Complex engineered bacteria and method for producing D-allulose

Abstract

The present invention relates to the technical field of biotechnology, particularly to a modified bacterium and a method for producing D-allulose. The present invention involves expressing glucose isomerase and D-allulose 3-epimerase from a specific source in Bacillus subtilis, respectively, and then fermenting the resulting enzyme combination containing glucose isomerase and D-allulose 3-epimerase. The enzyme combination is then used to catalyze the isomerization of the substrate glucose, ultimately producing D-allulose. The method provided by the present invention significantly improves the conversion rate of D-allulose and significantly shortens the conversion time from glucose to D-allulose. At the same time, the present invention allows the use of inexpensive glucose as a raw material, significantly reducing the production cost of D-allulose. The present invention also allows the synchronous production of fructose glucose syrup, thereby saving the input required for the fructose glucose 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 to a complex modified bacterium and a method for producing 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 studies have also found that D-allulose also has the ability to lower blood lipids and blood glucose levels. The U.S. Food and Drug Administration (FDA) has designated D-allulose as generally recognized as safe (GRAS), allowing it to be used as a food additive. D-Allulose is expected to have a wide range of applications in food and health products. However, existing processes primarily use fructose as a raw material, resulting in high raw material costs. Although some processes use glucose as a raw material, they require a long production cycle, consume a large amount of labor and materials, and are therefore expensive, making them unsuitable for industrial production. Summary of the Invention

[0003] In view of this, the present invention provides a composite modified bacterium and a method for producing D-allulose. The present invention uses glucose isomerase and D-allulose 3-epimerase from a specific source to catalyze the isomerization of glucose to produce D-allulose, which can significantly improve the conversion rate of D-allulose, shorten the production time of D-allulose, and reduce production costs.

[0004] In order to achieve the above objectives of the invention, the present invention provides the following technical solutions:

[0005] The method for producing D-allulose is as follows: 1) introducing genes encoding glucose isomerase derived from Thermus thermophilus and D-allulose 3-epimerase derived from Ruminococcus sp. into Bacillus subtilis to obtain GI-modified bacteria and DPE-modified bacteria, respectively; 2) mixing the GI-modified bacteria and the DPE-modified bacteria, inoculating the mixture into a fermentation medium, and fermenting the mixture; disrupting the bacterial cells to obtain a crude enzyme solution containing glucose isomerase and D-allulose 3-epimerase; 3) catalyzing the isomerization reaction of glucose using the crude enzyme solution as a catalyst to obtain D-allulose.

[0006] In step 1) of the present invention, the amino acid sequence of the glucose isomerase is a) as shown in SEQ ID NO:1; b) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the sequence shown in SEQ ID NO: 1, without altering the protein activity, or c) 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 the sequence shown in SEQ ID NO: 1; 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.

[0007] In step 1) of the present invention, the gene encoding the glucose isomerase is optimized according to Bacillus subtilis codon preference, and the optimized nucleotide sequence is shown in SEQ ID NO:3; The gene encoding the D-allulose 3-epimerase was optimized according to Bacillus subtilis codon preference, and the optimized nucleotide sequence is shown in SEQ ID NO:4.

[0008] In the present invention, the glucose isomerase GI is derived from Thermus thermophilus, has 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 registration number MBS6425357.1, and its amino acid sequence is SEQ ID NO: 2.

[0009] 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.

[0010] In step 2) of the present invention, the mixed fermentation was specifically cultured at 37°C, 200 rpm for 48 hours.

[0011] In step 2) of the present invention, the fermentation medium is prepared by adding 10 g of peptone, 5 g of yeast powder, 2.5 g of potassium dihydrogen phosphate, 15 g of dipotassium hydrogen phosphate, 0.1 g of manganese chloride tetrahydrate, 0.1 g of magnesium sulfate heptahydrate, 6 g of glucose, and water to a volume of 1 L.

[0012] In step 2) of the present invention, before inoculating the modified bacteria into a fermentation medium and mixing to ferment, the modified bacteria are further prepared into a seed solution, i.e., the modified bacteria are inoculated into an LB seed medium and cultured overnight at 37°C and 200 rpm to obtain a seed solution.

[0013] In step 2) of the present invention, the disruption of the bacterial cells is specifically carried out by mixing lysozyme and the fermentation liquid at a mass / volume ratio of 0.1‰ (i.e., a final concentration of lysozyme of 0.1 g / L) and reacting them at 37°C, 200 rpm for 0.5 to 4 hours. In some specific embodiments, the conditions for disrupting the reacted bacterial cells are 37°C, 200 rpm, and 1 hour.

[0014] In step 3) of the present invention, the isomerization reaction temperature is 60°C and the time is 2 to 24 hours. In some specific embodiments, the isomerization reaction temperature is 60°C and the time is 2 hours, 4 hours, 6 hours, 12 hours, or 24 hours.

[0015] After the isomerization reaction described in the present invention, the reaction product can be successively filtered, purified, chromatographically separated, and concentrated to obtain D-allulose and fructose, respectively. The filtration involves sequentially using a ceramic membrane with a pore size of 8-10 nm and a nanofiltration membrane with a pore size of 100-300 Daltons. The purification involves first passing through a cation resin and then through an anion resin to remove anions and cations. In a specific embodiment, the cation resin type is D001-FD and the anion resin type is D354-FD.

[0016] The present invention further provides a composite engineered bacterium, including a Bacillus subtilis strain expressing glucose isomerase from Thermus thermophilus and a Bacillus subtilis strain expressing D-allulose 3-epimerase from Ruminococcus sp. In some specific embodiments, the Bacillus subtilis strain is Bacillus subtilis WB600.

[0017] The method for constructing the composite engineered bacterium according to the present invention 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 and fully synthesized by Kingsley Biotechnology. The optimized GI gene sequence is SEQ ID NO:3, and the optimized DPE gene sequence is SEQ ID NO:4. These were then ligated into the vector pWB980 by homologous recombination to obtain the recombinant plasmids pWB980-GI and pWB980-DPE. The SacB signal peptide gene fragment on the plasmids was then removed during recombination, and the GI and DPE genes were constitutively expressed intracellularly under the control of the p43 promoter. 2) The recombinant plasmids pWB980-GI and pWB980-DPE are transformed into Bacillus subtilis WB600, respectively, to obtain recombinant Bacillus subtilis WB600 / GI and WB600 / DPE.

[0018] The present invention further provides the use of the complex modified bacteria in the production of D-allulose or in the preparation of hypoglycemic and hypolipidemic products.

[0019] In a specific embodiment of the present invention, the method for preparing D-allulose includes: (1) Inoculate each of the two modified bacteria (WB600 / GI and WB600 / DPE) into LB seed medium, cultivate overnight at 37°C and 200 rpm, and concentrate to obtain a seed solution with an OD600 value of 3 to 6. (2) The seed solutions of the two recombinant strains obtained in step (1) were co-inoculated into the same fermentation medium at a ratio of 0.1% (v / v), respectively, and mixed fermentation was carried out. The mixture was 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) at 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 resin and then through an anion resin to remove anions and cations, the type of the cation resin is D001-FD, and the type of the anion resin is D354-FD, to obtain a desalted sugar solution. (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.

[0020] The present invention involves expressing glucose isomerase and D-allulose 3-epimerase from a specific source in Bacillus subtilis, respectively, and then fermenting the resulting enzyme combination to produce D-allulose, which is then used to catalyze the isomerization of the substrate glucose, ultimately producing D-allulose. The method provided by the present invention significantly improves the conversion rate of D-allulose and significantly shortens the conversion time from glucose to D-allulose. At the same time, the present invention uses inexpensive glucose as a raw material, significantly reducing the production cost of D-allulose. The present invention also allows for the synchronous production of fructose glucose syrup, saving the input of fructose glucose syrup production lines and significantly improving economic benefits. [Effects of the Invention]

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The present invention uses an enzyme from a specific source to catalyze the conversion of glucose into D-allulose in a short time, thereby significantly shortening the production time and significantly improving economic benefits, making it suitable for industrial production.

[0023] (2) The present invention uses glucose, an inexpensive raw material, as a substrate, thereby reducing the production cost of D-allulose.

[0024] (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 profiles of recombinant plasmids pWB980-GI and pWB980-DPE. [Figure 2]An SDS-PAGE electrophoresis photograph is shown, in which lanes 1 to 4 represent: 1: WB600 / pWB980, 2: WB600 / pWB980-GI, 3: WB600 / pWB980-DPE, and 4: WB600 / pWB980-GI + WB600 / pWB980-DPE, respectively. [Figure 3] 1 shows a liquid phase detection profile. [Figure 4] 1 shows a liquid phase detection profile. [Figure 5] 1 shows a liquid phase detection profile. DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention provides a complex modified bacterium and a method for producing 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 invention 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 the Glucose Isomerase (GI)-Modified Bacterium of the Present Invention

[0030] 1) The glucose isomerase protein sequence (GI, SEQ ID NO: 1) from Thermus thermophilus was selected, and its coding gene was optimized according to the Bacillus subtilis codon. The optimized GI gene sequence is SEQ ID NO: 3. Kingsley Biotechnology completely synthesized the gene, ligated it into the pUC57 vector, and named pUC57-GI.

[0031] 2) Using the synthesized recombinant plasmid pUC57-GI as a template, PCR amplification is performed using GI-F and GI-R as primers, and the GI fragment is obtained by gel purification.

[0032] 3) Using the plasmid vector pWB980 as a template and pWB980-F1 and pWB980-R1 as primers, PCR amplification is carried out, and the linearized gene fragment P1 from which the SacB signal peptide has been removed is obtained by gel purification.

[0033] 4) The GI fragment and the pWB980 linearized gene fragment P1 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 / GI, and the recombinant plasmid was designated pWB980-GI (the plasmid profile is shown in Figure 1). The accuracy of the transformant was verified by colony PCR and sequence analysis (primer sequences are shown in Table 1). [Table 1]

[0034] 5) Positive transformants that appeared on the kanamycin-containing LB medium plate were extracted 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 a crude GI-DPE enzyme solution (the results of SDS-PAGE electrophoresis are shown in Figure 2).

[0035] 6) Glucose was added to the crude enzyme solution obtained above to a final concentration of 600 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 membrane, and the filtrate was subjected to high-performance liquid chromatography analysis.

[0036] Bacillus subtilis WB600 spiked with empty plasmid pWB980 was used as the blank control, and other operating conditions were the same.

[0037] 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.

[0038] As can be seen from the liquid chromatography analysis results, the peak times for glucose, fructose, and D-allulose were 14.1 min, 17.9 min, and 26.9 min, respectively.

[0039] The liquid phase profile of the blank control (B. subtilis WB600 / pWB980) contained only glucose, while the liquid phase profile of the experimental group (B. subtilis WB600 / pWB980-GI) contained both glucose and fructose (the liquid phase profiles are shown in Figure 3). These results indicate that the GI crude enzyme solution produced by the above method converted glucose to fructose, and peak area calculations showed that the converted sugar solution contained 330 g / L of glucose and 270 g / L of fructose, with an occupancy ratio of 55:45.

[0040] Example 2: Construction of modified bacteria expressing D-allulose 3-epimerase (DPE) and functional verification

[0041] 1) D-allulose 3-epimerase (DPE, SEQ ID NO: 2) derived from Ruminococcus sp. was selected, and its coding gene was optimized according to Bacillus subtilis codons. The optimized DPE gene sequence is SEQ ID NO: 4. Kingsley Biotechnology completely synthesized the gene, ligated it into the pUC57 vector, and named pUC57-DPE.

[0042] 2) The coding gene was codon-optimized and then completely synthesized by Kingsley Biotechnology, and the synthesized gene was ligated into the pUC57 vector and named pUC57-DPE.

[0043] 3) Using the synthesized recombinant plasmid pUC57-DPE as a template, PCR amplification is performed using DPE-F and DPE-R as primers, and the DPE fragment is obtained by gel recovery and purification.

[0044] 4) Using the plasmid vector pWB980 as a template and pWB980-F2 and pWB980-R2 as primers, PCR amplification is carried out, and the linearized gene fragment P2 from which the SacB signal peptide has been removed is obtained by gel purification.

[0045] 5) 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 recombinant B. subtilis was designated WB600 / DPE, and the recombinant plasmid was designated pWB980-DPE (the plasmid profile is shown in Figure 1). The accuracy of the transformant was verified by colony PCR and sequence analysis (primer sequences are shown in Table 1).

[0046] 6) Transformants with accurate colony PCR and sequencing 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 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 DPE enzyme solution (SDS-PAGE electrophoresis results are shown in Figure 2).

[0047] 7) Fructose was added to the crude enzyme solution obtained above at a final concentration of 600 g / L, and the mixture was allowed to react for 24 hours at 60°C. 0.02 ml of the reaction mixture was sampled and diluted 50-fold with pure water, then inactivated by placing it in a 100°C water bath for 10 minutes. The mixture was then 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.

[0048] Bacillus subtilis WB600 spiked with empty plasmid pWB980 was used as the blank control, and other operating conditions were the same.

[0049] 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.

[0050] The results of liquid chromatography analysis (see FIG. 4) showed that the peak times for glucose, fructose, and D-allulose were 14.1 min, 17.9 min, and 26.9 min, respectively.

[0051] The liquid phase profile of the blank control (Bacillus subtilis WB600 / pWB980) contained only fructose, while the liquid phase profile of the experimental group (Bacillus subtilis WB600 / pWB980-DPE) contained both fructose and D-allulose (the liquid phase profiles are shown in Figure 5). From these results, it was determined that the DPE crude enzyme solution produced by the above method converted fructose to D-allulose, and the peak area calculation showed that the post-conversion sugar solution contained 426 g / L of fructose and 174 g / L of D-allulose, with an occupancy ratio of 71:29.

[0052] Example 3: Co-fermentation of GI-modified bacteria and DPE-modified bacteria to produce fructoglucose syrup and D-allulose

[0053] The specific steps are as follows: (1) The two modified bacteria described in Example 1 and Example 2, respectively, are 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 solutions of the two recombinant strains obtained in step (1) were co-inoculated into the same fermentation medium at a ratio of 0.1% (v / v), respectively, and mixed fermentation was carried out. The mixture was 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) at 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 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.

[0054] Comparative Example 1

[0055] 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 modified Bacillus subtilis bacteria expressing glucose isomerase and D-allulose 3-epimerase were constructed according to the methods of Examples 1 and 2 of the present invention, respectively. Then, an invert syrup containing glucose, fructose, and allulose was prepared according to the method of Example 3 of the present invention.

[0056] Comparative Example 2 (GI is not of the same genus (56% homology with the GI of the present invention), DPE is the same)

[0057] Glucose isomerase (GI, the nucleic acid sequence of which can be found in SEQ ID NO: 1 of CN113980880) from the CN113980880 patent was selected, and a modified Bacillus subtilis bacterium expressing GI was constructed according to the method of Example 1 of the present invention. The constructed GI expression strain and the DPE expression strain constructed in the present invention were used to prepare a conversion syrup containing glucose, fructose, and allulose according to the method of Example 3 of the present invention.

[0058] Comparative Example 3 (GI is not of the same genus (GI homology with the present invention is 97%), DPE is the same)

[0059] Glucose isomerase (GI, registration number WP_126200404.1) from the thermophilic black amphibian Thermus scotoductus was selected and codon-optimized (see SEQ ID NO: 5 for the GI nucleic acid sequence), and modified Bacillus subtilis bacteria expressing GI were constructed according to the method of Example 1 of the present invention. Using the constructed GI-expressing strain and the DPE-expressing strain constructed in the present invention, a converted syrup containing glucose, fructose, and allulose was prepared according to the method of Example 3 of the present invention.

[0060] Comparative Example 4 (GI is the same, DPE is different (40% homology with the present invention)

[0061] D-allulose 3-epimerase (DPE, the nucleic acid sequence of which can be found in SEQ ID NO: 3 of CN113980880) was selected from the CN113980880 patent, and a modified Bacillus subtilis bacterium expressing DPE was constructed according to the method of Example 2 of the present invention. The constructed DPE-expressing strain and the GI-expressing strain constructed in the present invention were used to prepare a converted syrup containing glucose, fructose, and allulose according to the method of Example 3 of the present invention.

[0062] Comparative Example 5 (same GI, different DPE (69% homology with the present invention)

[0063] D-allulose 3-epimerase (DPE, WP_183684385.1) from Oribacterium sinus was selected and codon-optimized (see SEQ ID NO: 6 for the nucleic acid sequence), and then modified Bacillus subtilis bacteria expressing DPE were constructed according to the method of Example 2 of the present invention. Using the constructed DPE-expressing strain and the GI-expressing strain constructed in the present invention, a conversion syrup containing glucose, fructose, and allulose was prepared according to the method of Example 3 of the present invention.

[0064] Test Example

[0065] The conversion syrup was prepared according to the methods of Example 3 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.

[0066] [Table 2] TIFF2025532204000004.tif42168

[0067] As can be seen from the above results, the enzyme catalysts produced from the modified bacterial strains of Comparative Examples 1 to 5 and the present invention produced comparable allulose production efficiencies from glucose, ranging from 15 to 18%, but there were significant differences in the time required to reach equilibrium conversion: 6 h, 6 h, 12 h, 4 h, 12 h, and 2 h, respectively. Among these, the enzyme combination from the specific source of the present invention produced the shortest time, significantly lower than the other comparative examples, and the highest allulose production rate, significantly higher than 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 can be determined to be the optimal combination with the highest catalytic activity toward glucose.

[0068] It should be noted that the above are only preferred embodiments of the present invention, and those skilled in the art may make many improvements and modifications without departing from the principle of the present invention, and all of these improvements and modifications shall fall within the protection scope of the present invention.

Claims

1. 1) introducing genes encoding glucose isomerase derived from Thermus thermophilus and D-allulose 3-epimerase derived from Ruminococcus sp. into Bacillus subtilis to obtain GI-modified bacteria and DPE-modified bacteria, respectively; 2) mixing the GI-modified bacteria and the DPE-modified bacteria, inoculating the mixture into a fermentation medium, and fermenting the mixture; disrupting the bacteria to obtain a crude enzyme solution containing glucose isomerase and D-allulose 3-epimerase; 3) A method for producing D-allulose, comprising the step of catalyzing the isomerization reaction of glucose using the crude enzyme solution as a catalyst to obtain D-allulose.

2. In step 1), the amino acid sequence of the glucose isomerase is a) the sequence shown in SEQ ID NO: 1; 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: 1, 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: 1 and having the same or similar protein activity as the sequence shown in SEQ ID NO: 1; 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 substituting, deleting or adding one or more amino acids to the sequence shown in SEQ ID NO: 2, which does not alter the protein activity; or C) The method of claim 1, characterized in that the amino acid sequence is 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.

3. The encoding glucose isomerase gene was optimized according to Bacillus subtilis codon preference, and the optimized nucleotide sequence is the sequence shown in SEQ ID NO: 3: The method of claim 2, wherein the gene encoding D-allulose 3-epimerase is optimized according to Bacillus subtilis codon preference, and the optimized nucleotide sequence is shown in SEQ ID NO:

4.

4. 2. The method according to claim 1, wherein the fermentation in step 2) is specifically cultured at 37°C, 200 rpm for 48 hours.

5. 2. The method of claim 1, wherein the fermentation medium in step 2) comprises water, 10 g / L peptone, 5 g / L yeast powder, 2.5 g / L potassium dihydrogen phosphate, 15 g / L dipotassium hydrogen phosphate, 0.1 g / L manganese chloride tetrahydrate, 0.1 g / L magnesium sulfate heptahydrate, and 6 g / L glucose.

6. The method of claim 1, further comprising the step of preparing a seed solution from the modified bacteria in step 2), i.e., inoculating the modified bacteria into an LB seed medium and culturing overnight at 37°C and 200 rpm, before inoculating the modified bacteria into a fermentation medium and performing mixed fermentation.

7. 2) The method according to claim 1, characterized in that in step 2), the disruption of the bacterial cells is specifically carried out by mixing lysozyme and fermentation liquid in a mass / volume ratio of 0.1‰, and reacting at 37°C, 200 rpm for 1 hour.

8. 2. The method according to claim 1, wherein in step 3), the temperature of the isomerization reaction is 60°C and the time is 2 to 24 hours.

9. The method according to claim 1, further comprising, in step 3), sequentially filtering, purifying, separating by chromatography, and concentrating the reaction product after the isomerization reaction to obtain D-allulose and fructose, respectively.

10. A composite modified bacterium comprising Bacillus subtilis expressing glucose isomerase derived from Thermus thermophilus and Bacillus subtilis expressing D-allulose 3-epimerase derived from Ruminococcus sp.

11. 11. Use of the composite engineered bacterium of claim 10 in the production of D-allulose or in the preparation of hypoglycemic and hypolipidemic products.

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