7%, the purity of the mannose finished product crystals was 95.6%, and the water content was 0.34%.

[0131] Comparative Example 4

[0132] In this comparative example, mannose was prepared according to the preparation method of Example 1, except that in step 6, the concentration of the second refined liquid was further concentrated to increase the fixed matter content to 77%, and the temperature was increased to 53°C.

[0133] Under the conditions of this comparative example, the enzyme conversion rate was 25.8%, and mannose containing crystal water was obtained. The crystalline purity of the mannose finished product was 98.7%, and the water content was 8.7%.

[0134] Comparative Example 5

[0135] In this comparative example, mannose was prepared according to the preparation method of Example 1, except that, in step 6, the second refined liquid was cooled and crystallized in a uniform decreasing manner, and the temperature was uniformly reduced from 40° C. to 28° C. at a rate of 0.5° C. per hour.

[0136] Under the conditions of this comparative example, the enzyme conversion rate was 25.8%, and mannose containing crystal water was obtained. The crystal purity of the mannose finished product was 98.4%, and the water content was 9.3%.

[0137] Comparative Example 6

[0138] This comparative example prepared mannose according to the preparation method of Example 1, except that in step 2, the enzymatic conversion reaction: pH 5.0

[0139] After the reaction, the purity of mannose was 19.8%, the purity of mannose in the mannose solution after chromatographic separation was 88.6%, the purity of mannose finished product crystals was 94.2%, and the water content was 0.33%.

[0140] Experimental Example 1

[0141] The enzyme conversion rates and crystal purity of Examples 1-2 and Comparative Examples 1-6 were statistically analyzed.

[0142] Table 1 Enzyme conversion rate and crystal purity of Examples 1-2 and Comparative Examples 1-6

[0143] Serial number Enzyme conversion rate Purity of product after chromatographic separation Finished product crystal purity Finished product moisture Example 1 25.8% 98.4% 99.4% 0.26% Example 2 29.1% 98.9% 99.7% 0.21% Comparative Example 1 18.2% 87.8% 93.7% 0.34% Comparative Example 2 15.4% 85.3% 91.4% 0.36% Comparative Example 3 21.4% 89.7% 95.6% 0.30% Comparative Example 4 25.8% 98.4% 98.7% 8.7% Comparative Example 5 25.8% 98.4% 98.4% 9.3% Comparative Example 6 19.8% 88.6% 94.2% 0.33%

[0144] As can be seen from Table 1, the effects of enzyme conversion rate and product purity of Comparative Examples 1 and 2 are lower than those of Example 1. This is because the components in the culture medium, potassium dihydrogen phosphate and diammonium hydrogen phosphate, can be used as a pair of buffer systems to ensure mild conditions for bacterial strain growth and enzyme production. At the same time, the addition of diammonium hydrogen phosphate provides an inorganic nitrogen source for bacterial growth and enzyme production. Therefore, the addition of potassium dihydrogen phosphate and diammonium hydrogen phosphate is small, and it does not play the role of a buffer system, resulting in low enzyme activity and poor ability to convert substrates, so the effect is lower than that of Example 1. Comparative Example 3 reduces the content of magnesium sulfate heptahydrate in the feed medium, resulting in a low enzyme conversion rate, indicating that magnesium ions have a promoting effect on improving enzyme activity. The amount of magnesium ions is small, and the reaction activity of the enzyme is reduced, resulting in poor effect. In Comparative Example 4, the solid content and temperature were lower than those in Example 1, and an optimal parameter of the crystallization step was not reached, resulting in low product purity and high water content, and mannose containing crystal water was obtained; in Comparative Example 5, the crystallization method was changed, and cooling crystallization was adopted to obtain mannose containing crystal water; in Comparative Example 6, the pH value during the conversion process was reduced, so that the mannose 2-isomerase did not react at the optimal enzyme activity, so the enzyme conversion efficiency was low, and the purity of the obtained product was naturally low.

[0145] Experimental Example 2

[0146] Taking angle of repose measurements

[0147] The mannose prepared in Examples 1-2 and Comparative Examples 1-6 was subjected to repose angle test.

[0148] Angle of repose detection method: The fixed funnel method is used to determine the angle of repose of the particles. A dry and clean funnel is fixed on an iron frame, and the bottom funnel mouth is 2.5 cm (H) away from the horizontal coordinate paper. Mannose is poured into the funnel along the funnel wall until the tip of the particle cone formed by the last funnel touches the funnel mouth. The cone diameter (2R) is measured from the coordinate paper, then tanθ = H / R, and the angle θ is the angle of repose. The test results are shown in Table 2.

[0149] Table 2 Particle fluidity test

[0150] Serial number Angle of repose θ / ° Example 1 21 Example 2 20 Comparative Example 1 24 Comparative Example 2 25 Comparative Example 3 22 Comparative Example 4 36 Comparative Example 5 39 Comparative Example 6 23

[0151] Comparative Example 4 reduces the substrate concentration and temperature before crystallization, and obtains mannose containing crystal water. The crystals of the mannose crystallization product are partially adhered, the water activity is high, and the particle fluidity is poor. Comparative Example 5 adopts a uniform decreasing method to cool and crystallize, and obtains mannose containing crystal water. The product has adhesion and high water activity. The enzyme preparations obtained by fermentation of other strains in the prior art use fructose as a substrate, and the conversion rate is up to 25%. The strains in the present invention can directly use glucose as a substrate, and the conversion rate reaches 25-29%.

[0152] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A recombinant Bacillus subtilis ( Bacillus subtilis ) BLCY-013, characterized in that, The recombinant Bacillus subtilis BLCY-013 was deposited at the General Microbiology Center of the China Microorganism Culture Collection Administration on November 25, 2024, with the address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the culture collection number is CGMCC No.32760.

2. Use of the recombinant Bacillus subtilis according to claim 1 in preparing mannose using glucose as raw material.

3. A method for preparing mannose using glucose as raw material, characterized in that: The following steps are involved: S1. The recombinant Bacillus subtilis BLCY-013 according to claim 1 is subjected to primary seed culture, secondary seed culture, and fermentation culture to obtain a crude solution of mannose 2-epimerase; S2. adding mannose 2-epimerase crude solution to the glucose solution to prepare mannose crude solution through conversion; S3. The crude mannose liquid is sequentially subjected to first refining, chromatographic separation, second refining, evaporative crystallization, centrifugation, and drying to obtain mannose.

4. The method according to claim 3, characterized in that The specific method of primary seed cultivation in S1 is: The recombinant Bacillus subtilis was streaked on an LB plate and incubated upside down at 33-37°C for 18-23 h; Then, a single colony was picked and inoculated into an LB shake flask, and then cultured at 170-220 rpm and 33-37°C for 8-12 h to obtain a primary seed solution; The liquid volume of the LB shake flask is 1 / 10 to 1 / 5 of the container volume; The specific method of secondary seed cultivation is: The first-level seed solution was inoculated into a seed tank at an inoculation rate of 0.5% to 1%, and then cultured at a speed of 150 to 250 rpm and 33 to 37°C. 600 When the temperature reaches 8-12, stop the culture and obtain the secondary seed solution; The specific method of fermentation culture is: The secondary seed liquid is inoculated into the fermentation medium at an inoculation rate of 5-15%, and then fermented at 35-37° C., pH 6.8-7.2, and a dissolved oxygen rate of 25-35%; When dissolved oxygen rebound occurs during fermentation, feed addition is started to control the glucose concentration to 5-8 g / L. During the fermentation process, the OD 600 When the temperature reaches 45-55°C, lower the temperature to 25-28°C and add ITPG at a final concentration of 0.2-0.4 mM for induction; When the fermentation liquid OD 600 When the count reached 100-120, ITPG was added for the second time with a final concentration of 0.2-0.4 mM for induction; When the fermentation liquid OD 600 When the concentration reaches 170-190, the fermentation culture is stopped to obtain the crude mannose 2-isomerase solution.

5. The method according to claim 3, characterized in that: The parameters in S2 are as follows: The concentration of glucose solution is 550~600g / L; The amount of enzyme added is 0.7~1.0g / L; The conversion reaction temperature is 40~45℃; The conversion reaction pH is 6.0~7.0; The conversion reaction time is 22~26h; The purity of the obtained mannose crude liquid is 25-29%.

6. The method according to claim 3, characterized in that The specific method of the first refining in S3 is: The crude mannose solution is sequentially subjected to decolorization, ion exchange, and concentration to obtain a first refined solution; The solid content of the first refined liquid is 55-60%.

7. The method according to claim 6, characterized in that The specific method of chromatographic separation in S3 is: The first refined liquid is separated by continuous simulated mobile chromatography to obtain mannose liquid and glucose liquid; the continuous simulated mobile chromatography separation material-water ratio is 1:3.5-1:4.0, and the processing capacity is 0.025-0.028T.DS / h / m 3 Resin, yield 80~83%; The purity of the mannose solution obtained by chromatographic separation is above 98%.

8. The method according to claim 7, characterized in that The specific method of the second refining in S3 is: The mannose solution is sequentially subjected to decolorization, ion exchange, and concentration to obtain a second refined solution; The second refined liquid has a solid content of 75-78%.

9. The method according to claim 6 or 8, characterized in that: The decolorization process is as follows: decolorization is performed using a granular activated carbon column, wherein the material temperature is 55-65°C; and then filtered through an Ama filter, wherein the material temperature is 50-60°C, and the working pressure of the Ama filter is 0.2-0.4Mpa; The ion exchange process is as follows: the decolorized material is sequentially passed through a cation exchange column, an anion exchange column, a cation exchange column, an anion exchange column, and a cation exchange column; wherein the material temperature is 35-45° C., and the flow rate is 2-4 BV / h; The concentration process is as follows: using a six-effect evaporator to concentrate the material after ion exchange; wherein the concentration temperature is 65-75° C. and the pressure is -0.06--0.1Mpa.

10. The method according to claim 3, characterized in that: The specific method of the evaporative crystallization is: The second refined liquid is concentrated to increase the fixed matter content to 78-81%, the temperature is increased to 55-60°C, 0.1-0.5‰ of dry basis weight of 180-250 mesh mannose is added as a seed crystal, and after 60-120 minutes, continuous feeding is started. During the feeding process, the temperature is controlled at 57-60°C, the fixed matter content is 79-81%, and the crystallization liquid is obtained after 16-20 hours; The feeding liquid used in the feeding is a mannose solution with a solid content of 45%-50%.