Enzyme combinations, genetically modified bacteria and their use in the production of D-allulose

Abstract

The present invention relates to the technical field of biotechnology, particularly to an enzyme combination, a genetically modified bacterium, and its use in the production of D-allulose. The present invention relates to Bacillus subtilis co-expressing glucose isomerase and D-allulose 3-epimerase from a specific source. The resulting modified bacterium is fermented to obtain a crude enzyme solution, which is then isomerized using a high concentration of glucose as a substrate. After separation, purification, and concentration, fructoglucose syrup and D-allulose syrup are obtained. The enzyme combination provided by the present invention has a high catalytic rate relative to the substrate, significantly improving the conversion rate of D-allulose. At the same time, the present invention reduces the production costs of D-allulose by using inexpensive glucose as a raw material, and simultaneously produces fructoglucose syrup while producing D-allulose, thereby saving on the input of the fructoglucose syrup production line and significantly improving economic benefits.

Description

[Technical Field]

[0001] The present invention relates to the technical field of biotechnology, in particular to enzyme combinations, genetically modified bacteria and their use in the production of D-allulose. [Background technology]

[0002] D-Allulose is a rare sugar with 70% the sweetness of sucrose but very low calories, making it suitable as a sucrose substitute. Furthermore, because D-allulose has a lower absorption rate than other sweeteners, it can suppress the absorption of fructose and glucose in the body and reduce fat accumulation, thereby reducing the risk of conditions such as type 2 diabetes and obesity. Some research has now found that D-allulose also has the ability to lower blood lipids and blood sugar levels. The U.S. Food and Drug Administration (FDA) has recognized D-allulose as generally recognized as safe (GRAS), meaning it can be used as a food additive. D-Allulose is expected to have a wide range of applications in food and health products.

[0003] Currently, allulose is mainly produced using fructose as a raw material through the enzymatic catalysis of D-allulose 3-epimerase. However, the high market price of fructose inevitably increases the production costs of allulose. Meanwhile, fructose is produced by the epimerization of glucose using glucose isomerase as a catalyst. A one-step method for producing D-allulose from glucose can be achieved through the fermentation application of modified bacteria co-expressing glucose isomerase and D-allulose 3-epimerase. Furthermore, this method uses glucose as a raw material, reducing raw material costs, eliminating the need for a production line for producing fructose from glucose, and reducing equipment investment, making it of great industrial value. Summary of the Invention [Problem to be solved by the invention]

[0004] In view of the above, the present invention provides an enzyme combination, a genetically modified bacterium, and its use in the production of D-allulose. The present invention uses glucose isomerase and D-allulose 3-epimerase from a specific source as catalysts to convert the substrate glucose into D-allulose, significantly improving the conversion efficiency of D-allulose and shortening the production time, making it suitable for industrial production. [Means for solving the problem]

[0005] In order to achieve the above invention objectives, the present invention provides the following technical solutions:

[0006] The present invention provides an enzyme combination comprising glucose isomerase from Thermus thermophilus and D-allulose 3-epimerase from Ruminococcus sp.

[0007] The amino acid sequence of the glucose isomerase is 1) as shown in SEQ ID NO:1; 2) an amino acid sequence that does not alter function, resulting from the substitution, deletion, or addition of one or more amino acids to the sequence shown in SEQ ID NO:1; or 3) selected from any of amino acid sequences having at least 90% homology to the sequence shown in SEQ ID NO:1 and having the same or similar protein activity as SEQ ID NO:1.

[0008] The amino acid sequence of the D-allulose 3-epimerase is a) as shown in SEQ ID NO:2; b) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the sequence shown in SEQ ID NO:2, without altering the protein activity; or c) selected from any of the amino acid sequences having at least 90% homology to the sequence shown in SEQ ID NO:2 and having the same or similar protein activity as the sequence shown in SEQ ID NO:2.

[0009] In a specific embodiment, the glucose isomerase GI described in the present invention is derived from Thermus thermophilus, has the registration number WP_244348257.1, and its amino acid sequence is SEQ ID NO: 1. The D-allulose 3-epimerase DPE is derived from Ruminococcus sp., has the registration number MBS6425357.1, and its amino acid sequence is SEQ ID NO: 2.

[0010] The present invention has been developed through a long period of research, and has found that combinations of enzymes from different origins catalyze the conversion of the substrate glucose to allulose at different rates. Finally, a combination with the best conversion rate was found, namely, a combination of glucose isomerase from Thermus thermophilus and D-allulose 3-epimerase from Ruminococcus sp. Specifically, the amino acid sequence of the glucose isomerase is SEQ ID NO: 1, and the amino acid sequence of the D-allulose 3-epimerase is SEQ ID NO: 2.

[0011] The present invention further provides a nucleic acid combination encoding the enzyme combination, which comprises a GI gene and a DPE gene, wherein the GI gene and the DPE gene are optimized according to the codon preference of Bacillus subtilis, and the optimized sequence of the GI gene is SEQ ID NO:3, and the optimized sequence of the DPE gene is SEQ ID NO:4.

[0012] The present invention further provides a gene expression framework, which includes a promoter, one of the genes in the nucleic acid combination, and a terminator. The GI gene and the DPE gene in the nucleic acid combination are each initiated by a constitutive promoter, and in some embodiments, the constitutive promoter is a p43 promoter.

[0013] The present invention further provides an expression vector, comprising a backbone vector and the nucleic acid combination, wherein the genes encoding glucose isomerase and D-allulose 3-epimerase are located in the same backbone vector, and each gene is driven by a promoter, which may be the same (e.g., p43 promoter) or different.

[0014] In the expression vector according to the present invention, the backbone vector can be, but is not limited to, pWB980, pP43NMK, pYH-P43, etc., and can also be other vector types common in the art.

[0015] The present invention further provides modified bacteria transfected or transformed with said expression vectors.

[0016] The starting bacterium for the modified bacteria according to the present invention is Bacillus subtilis, and in a specific embodiment Bacillus subtilis WB600.

[0017] The present invention further provides a method for constructing the modified bacterium, which comprises ligating the glucose isomerase gene and the D-allulose 3-epimerase gene into a backbone vector to obtain a recombinant vector, and then introducing the recombinant vector into Bacillus subtilis to obtain the modified bacterium, wherein the sequence of the glucose isomerase gene is set forth in SEQ ID NO:3 and the sequence of the D-allulose 3-epimerase gene is set forth in SEQ ID NO:4.

[0018] In some specific embodiments, the method for constructing the modified bacteria is as follows: 1) The glucose isomerase protein sequence (GI, SEQ ID NO:1) from Thermus thermophilus and the D-allulose 3-epimerase protein sequence (DPE, SEQ ID NO:2) from Ruminococcus sp. were selected, and their encoding genes were codon-optimized. The optimized GI gene sequence is SEQ ID NO:3, and the DPE gene sequence is SEQ ID NO:4. These genes were completely synthesized by Kingsley Biotechnology. The two genes were ligated into the vector pWB980 by homologous recombination to obtain the recombinant plasmids pWB980-GI and pWB980-DPE. 2) Next, the p43-RBS-DPE fragment was amplified by PCR using the pWB980-DPE plasmid as a template, and the fragment was ligated into the vector pWB980-GI by homologous recombination to obtain the recombinant plasmid pWB980-GI-DPE, in which the GI and DPE genes were constitutively expressed intracellularly under the control of the p43 promoter. 3) The recombinant plasmid pWB980-GI-DPE is transformed into Bacillus subtilis WB600 to obtain recombinant Bacillus subtilis WB600 / GI-DPE.

[0019] Experiments have shown that this strain can directly ferment glucose to catalyze the production of fructose syrup and D-allulose, with a high glucose catalytic rate and a significantly shorter reaction time, facilitating the industrial production of allulose and resulting in high economic benefits.

[0020] The present invention further provides an enzyme combination, and further provides the use of the nucleic acid combination, the expression vector or the modified bacterium in the production of D-allulose or in the preparation of a blood glucose and lipid lowering product.

[0021] The present invention further provides a method for preparing allulose, which comprises reacting the substrate glucose with a fermentation culture, extract, or isolated enzyme solution of an engineered bacterium co-expressing glucose isomerase and D-allulose 3-epimerase of the present invention as a catalyst to produce D-allulose, specifically comprising the following steps:

[0022] a step of fermenting and culturing the modified bacterium and disrupting the cells to obtain a crude enzyme solution containing glucose isomerase and D-allulose 3-epimerase; adding a substrate glucose to the crude enzyme solution to obtain a conversion syrup; The conversion syrup is successively filtered, purified, chromatographically separated, and concentrated to obtain D-allulose and fructose, respectively.

[0023] In a specific embodiment, the method for preparing D-allulose comprises the following steps: (1) The recombinant strain is inoculated into LB seed medium, cultured overnight at 37°C and 200 rpm, and concentrated to obtain a seed solution with an OD600 value of 3 to 6. (2) The seed solution of the recombinant strain obtained in step (1) is inoculated into a fermentation medium (peptone 10 g, yeast powder 5 g, potassium dihydrogen phosphate 2.5 g, dipotassium hydrogen phosphate 15 g, manganese chloride tetrahydrate 0.1 g, magnesium sulfate heptahydrate 0.1 g, glucose 6 g, and water to a volume of 1 L) at a ratio of 0.1% (v / v) and fermented. The mixture is cultured at a fermentation temperature of 37°C and 200 rpm for 48 hours to obtain a fermentation liquid. (3) Add lysozyme at 0.1‰ (w / v) to the fermentation liquid obtained in step (2), and react at 37°C and 200 rpm for 1 hour. Disrupt the cells to release the intracellular enzymes and obtain a crude enzyme solution. (4) Glucose is added to the crude enzyme solution obtained in step (3) to a final concentration of 600 g / L, and the mixture is reacted at 60°C for 24 hours to obtain a conversion syrup. (5) The converted syrup obtained in step (4) is filtered through a ceramic membrane with a pore size of 8-10 nm to a flux of 50 L / m 2 / h to remove impurities and obtain the permeate. (6) The permeate obtained in step (5) is filtered through a nanofiltration membrane with a cutoff molecular weight of 100 to 300 Daltons, and the flux is 10 L / m 2 / h to remove impurities and decolorize, and obtain the permeate. (7) The permeate obtained in step (6) is first passed through a cation exchange resin and then through an anion exchange resin to remove anions and cations, and a desalted sugar solution is obtained. The cation exchange resin type is D001-FD, and the anion exchange resin type is D354-FD. (8) The sugar solution after desalting in step (7) is subjected to chromatographic separation, resulting in an allulose sugar solution (AD solution) and a glucose and fructose mixed sugar solution (BD solution). The chromatographic separation process is controlled to achieve a sugar solution concentration of 15-25%, a temperature of 50-60°C, a pressure of 0.2-0.3 MPa, a water to raw material mass ratio of 1:2, and a throughput of 2.2 kg / kg / d. (9) The AD liquid and BD obtained in step (8) are concentrated to a solid content of 75% to obtain D-allulose syrup and fructoglucose syrup. [Effects of the Invention]

[0024] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention uses a combination of enzymes from specific sources to convert glucose into D-allulose in a short period of time, thereby significantly shortening production time and significantly improving economic benefits, making it suitable for industrial production. (2) The present invention uses glucose, an inexpensive raw material, as a substrate, thereby reducing the production cost of D-allulose. (3) The present invention can produce D-allulose and fructoglucose syrup simultaneously, thereby saving the input of fructoglucose syrup production line. [Brief explanation of the drawings]

[0025] [Figure 1] 1 shows the profile of the recombinant plasmid pWB980-GI-DPE. [Figure 2]This is a photograph of SDS-PAGE electrophoresis, with lanes 1 and 2 representing 1:WB600 / pWB980 and 2:WB600 / pWB980-GI-DPE, respectively. [Figure 3] This is a liquid detection image. DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention provides an enzyme combination, a genetically modified bacterium, and its use in the production of D-allulose. Those skilled in the art can refer to the contents of this specification and appropriately modify the process parameters to achieve this. In particular, it should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The method and application of the present invention have been described by preferred embodiments, but it is clear that those skilled in the art can make modifications or appropriate changes and combinations to the method and application of the present specification to realize and apply the technology of the present invention without departing from the content, spirit, and scope of the present invention.

[0027] All of the test materials used in the present invention are common commercial products and can be purchased from the market.

[0028] The present invention will now be further described with reference to the following examples.

[0029] Example 1 Construction and Functional Verification of Modified Bacteria Co-Expressing Glucose Isomerase (GI) and D-Allulose 3-Epimerase (DPE) of the Present Invention

[0030] The glucose isomerase protein sequence (GI, SEQ ID NO:1) from Thermus thermophilus and the D-allulose 3-epimerase protein sequence (DPE, SEQ ID NO:2) from Ruminococcus sp. were selected, and after codon optimization of the coding genes, the GI gene sequence was SEQ ID NO:3 and the DPE gene sequence was SEQ ID NO:4. These genes were then fully synthesized by Kingsley Biotechnology, and the synthesized genes were ligated into the pUC57 vector and named pUC57-GI and pUC57-DPE, respectively.

[0031] PCR amplification is performed using the synthesized recombinant plasmid pUC57-GI as a template and GI-F and GI-R as primers, and the GI fragment is obtained by gel recovery and purification. PCR amplification is performed using the synthesized recombinant plasmid pUC57-DPE as a template and DPE-F and DPE-R as primers, and the DPE fragment is obtained by gel recovery and purification.

[0032] PCR amplification was performed using the plasmid vector pWB980 as a template and pWB980-F1 and pWB980-R1 as primers, followed by gel recovery and purification to obtain linearized plasmid gene fragment P1. PCR amplification was performed using the plasmid vector pWB980 as a template and pWB980-F2 and pWB980-R2 as primers, followed by gel recovery and purification to obtain linearized plasmid gene fragment P2.

[0033] The GI fragment and the pWB980 linearized gene fragment P1 were ligated according to the instructions of the homologous recombination kit. The ligation product was electrotransformed into B. subtilis WB600 competent cells. The resulting recombinant B. subtilis was designated WB600 / GI, and the recombinant plasmid was designated pWB980-GI. The accuracy of the transformant was verified by colony PCR and sequence analysis.

[0034] [Table 1]

[0035] The DPE fragment and the pWB980 linearized gene fragment P2 were ligated according to the instructions in the homologous recombination kit. The ligation product was electrotransformed into B. subtilis WB600 competent cells. The resulting recombinant B. subtilis was designated WB600 / DPE, and the recombinant plasmid was designated pWB980-DPE. The accuracy of the transformant was verified by colony PCR and sequence analysis.

[0036] PCR amplification was performed using the recombinant plasmid pWB980-DPE as a template and p43-RBS-DPE-F and p43-RBS-DPE-R as primers to obtain the p43-RBS-DPE fragment. PCR amplification was performed using the recombinant plasmid pWB980-GI as a template and pWB980-GI-F and pWB980-GI-R as primers to obtain the linearized plasmid gene fragment P3.

[0037] The p43-RBS-DPE fragment and the pWB980-GI linearized gene fragment P3 were ligated according to the instructions of the homologous recombination kit, and the ligation product was electrotransformed into B. subtilis WB600 competent cells. The resulting recombinant B. subtilis was named WB600 / GI-DPE, and the recombinant plasmid was named pWB980-GI-DPE (the plasmid profile is shown in Figure 1). The accuracy of the transformant was verified by colony PCR and sequence analysis.

[0038] Positive transformants that showed accurate sequencing and colony PCR results were selected and inoculated into 5 ml of LB liquid medium containing 50 mg / L kanamycin. Cultured overnight at 37°C and 200 rpm. A 0.1% inoculum was then added to 1 L or more of fermentation medium containing 50 mg / L kanamycin and cultured at 37°C and 200 rpm for 48 hours. Lysozyme powder was added at 0.1‰, and the mixture was allowed to react continuously at 37°C and 200 rpm for 1 hour to obtain the crude GI-DPE enzyme solution (SDS-PAGE electrophoresis results are shown in Figure 2).

[0039] Glucose was added to the crude enzyme solution obtained above to a final concentration of 700 g / L, and the mixture was allowed to react for 24 hours at 60°C. 0.02 ml of the reaction solution was diluted 50-fold with pure water and then inactivated in a 100°C water bath for 10 minutes. The mixture was centrifuged at 10,000 rpm for 10 minutes, filtered through a 0.22 μm micropore filter, and the filtrate was subjected to high-performance liquid chromatography analysis.

[0040] Bacillus subtilis WB600 spiked with empty plasmid pWB980 was used as the blank control, and other operating conditions were the same.

[0041] High-performance liquid chromatography was performed under the following conditions: Agilent high-performance liquid chromatography system 1200, analytical column: water sugar pak I chromatography column, mobile phase: pure water, flow rate: 0.3 ml / min, column temperature: 80 °C, detector: differential refractive index detector. The above samples were analyzed using pure glucose, fructose, and D-allulose produced by Sigma as standards, and the sample loading volume was 10 μL.

[0042] As can be seen from the liquid chromatography analysis results (FIG. 3), the peak times for glucose, fructose, and D-allulose were 14.1 min, 17.9 min, and 26.9 min, respectively.

[0043] The liquid phase profile of the blank control (Bacillus subtilis WB600 / pWB980) contained only glucose, while the liquid phase profile of the experimental group (Bacillus subtilis WB600 / pWB980-GI-DPE) contained glucose, fructose, and D-allulose. These results demonstrate that the GI-DPE crude enzyme solution produced by the above method can convert glucose to fructose and further convert fructose to D-allulose.

[0044] From the calculation of the peak areas, after 24 hours of reaction, the sugar solution after conversion contained 284 g / L of glucose, 209 g / L of fructose, and 107 g / L of D-allulose in a ratio of 47.33:34.83:17.83.

[0045] Example 2: Fermentative production method of fructoglucose syrup and D-allulose using the co-expression modified bacterium of the present invention

[0046] The specific steps are as follows: (1) The co-expression modified bacteria of Example 1 above was inoculated into an LB liquid seed medium containing 50 mg / L kanamycin and cultured overnight at 37°C and 200 rpm to obtain a co-expression modified bacteria seed solution; (2) The modified bacterial seed solution obtained above is inoculated into 1 L or more of fermentation medium containing 50 mg / L kanamycin at a ratio of 0.1% (v / v) (peptone 10 g, yeast powder 5 g, potassium dihydrogen phosphate 2.5 g, potassium dihydrogen phosphate 15 g, manganese chloride tetrahydrate 0.1 g, magnesium sulfate heptahydrate 0.1 g, glucose 6 g, and water to a constant volume of 1 L), and fermented. The culture is then incubated at 37°C and 200 rpm for 48 hours to obtain a fermentation liquid. (3) Lysozyme is added to the fermentation liquid obtained in step (2) at 0.1‰ (w / v), and the mixture is reacted at 37°C and 200 rpm for 1 hour. The cells are disrupted to release the intracellular enzymes, and a crude GI-DPE enzyme solution is obtained. (4) Glucose is added to the crude enzyme solution obtained in step (3) at a final concentration of 600 g / L, and the mixture is reacted at 60°C for 2 to 24 hours to obtain a conversion syrup. (5) The converted syrup obtained in step (4) is filtered through a ceramic membrane with a pore size of 8-10 nm and a flux of 50 L / m 2 / h to remove impurities and obtain the permeate. (6) The permeate obtained in step (5) is filtered through a nanofiltration membrane with a cutoff molecular weight of 100 to 300 Daltons, and the flux is 10 L / m 2 / h to remove impurities and decolorize, and obtain the permeate. (7) The permeate obtained in step (6) is first passed through a cation resin, and then through an anion resin to remove anions and cations, the cation resin type is D001-FD, and the anion resin type is D354-FD, to obtain a desalted sugar solution. (8) The sugar solution after desalting in step (7) is subjected to chromatographic separation, and the resulting AD solution is an allulose sugar solution, and the BD solution is a glucose and fructose mixed sugar solution. During the chromatographic separation process, the sugar solution concentration is controlled to 15-25%, the temperature to 50-60°C, the pressure to 0.2-0.3 MPa, the water to raw material mass ratio to 1:2, and the processing rate to 2.2 kg / kg / d. (9) The AD liquid and BD obtained in step (8) are concentrated to a solid content of 75% to obtain D-allulose syrup and fructoglucose syrup.

[0047] Comparative Example 1

[0048] Glucose isomerase (GI, the nucleic acid sequence of which is SEQ ID NO: 1 in CN113980880) and D-allulose 3-epimerase (DPE, the nucleic acid sequence of which is SEQ ID NO: 3 in CN113980880) from the CN113980880 patent were selected, and Bacillus subtilis co-expressing glucose isomerase and D-allulose 3-epimerase was constructed according to the method of Example 1 of the present invention. A conversion syrup containing glucose, fructose, and allulose was prepared according to the method of Example 2 of the present invention.

[0049] Comparative Example 2 (GI is not of the same genus (GI homology with the present invention is 56%), DPE is the same)

[0050] Glucose isomerase (GI, the nucleic acid sequence of which is SEQ ID NO: 1 in CN113980880) from patent CN113980880 and D-allulose 3-epimerase (DPE, the nucleic acid sequence of which is SEQ ID NO: 4) from Ruminococcus sp. of the present application were selected, and Bacillus subtilis co-expressed GI and DPE were constructed according to the method of Example 1 of the present invention. A conversion syrup containing glucose, fructose, and allulose was prepared according to the method of Example 2 of the present invention.

[0051] Comparative Example 3 (GI is not of the same genus (homology 97%), DPE is the same) Glucose isomerase (GI, registration number WP_126200404.1, SEQ ID NO: 5 after codon optimization) from Thermus scotoductus and D-allulose 3-epimerase (DPE, nucleic acid sequence SEQ ID NO: 4) from Ruminococcus sp. of the present invention were selected, and Bacillus subtilis was constructed to co-express GI and DPE according to the method of Example 1 of the present invention. A conversion syrup containing glucose, fructose, and allulose was prepared according to the method of Example 2 of the present invention.

[0052] Comparative Example 4 (same GI, different DPE (40% homology with the present invention) The glucose isomerase (GI, nucleic acid sequence see SEQ ID NO: 3) from Thermus thermophilus of the present invention and D-allulose 3-epimerase (DPE, nucleic acid sequence see SEQ ID NO: 3 in CN113980880 patent) were selected, and Bacillus subtilis co-expressing GI and DPE was constructed according to the method of Example 1 of the present invention. A conversion syrup containing glucose, fructose and allulose was prepared according to the method of Example 2 of the present invention.

[0053] Comparative Example 5 (same GI, different DPE (69% homology with the present invention) Thermus thermophilus glucose isomerase (GI, see SEQ ID NO: 3 for the nucleic acid sequence) and Oribacterium sinus D-allulose 3-epimerase (DPE, WP_183684385.1, see SEQ ID NO: 6 for the nucleic acid sequence) of the present invention were selected, and Bacillus subtilis co-expressing GI and DPE was constructed according to the method of Example 1. A conversion syrup containing glucose, fructose, and allulose was prepared according to the method of Example 2 of the present invention.

[0054] Test Example

[0055] The conversion syrups were prepared according to the methods of Example 2 and Comparative Examples 1 to 5. Samples were taken and reacted for 2 hours, 4 hours, 6 hours, 12 hours, and 24 hours, and the contents of glucose, fructose, and allulose were detected by HPLC, and the allulose production rate was calculated. The results are shown in Table 2.

[0056] [Table 2] TIFF2025532205000004.tif24168

[0057] As can be seen from the above results, the conversion rates of allulose production using enzyme-catalyzed glucose produced by the modified bacterial strains of Comparative Examples 1 to 5 and the present invention were all similar, ranging from 14 to 18%, but the time required to reach equilibrium conversion was significantly different: 6 h, 6 h, 12 h, 4 h, 12 h, and 2 h, respectively. As a result, the maximum allulose production rates were calculated to be 21.5 g / L / h, 25 g / L / h, 16 g / L / h, 25 g / L / h, 11.5 g / L / h, and 53.5 g / L / h, respectively. Among these, the equilibrium catalysis time of the enzyme combination of the present invention was the shortest, significantly lower than that of the other comparative examples, and the allulose production rate of the enzyme combination of the present invention was also the highest, significantly higher than that of the other comparative examples. Therefore, the combination of Thermus thermophilus-derived glucose isomerase and Ruminococcus sp.-derived D-allulose 3-epimerase provided by the present invention has the highest catalytic rate for glucose and the highest allulose production rate in the conversion reaction, and can be determined to be the optimal combination.

[0058] It should be noted that the above are only preferred embodiments of the present invention, and many improvements and modifications may be made by those skilled in the art without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the present invention.

Claims

1. An enzyme combination comprising glucose isomerase derived from Thermus thermophilus and D-allulose 3-epimerase derived from Ruminococcus sp.

2. The amino acid sequence of the glucose isomerase is 1) the sequence shown in SEQ ID NO: 1; 2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the sequence shown in SEQ ID NO: 1, which does not alter the protein activity; or 3) an amino acid sequence having at least 90% homology to the sequence shown in SEQ ID NO: 1 and having the same or similar protein activity as SEQ ID NO:

1. The enzyme combination according to claim 1, characterized in that the enzyme combination is selected from the group consisting of:

3. The amino acid sequence of the D-allulose 3-epimerase is a) the sequence shown in SEQ ID NO: 2; b) an amino acid sequence obtained by substitution, deletion or addition of one or more amino acids to the sequence shown in SEQ ID NO: 2, which does not alter the protein activity; or c) an amino acid sequence having at least 90% homology to the sequence shown in SEQ ID NO: 2 and having the same or similar protein activity as the sequence shown in SEQ ID NO:

2.

4. A nucleic acid combination encoding the enzyme combination according to any one of claims 1 to 3.

5. The glucose isomerase gene is a gene after being optimized according to the codon preference of Bacillus subtilis, and its nucleotide sequence is the sequence shown in SEQ ID NO: 3: The nucleic acid combination of claim 4, wherein the D-allulose 3-epimerase gene is a gene that has been optimized according to the codon preference of Bacillus subtilis, and its nucleotide sequence is set forth in SEQ ID NO:

4.

6. An expression vector comprising a backbone vector and the nucleic acid combination of claim 4 or 5.

7. A modified bacterium comprising the expression vector of claim 6.

8. The modified bacterium of claim 7, wherein the starting bacterium is Bacillus subtilis.

9. A method for constructing the modified bacterium according to claim 7 or 8, comprising ligating the glucose isomerase gene and the D-allulose 3-epimerase gene into a backbone vector to obtain a recombinant vector, and introducing the recombinant vector into Bacillus subtilis to obtain the modified bacterium.

10. Use of an enzyme combination according to any one of claims 1 to 3, a nucleic acid combination according to claim 4 or 5, an expression vector according to claim 6 or a modified bacterium according to claim 7 or 8 in the production of D-allulose or in the preparation of a hypoglycemic and hypolipidemic product.

11. A method for preparing D-allulose, comprising catalyzing a reaction of substrate glucose to produce D-allulose using a fermentation culture, extract, or extracted and separated enzyme liquid of the modified bacterium according to claim 7 or 8 as a catalyst.

12. a step of fermenting and culturing the modified bacterium according to claim 7 or 8 and disrupting the cells to obtain a crude enzyme solution containing glucose isomerase and D-allulose 3-epimerase; adding a substrate glucose to the crude enzyme solution to obtain a conversion syrup; The method of claim 11, further comprising the steps of sequentially filtering, purifying, chromatographically separating and concentrating the converted syrup to obtain D-allulose and fructose, respectively.

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