Enzyme combinations, genetically modified bacteria, and their applications in D-allulose production

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, and in particular relates to an enzyme combination, a genetically modified bacterium, and use thereof in the production of D-allulose. BACKGROUND ART

[0002] D-allulose is one of the rare sugars, has 70% of the sweetness of sucrose, but has very low calories, and is suitable as a substitute for sucrose. Furthermore, since D-allulose has a lower absorption rate compared with other sweeteners, it can inhibit the in vivo absorption of fructose and glucose and reduce the amount of fat accumulation, thereby reducing the risk of conditions such as type II diabetes and excessive obesity. At present, some studies have found that D-allulose also has the function of lowering blood lipids and blood glucose levels. The U.S. Food and Drug Administration (FDA) has classified D-allulose as GRAS (Generally Recognized as Safe), and it can be used as a constituent component of food additives. D-allulose is expected to be widely applied in foods and health products.

[0003] At present, allulose is mainly produced by using fructose as a raw material and carrying out enzymatic catalysis with D-allulose 3-epimerase to generate allulose. However, due to the high market price of fructose, the production cost of allulose is inevitably high. On the other hand, fructose is produced by the epimerization reaction of glucose using glucose isomerase as a catalyst. Through the fermentation application of a modified bacterium with co-expression of two genes, namely glucose isomerase and D-allulose 3-epimerase, a method for producing D-allulose from glucose in one step can be achieved. In addition, this method uses glucose as the raw material, can reduce raw material costs, omit the production line for producing fructose from glucose, and reduce equipment investment, thus having important industrial application value. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

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

[0005] To achieve the above-mentioned objectives of the invention, the present invention provides the following technical solutions.

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

[0007] The amino acid sequence of the glucose isomerase is, 1) As indicated by SEQ ID NO:1, 2) An amino acid sequence whose function is not altered by the substitution, deletion, or addition of one or more amino acids to the sequence shown in SEQ ID NO:1, or 3) Select from one of the amino acid sequences that has at least 90% homology to the sequence shown in SEQ ID NO:1 and has the same or similar protein activity as SEQ ID NO:1.

[0008] The amino acid sequence of the aforementioned D-allulose 3-epimerase is, a) As shown in SEQ ID NO:2, b) An amino acid sequence in which the protein activity obtained by substituting, deleting, or adding one or more amino acids to the sequence shown in SEQ ID NO:2 does not change, or c) Selected from one of the amino acid sequences that has at least 90% homology to the sequence shown in SEQ ID NO:2 and has the same or similar protein activity as the sequence shown in SEQ ID NO:2.

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

[0010] The present invention has been studied over a long period of time, and different enzyme combinations from different origins catalyze the conversion of substrate glucose to allulose at different rates. Ultimately, the best combination with the best conversion rate was obtained, 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 nucleic acid combinations encoding the enzyme combination, including a GI gene and a DPE gene. The present invention optimizes the GI gene and DPE gene according to the codon preference of Bacillus subtilis, with the optimized GI gene sequence being SEQ ID NO:3 and the optimized DPE gene sequence being SEQ ID NO:4.

[0012] The present invention further provides a gene expression frame comprising a promoter, one gene 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 the p43 promoter.

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

[0014] In the expression vector described in the present invention, the backbone vector may be pWB980, pP43NMK, pYH-P43, or other vector types common in the art, but is not limited to these.

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

[0016] The starting organism for the modified bacteria described in this invention is Bacillus subtilis, and in specific examples, Bacillus subtilis WB600 is used.

[0017] The present invention further provides a method for constructing the modified bacteria, comprising ligating a glucose isomerase gene and a 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 bacteria. The sequence of the glucose isomerase gene is shown in SEQ ID NO:3, and the sequence of the D-allulose 3-epimerase gene is shown 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 coding genes were codon-optimized. The optimized GI gene sequence is SEQ ID NO:3, and the DPE gene sequence is SEQ ID NO:4, and these genes were completely synthesized by Kingsley Biotechnology. The two genes were ligated onto the vector pWB980 by homologous recombination to obtain recombinant plasmids pWB980-GI and pWB980-DPE. 2) Next, the p43-RBS-DPE fragment is amplified by PCR using the pWB980-DPE plasmid as a template, and this fragment is ligated onto the vector pWB980-GI by homologous recombination to obtain the recombinant plasmid pWB980-GI-DPE. In this plasmid, the GI and DPE genes are constitutively regulated intracellularly by the P43 promoter. 3) Convert the recombinant plasmid pWB980-GI-DPE to Bacillus subtilis WB600 to obtain recombinant Bacillus subtilis WB600 / GI-DPE.

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

[0020] The present invention further provides enzyme combinations and further provides applications of the nucleic acid combination, the expression vector, or the modified bacteria in the production of D-allulose or in the preparation of blood glucose / lipid-lowering products.

[0021] The present invention further provides a method for preparing allulose, wherein a fermentation culture, extract or extracted and separated enzyme solution of a modified bacterium co-expressing glucose isomerase and D-allulose 3-epimerase of the present invention is used as a catalyst to react the substrate glucose to generate D-allulose. Specifically, the method includes the following steps.

[0022] a step of subjecting the modified bacterium to fermentation culture and cell disruption treatment to obtain a crude enzyme solution containing glucose isomerase and D-allulose 3-epimerase, a step of adding the substrate glucose to the crude enzyme solution to obtain a conversion syrup, a step of sequentially subjecting the conversion syrup to filtration, purification, chromatographic separation and concentration to obtain D-allulose and fructose respectively.

[0023] In a specific example, the method for preparing D-allulose includes the following steps: (1) The above recombinant strain is inoculated into an LB seed medium, cultured overnight at 37°C and 200rpm for concentration, 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 at a ratio of 0.1% (v / v) into a fermentation medium (10g of peptone, 5g of yeast powder, 2.5g of potassium dihydrogen phosphate, 15g of dipotassium hydrogen phosphate, 0.1g of manganese chloride tetrahydrate, 0.1g of magnesium sulfate heptahydrate, 6g of glucose, water is added to make the constant volume to 1L) for fermentation, and cultured at a fermentation temperature of 37°C and 200rpm for 48h to obtain a fermentation broth. (3) Lysozyme is added to the fermentation broth obtained in step (2) at 0.1‰ (w / v), the mixture is reacted at 37°C and 200rpm for 1h to disrupt the cells and release the intracellular enzymes, so as to obtain a crude enzyme solution. (4) Glucose is added to the crude enzyme solution obtained in step (3) to a final concentration of 600g / L, and the mixture is reacted at 60°C for 24h to obtain a conversion syrup. (5) The conversion syrup obtained in step (4) is filtered through a ceramic membrane with a pore size of 8 to 10nm at a flux of 50L / m 2 / h to remove impurities, so as to obtain a permeate. (6) The permeate obtained in step (5) is filtered through a nanofiltration membrane with a cutoff molecular weight of 100-300 daltons, and the flux is 10 L / m 2 The impurities are removed and the color is decolorized at / h to obtain the permeate. (7) The permeate obtained in step (6) is first passed through a cationic resin, then through an anionic resin to remove the anions and cations, and a sugar solution is obtained after desalting. The cationic resin is of type D001-FD and the anionic resin is of type D354-FD. (8) The desalted sugar solution from step (7) is chromatographically separated, and the obtained solution AD is allulose sugar solution, and solution BD is a glucose and fructose mixed sugar solution. During the chromatographic separation process, the sugar solution concentration is controlled to be 15-25%, the temperature to be 50-60°C, the pressure to be 0.2-0.3 MPa, the water to raw material mass ratio to be 1:2, and the processing rate to be 2.2 kg / kg / d. (9) The AD solution and BD obtained in step (8) are concentrated until the solid content reaches 75% to obtain D-allulose syrup and fructoglucose syrup. [Effects of the Invention]

[0024] Compared to prior art, the present invention has the following beneficial effects. (1) The present invention converts glucose to D-allulose in a short time using an enzyme combination from a specific source, thereby significantly reducing production time and greatly improving economic benefits, making it suitable for industrial production. (2) Since the present invention uses glucose, which is an inexpensive raw material, as a substrate, the production cost of D-allulose can be reduced. (3) The present invention allows for the simultaneous production of D-allulose and fructoglucose syrup, thereby saving input to the fructoglucose syrup production line. [Brief explanation of the drawing]

[0025] [Figure 1] This is the profile of the recombinant plasmid pWB980-GI-DPE. [Figure 2]These are SDS-PAGE electrophoresis images, with lanes 1 and 2 representing 1: WB600 / pWB980 and 2: WB600 / pWB980-GI-DPE, respectively. [Figure 3] This is a liquid detection image. [Modes for carrying out the invention]

[0026] The present invention provides enzyme combinations, genetically modified bacteria, and applications in the production of D-allulose. Those skilled in the art can refer to the contents of this specification and appropriately modify 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. Although the methods and applications of the present invention have been described by preferred embodiments, it is obvious that those interested can realize and apply the technology of the present invention by modifying or appropriately changing and combining the methods and applications described herein without departing from the content, spirit and scope of the present invention.

[0027] All test materials used in this invention are standard commercially available products and can be purchased from the market.

[0028] The present invention will be further described below through examples.

[0029] Example 1: Construction and functional verification of a modified bacterium co-expressing glucose isomerase (GI) and D-allulose 3-epimerase (DPE) according to 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 became SEQ ID NO:3 and the DPE gene sequence became SEQ ID NO:4. These genes were completely synthesized by Kingsley Biotechnology, and the synthesized genes were ligated onto the pUC57 vector and named pUC57-GI and pUC57-DPE, respectively.

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

[0032] Using plasmid vector pWB980 as a template, PCR amplification was performed with pWB980-F1 and pWB980-R1 as primers, and linear plasmid gene fragment P1 was obtained by gel recovery and purification. Using plasmid vector pWB980 as a template, PCR amplification was performed with pWB980-F2 and pWB980-R2 as primers, and linear plasmid gene fragment P2 was obtained by gel recovery and purification.

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

[0034] [Table 1]

[0035] The DPE fragment and the pWB980 linearization gene fragment P2 were ligated according to the instructions of the homologous recombination kit. The ligation products were electrotransformed into Bacillus subtilis WB600 competent cells. The resulting recombinant Bacillus subtilis was named WB600 / DPE, and the recombinant plasmid was named pWB980-DPE. The accuracy of the ligation was verified by transformant colony PCR and sequencing analysis.

[0036] Using the recombinant plasmid pWB980-DPE as a template, PCR amplification was performed using p43-RBS-DPE-F and p43-RBS-DPE-R as primers, and the p43-RBS-DPE fragment was obtained by gel recovery and purification. Using the recombinant plasmid pWB980-GI as a template, PCR amplification was performed using pWB980-GI-F and pWB980-GI-R as primers, and the linearized plasmid gene fragment P3 was obtained by gel recovery and purification.

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

[0038] The above colony PCR and sequencing results were used to select transformation-positive cells, which were inoculated into 5 ml of LB liquid medium containing 50 mg / L kanamycin and incubated overnight at 37°C and 200 rpm. These cells were then inoculated at a 0.1% dose into 1 L or more of fermentation medium containing 50 mg / L kanamycin and incubated for 48 hours at 37°C and 200 rpm. Lysozyme powder was added at a dose of 0.1‰, and the mixture was reacted continuously for 1 hour at 37°C and 200 rpm to obtain a crude GI-DPE enzyme solution (SDS-PAGE electrophoresis results are shown in Figure 2).

[0039] To the crude enzyme solution obtained above, glucose was added according to a final concentration of 700 g / L, and the reaction was carried out at 60°C for 24 hours. 0.02 ml of the reaction solution was diluted 50-fold with pure water, then treated in a 100°C water bath for 10 minutes to inactivate the enzyme, followed by centrifugation at 10,000 rpm for 10 minutes. The solution was then filtered through a 0.22 μm micropore filter membrane, and the filtrate was subjected to high-performance liquid chromatography analysis.

[0040] Bacillus subtilis WB600 containing the empty plasmid pWB980 was used as a blank control, and all 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: waters sugar pak I chromatography column, mobile phase: pure water, flow rate: 0.3 ml / min, column temperature: 80°C, detector: differential refractive detector. Pure glucose, fructose, and D-allulose produced by Sigma were used as standards for the analysis of the above samples, with a sample loading volume of 10 μl.

[0042] As can be seen from the liquid chromatography analysis results (Figure 3), the peak time 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 indicate that the crude GI-DPE enzyme solution prepared by the above method can convert glucose to fructose, and further convert fructose to D-allulose.

[0044] Based on the peak area calculation, after a 24-hour reaction, the converted sugar solution 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: Method for fermenting and producing fructoglucose syrup and D-allulose using co-expression modified bacteria of the present invention

[0046] The specific steps are as follows: (1) The co-expression modified bacteria of Example 1 above were inoculated into LB liquid seed medium containing 50 mg / L kanamycin, and cultured overnight at 37°C and 200 rpm to obtain a co-expression modified bacterial seed solution. (2) The modified bacterial seed solution obtained above is inoculated at a ratio of 0.1% (v / v) into 1 L or more of fermentation medium containing 50 mg / L kanamycin (add 10 g peptone, 5 g yeast powder, 2.5 g potassium dihydrogen phosphate, 15 g dipotassium hydrogen phosphate, 0.1 g manganese chloride tetrahydrate, 0.1 g magnesium sulfate heptahydrate, 6 g glucose, and water to a constant volume of 1 L), and fermented, and cultured at a fermentation temperature of 37°C and 200 rpm for 48 hours to obtain the fermentation liquid. (3) Add lysozyme at 0.1‰(w / v) to the fermentation liquid obtained in step (2), react at 37°C and 200 rpm for 1 hour, disrupt the microbial cells to release intracellular enzymes, and obtain crude GI-DPE enzyme solution. (4) Add glucose to the crude enzyme solution obtained in step (3) at a final concentration of 600 g / L, and react at 60°C for 2 to 24 hours to obtain the conversion syrup. (5) The conversion 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 / m² is applied. 2 Remove impurities using a 2000-hour setting to obtain a permeate. (6) The permeate obtained in step (5) is filtered through a nanofiltration membrane with a cutoff molecular weight of 100-300 daltons, and the flux is 10 L / m 2 The impurities are removed and the color is decolorized at / h to obtain the permeate. (7) The permeate obtained in step (6) is first passed through a cationic resin, and then through an anionic resin to remove the anions and cations. The cationic resin type is D001-FD and the anionic resin type is D354-FD, and a sugar solution is obtained after desalting. (8) The desalted sugar solution from step (7) is chromatographically separated, and the obtained solution AD is allulose sugar solution, and solution BD 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 solution and BD obtained in step (8) are concentrated until the solid content reaches 75% to obtain D-allulose syrup and fructoglucose syrup.

[0047] Comparative Example 1

[0048] Glucose isomerase (GI, see SEQ ID NO:1 in CN113980880 for the nucleic acid sequence) and D-allulose 3-epimerase (DPE, see SEQ ID NO:3 in CN113980880 for the nucleic acid sequence) were selected from the patent CN113980880, 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, and 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 (56% GI homology with the present invention), DPE is the same)

[0050] Glucose isomerase (GI, see SEQ ID NO:1 in CN113980880 for the nucleic acid sequence) from the patent CN113980880 and D-allulose 3-epimerase (DPE, see SEQ ID NO:4 for the nucleic acid sequence) from Ruminococcus sp. of this application were selected, and Bacillus subtilis cells expressing co-expression of 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 (97% homology), DPE is the same) Glucose isomerase (GI, registration number WP_126200404.1, SEQ ID NO: 5 after codon optimization of the nucleic acid sequence) derived from the water pipe-causing black amphibian thermophilic bacterium Thermus Scotoductus and D-allulose 3-epimerase (DPE, nucleic acid sequence SEQ ID NO: 4) derived from Ruminococcus sp. of the present invention were selected, and Bacillus subtilis with co-expression of 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.

[0052] Comparative Example 4 (GI is the same, but DPE is different (40% DPE homology with the present invention)) The present invention selected glucose isomerase (GI, see SEQ ID NO:3 for nucleic acid sequence) derived from Thermus thermophilus and D-allulose 3-epimerase (DPE, see SEQ ID NO:3 described in CN113980880 for nucleic acid sequence) from the present invention, and Bacillus subtilis with co-expression of 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 (GI is the same, but DPE is different (69% DPE homology with the present invention)) The present invention selected glucose isomerase (GI, see SEQ ID NO:3 for nucleic acid sequence) from Thermus thermophilus and D-allulose 3-epimerase (DPE, WP_183684385.1, see SEQ ID NO:6 for nucleic acid sequence) from Oribacterium sinus, and Bacillus subtilis with co-expression of 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] Conversion syrups were prepared according to the methods of Example 2 and Comparative Examples 1-5. Samples were taken from each and reacted for 2h, 4h, 6h, 12h, and 24h. The content of glucose, fructose, and allulose was detected by HPLC, and the allulose production rate was calculated. The results are shown in Table 2.

[0056] [Table 2] TIFF0007943706000003.tif24168

[0057] As can be seen from the results above, although the conversion rates of enzyme-catalyzed glucose for allulose production produced by Comparative Examples 1-5 and the modified bacterial strain of the present invention were all similar, in the range of 14-18%, the time required to reach the equilibrium conversion rate differed significantly, at 6h, 6h, 12h, 4h, 12h, and 2h, respectively. Based on this, the maximum allulose production rates were calculated to be 21.5g / L / h, 25g / L / h, 16g / L / h, 25g / L / h, 11.5g / L / h, and 53.5g / L / h, respectively. Among these, the equilibrium catalytic time for the enzyme combination of the present invention was the shortest, significantly lower than the other comparative examples, and the allulose production rate for the enzyme combination of the present invention was also the highest, significantly higher than the other comparative examples. Therefore, the combination of glucose isomerase derived from Thermus thermophilus and D-allulose 3-epimerase derived from Ruminococcus sp. provided by the present invention exhibits the highest catalytic efficiency for glucose and the highest allulose production rate in the conversion reaction, making it the optimal combination.

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

Claims

1. A nucleic acid combination comprising a nucleic acid encoding glucose isomerase from Thermus thermophilus and a nucleic acid encoding D-allulose 3-epimerase from Ruminococcus sp., The nucleotide sequence of the nucleic acid encoding the glucose isomerase is shown in SEQ ID NO:

3. A nucleic acid combination characterized in that the nucleotide sequence of the nucleic acid encoding D-allulose 3-epimerase is shown in SEQ ID NO:

4.

2. An expression vector comprising a backbone vector and the nucleic acid combination described in claim 1.

3. A modified bacterium comprising the expression vector described in claim 2.

4. The modified bacterium according to claim 3, characterized in that the starting organism for the modified bacterium is Bacillus subtilis.

5. A method for constructing a modified bacterium according to claim 3 or 4, wherein the nucleic acid encoding glucose isomerase and the nucleic acid encoding D-allulose 3-epimerase are ligated to a backbone vector to obtain the expression vector, The expression vector is introduced into Bacillus subtilis to obtain modified bacteria. A construction method characterized by the following.

6. A method for preparing D-allulose, characterized by using a fermented culture, extract, or extracted enzyme solution of the modified bacteria described in claim 3 or 4 as a catalyst to catalyze the reaction of substrate glucose to produce D-allulose.

7. The modified bacteria are fermented and cultured, and the cells are subjected to lysing to obtain a crude enzyme solution containing glucose isomerase and D-allulose 3-epimerase. A step of adding substrate glucose to the crude enzyme solution to obtain a conversion syrup, The preparation method according to claim 6, characterized by comprising the steps of sequentially filtering, purifying, chromatographically separating, and concentrating the conversion syrup to obtain D-allulose and fructose, respectively.

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