Method for converting glucose or high fructose corn syrup into D-psicose

Abstract

The invention relates to a method for converting glucose or high fructose corn syrup into D-psicose. A catalyst used in the method is a microorganism containing an artificial polypeptide (ARG, SEQ ID NO: 3, AFG, SEQ ID NO: 4), a plasmid containing encoding genes (pET-22b-ARG, SEQ ID NO: 16, pET-22b-AFG, SEQ ID NO: 17, pMA5-ARG, SEQ ID NO: 18) of the polypeptide, a recombinant microorganism for expressing the polypeptide, and a method for producing D-psicose by using the polypeptide or the recombinant microorganism with D-glucose or high fructose corn syrup as a substrate.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a method for converting D-glucose or fructose syrup into D-psicose. Background Art

[0002] D-allulose (D-allulose or D-psicose) is a functional sugar substitute with high added value. It is a diastereomer at the C-3 position of D-fructose. It has good taste, no odor, low calories, and anti-hypertension and hyperlipidemia. It has important application value in the fields of food and medicine. The content of D-psicose in nature is extremely low. In the early days, it was usually synthesized by chemical methods, but this method is costly, difficult to process, complex to purify, and easy to cause environmental pollution. It has been basically replaced by biotransformation. The biotransformation method mainly uses D-psicose 3-dimerase (DAE) or microorganisms containing the enzyme to catalyze the isomerization of D-fructose into D-psicose. However, due to the high cost of D-fructose raw materials, it is difficult to reduce the production cost of D-psicose. Therefore, finding low-value raw materials to replace D-fructose is of great significance for reducing the production cost of D-psicose.

[0003] D-glucose can be obtained by starch hydrolysis, and its price is much lower than fructose, making it an ideal raw material to replace D-fructose; fructose syrup is a mixture of D-fructose and D-glucose, which is obtained from starch sugar catalyzed by glucose isomerase, and its cost is also low, which has the potential value of replacing D-fructose. However, it is still difficult to convert these two raw materials into D-psicose. Recently, the patent "Method for producing D-psicose from glucose" was published.

[0004] (CN113667707B), patent "A method for producing D-tagatose and D-psicose from glucose" (CN117512033B), patent "A composite engineered bacteria and a method for producing D-psicose"

[0005] (CN116855553A) and "Recombinant bacteria for producing D-psicose by fermentation and preparation method thereof"

[0006] (CN118813432A) discloses some technical solutions for converting glucose into D-psicose, indicating that the conversion of glucose into D-psicose has become a hot topic in research. However, there are still some shortcomings in the above methods, such as low concentration of D-psicose produced or low conversion rate, long conversion pathway, significant increase in cost due to the use of multiple enzymes, increased difficulty in product separation and purification due to the use of cell disruption solution, or long microbial culture cycle, which means that this technology still has a lot of room for improvement. Summary of the invention

[0007] The object of the present invention is to provide a method for more efficiently catalyzing D-glucose or fructose syrup to synthesize D-psicose. To achieve the above object, the present invention provides the following technical solutions:

[0008] A method for converting glucose or fructose syrup into D-psicose, wherein a microorganism expressing an artificial polypeptide is added to a reaction system, wherein the amino acid sequence of the artificial polypeptide is selected from SEQ ID NO: 3 and SEQ ID NO: 4.

[0009] Wherein, the microorganism is one of Escherichia coli or Bacillus subtilis.

[0010] Wherein, the microorganism is a polynucleotide containing an artificial polypeptide, the sequence of which is selected from SEQ ID NO: 9 and SEQ ID NO: 10, or a plasmid containing an artificial polypeptide, the sequence of which is selected from SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

[0011] The reaction system contains D-glucose or F55 fructose syrup as substrate, wherein the amount of D-glucose added is 50-600 g / L, and the volume ratio of F55 fructose syrup added is 5%-60%; the pH value is 5.0-9.0; and the amount of Co 2+ , Mn 2 + Mg 2+ , Ca 2+ , Cu 2+ , Fe 2+ 、Ni 2+ 、Zn 2+ One or more of the following: the amount of microorganism added is 10-100 g / L, the reaction temperature is 45-75°C, and the reaction time is 0.5-12 hours.

[0012] The present invention designs an artificial polypeptide, which is fused with three parts: a glucose isomerase functional domain (GI), a linking peptide (LK) and a D-psicose 3-epimerase functional domain (DAE); the artificial polypeptide has the catalytic function of converting D-glucose into D-fructose and the catalytic function of converting D-fructose into D-psicose, thereby realizing the one-pot conversion of D-glucose to D-psicose. The artificial polypeptide is not a simple functional superposition. Different combinations and different linking peptides can affect the catalytic effect of the artificial polypeptide. A good design can shorten the distance between the functional domains, reduce the substrate transfer path, and play the role of "1+1>2". The artificial polypeptide-based bioconversion technology disclosed in the present invention not only has a simple process and a short microbial culture cycle, but also has a good catalytic effect. A higher D-psicose product concentration can be obtained using D-glucose as a substrate, and a mixed syrup composed of D-fructose and D-glucose can also be obtained, which is convenient for secondary conversion, and therefore has application value.

[0013] The artificial polypeptide (GRA, SEQ ID NO: 1, GFA, SEQ ID NO: 2, ARG, SEQ ID NO: 3, AFG, SEQ ID NO: 4) consists of three functional domains, including a glucose isomerase functional domain (GI), a linking peptide (LK) and a D-psicose 3-epimerase functional domain (DAE). The artificial polypeptide can convert D-glucose into a mixture of D-fructose and D-psicose.

[0014] A plasmid for expressing the artificial polypeptide is constructed, which contains a polynucleotide encoding the artificial polypeptide (pET-22b-GRA, SEQ ID NO: 14, pET-22b-GFA, SEQ ID NO: 15, pET-22b-ARG, SEQ ID NO: 16, pET-22b-AFG, SEQ ID NO: 17, pMA5-ARG, SEQ ID NO: 18), and can give the microorganism the ability to express the artificial polypeptide (GRA, SEQ ID NO: 1, GFA, SEQ ID NO: 2, ARG, SEQ ID NO: 3, AFG, SEQ ID NO: 4) of the present invention.

[0015] A recombinant microorganism was constructed, which has the ability to express artificial polypeptides (GRA, SEQ ID NO: 1, GFA, SEQ ID NO: 2, ARG, SEQ ID NO: 3, AFG, SEQ ID NO: 4). In order to verify the linking mode of different functional domains and the effect of the type of linking peptide on the catalytic activity of the four artificial polypeptides, relevant functional verification was carried out.

[0016] A method for converting D-glucose or fructose syrup into D-psicose using the artificial polypeptide of the present invention is established.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The present invention utilizes an artificial polypeptide to realize the bioconversion of D-glucose to D-psicose, and improves the original D-fructose as the raw material to D-glucose or fructose syrup as the raw material, thereby reducing the raw material cost.

[0019] (2) The artificial polypeptide of the present invention uses a linker peptide to pull in the GI and DAE functional domains, thereby improving the catalytic efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 、ARG structure diagram

[0021] Figure 2 , ARG expression plasmid map (E. coli)

[0022] Figure 3 , ARG expression plasmid map (Bacillus subtilis)

[0023] Figure 4 , SDS-PAGE gel electrophoresis of recombinant microorganisms expressing four artificial peptides, M: Maker, L1: GRA, L2: GFA, L4: ARG, L5: AFG, ck: control group without IPTG added to the fermentation broth

[0024] Figure 5 , 4 Comparison of HPLC results of D-glucose, D-fructose and D-psicose in the reaction system when the two artificial peptides were catalyzed by D-glucose as substrate for 12 hours

[0025] Figure 6 , 4 The GI and DAE activities of the artificial peptides were verified using D-glucose and D-fructose as substrates, a, glucose isomerase activity; b, D-psicose 3-epimerase activity

[0026] Figure 7 , 4 Trend of artificial peptides with different configurations catalyzing the production of D-psicose from D-glucose

[0027] Figure 8 Time curve of D-psicose production by recombinant Escherichia coli and recombinant Bacillus subtilis using D-glucose as substrate

[0028] Fig. 9 , the amount of D-psicose produced by ARG-catalyzed fructose syrup at different temperatures

[0029] Fig.10 , the amount of D-psicose produced by ARG-catalyzed fructose syrup under different buffers and pH

[0030] Fig.11 , the amount of D-psicose produced by ARG catalyzed by fructose syrup when different metal ions were added

[0031] Fig.12 , the amount of D-psicose produced by ARG-catalyzed fructose syrup under different cell addition amounts

[0032] Fig.13 , ARG catalyzes the production of D-psicose in different fructose syrups under optimal conditions

[0033] Fig.14 , the amount of D-psicose produced by D-glucose catalyzed by ARG at different temperatures

[0034] Fig.15 , the amount of D-psicose produced by D-glucose catalyzed by ARG under different buffers and pH

[0035] Fig.16 , the amount of D-psicose produced by D-glucose catalyzed by ARG when different metal ions were added

[0036] Fig.17 , the amount of D-psicose produced by D-glucose catalyzed by ARG under different cell addition amounts

[0037] Fig.18 , ARG catalyzes the production of D-psicose at different concentrations of D-glucose under optimal conditions DETAILED DESCRIPTION

[0038] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] A method for catalyzing D-glucose or fructose syrup to synthesize D-psicose, characterized in that D-glucose or fructose syrup is used as a substrate and an artificial polypeptide (GRA, SEQ ID NO: 1, GFA, SEQ ID NO: 2, ARG, SEQ ID NO: 3, AFG, SEQ ID NO: 4) expression strain is used to directly convert D-glucose or fructose syrup into D-psicose.

[0040] The artificial polypeptide consists of three functional domains, including a glucose isomerase domain (GI), a linker peptide (LK) and a D-psicose 3-epimerase domain (DAE). Two linker peptides (R, SEQ ID NO: 5 and F, SEQ ID NO: 6) are used to connect glucose isomerase (GI) SEQ ID NO: 11 and D-psicose 3-epimerase (DAE) SEQ ID NO: 12 to form four artificial polypeptides, which are named GRA, GFA, ARG, and AFG (GRA, SEQ ID NO: 1, GFA, SEQ ID NO: 2, ARG, SEQ ID NO: 3, and AFG, SEQ ID NO: 4).

[0041] The present invention constructs plasmids expressing the above artificial polypeptides, and the sequences thereof are pET-22b-GRA, SEQ ID NO: 14, pET-22b-GFA, SEQ ID NO: 15, pET-22b-ARG, SEQ ID NO: 16, pET-22b-AFG, SEQ ID NO: 17, pMA5-ARG, SEQ ID NO: 18

[0042] The invention constructs a recombinant strain containing the plasmid, and the host is Escherichia coli (BL21 (DE3)) or Bacillus subtilis (WB600).

[0043] In order to verify the linking mode of different functional domains and the effect of the type of linking peptide on the catalytic activity of the four polypeptides, relevant functional verification was carried out.

[0044] The present invention establishes a method for synthesizing D-psicose by catalyzing D-glucose or fructose syrup based on the above polypeptide and strain, specifically: pH = 5.0-9.0, containing Co 2+ , Mn 2+ Mg 2+ , Ca 2+ , Cu 2+ , Fe 2+ 、Ni 2+ 、Zn 2+ One or more of the above recombinant microorganisms, containing the above recombinant microorganisms, with a concentration of 10-100 g / L, a reaction temperature of 45-75 ° C, and a reaction time of 0.5-12 hours; preferably: pH = 7.5, containing 0.5 mmol / L Co 2+ and 0.5mmol / L Mn 2+ , containing 75g / L microorganisms, the reaction temperature is 65℃, and the reaction time is 12 hours.

[0045] The culture medium involved in the following examples:

[0046] (1) LB liquid culture medium, composed of 5.0 g / L yeast powder, 10 g / L tryptone, and 10 g / L sodium chloride.

[0047] (2) LB solid medium, composed of 5.0 g / L yeast powder, 10 g / L tryptone, 10 g / L sodium chloride, and 20 g / L agar powder.

[0048] (3) 10×Spizizen mother solution: 14 g K2HPO4, 6 g KH2PO4, 2 g (NH4)2SO4, 1 g Na3C6H5O7·7H2O, 0.2 g MgSO4·7H2O, 1 L tap water, autoclave at 121°C for 20 min.

[0049] (4) GMI solution: 1 mL 10× acid hydrolyzed casein, 0.1 mL 10% yeast extract solution, 0.25 mL 20% D-glucose solution, 0.2 mL 1% acid hydrolyzed casein, and add sterile water to 10 mL.

[0050] (5) GMII solution: 1 mL 10× acid hydrolyzed casein, 0.05 mL 10% yeast extract solution, 0.25 mL 20%

[0051] D-glucose solution, 0.04 mL 1% acid hydrolyzed casein, 0.05 mL 0.1 mol / L CaCl2 solution, 1 mL 25 mmol / L MgCl2 solution, add sterile water to 10 mL.

[0052] (6) PBS solution: concentration 50 mmol / L pH = 7, 5.43 g disodium hydrogen phosphate, 4.56 g sodium dihydrogen phosphate, adjust the solution pH to 7 with hydrochloric acid and sodium hydroxide, and make up to 1 L.

[0053] Example 1: Construction of a plasmid expressing an artificial polypeptide

[0054] The present invention uses two linking peptides (R, SEQ ID NO: 5 and F, SEQ ID NO: 6) to connect glucose isomerase (GI) SEQ ID NO: 11 and D-psicose 3-epimerase (DAE) SEQ ID NO: 12 to form four artificial polypeptides, which are named GRA, GFA, ARG, and AFG (GRA, SEQ ID NO: 1, GFA, SEQ ID NO: 2, ARG, SEQ ID NO: 3, and AFG, SEQ ID NO: 4). The structural diagram of ARG is shown in the attached figure. Figure 1As shown. The coding genes of GRA, GFA, ARG, and AFG (SEQ ID NO: 7, SEQ ID NO: 8, ARG, ID NO: 9, SEQ ID NO: 10) were artificially synthesized and inserted between the NdeI and XhoI sites in the Escherichia coli expression vector pET-22b to construct plasmids expressing the above-mentioned artificial polypeptides, which were named pET22b-GRA, pET22b-GFA, pET22b-ARG, and pET22b-AFG (SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 17), respectively. The coding sequence of ARG (SEQ ID NO: 9) was inserted between the NdeI and BamHI restriction sites in the Bacillus subtilis plasmid pMA5 to construct the plasmid pMA5-ARG (SEQ ID NO: 18) capable of expressing ARG in Bacillus subtilis. The plasmid schematic diagrams of pET22b-ARG and pMA5-ARG are shown in the attached figure. Figure 2 and attached Figure 3 shown.

[0055] Example 2: Construction of recombinant microorganisms expressing artificial polypeptides

[0056] The recombinant plasmids pET-22b-GRA, pET-22b-GFA, pET-22b-ARG and pET-22b-AFG (SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17) were transformed into Escherichia coli BL21 (DE3) to obtain recombinant Escherichia coli, named BL21-GRA, BL21-GFA, BL21-ARG, BL21-AFG. The recombinant plasmid pMA5-ARG (SEQ ID NO: 18) was transformed into Bacillus subtilis WB600 to obtain recombinant Bacillus subtilis, named WB600-ARG.

[0057] The transformation conditions may be conventional conditions. When the host cell is Escherichia coli, the transformation method is as follows: add the recombinant plasmid to 50 μL of Escherichia coli BL21 (DE3) competent cells, heat shock in a 42°C water bath for 90 seconds, and quickly put it back on ice for 2 minutes. Add 500 μL of LB medium without antibiotics, mix well, and culture at 37°C 200r / min for 90 minutes. Centrifuge at 10,000r / min for 1 minute, collect the bacteria, spread them on LB solid medium containing ampicillin antibiotics (100 μg / mL), invert the plate, and culture in a 37°C incubator overnight. After the colony grows on the plate, inoculate it into LBA medium, culture at 37°C 220r / min for 12 hours, and store it at -80°C using glycerol at a final concentration of 20%.

[0058] When the host cell is Bacillus subtilis, the transformation method is as follows: a single colony of Bacillus subtilis WB600 is inoculated into 2.5 mL of GM I solution and cultured overnight at 30°C and 150 r / min. The overnight culture is transferred to a new 2.5 mL of GM I solution at a ratio of 10%, and cultured at 37°C and 220 r / min for 3.5 h. The bacterial solution in the previous step is transferred to 5 mL of GMII solution at a ratio of 10%, and cultured at 37°C and 220 r / min for 1.5 h. 1 mL of bacterial solution is aspirated into a sterile centrifuge tube, centrifuged at 5000 r / min for 5 min to collect the bacteria, 900 μL of supernatant is discarded, and the remaining bacterial solution is blown evenly as a Bacillus subtilis competent cell. The recombinant plasmid pMA5-ARG is added to the above competent cells, kept at 37°C for 30 min, and then transferred to a 37°C shaker, and the bacterial solution is revived at 220 r / min for 3 h. Spread the revived bacteria on LB resistance plates and invert and culture until single colonies grow.

[0059] Example 3: Cultivation of recombinant microorganisms and expression of artificial polypeptides

[0060] The four artificial polypeptide-expressing recombinant E. coli were inoculated into LBA liquid medium and cultured at 37°C 220 r / min overnight to serve as the primary seed solution. The primary seed solution was inoculated into LBA liquid medium at a 2% (v / v) inoculation rate and cultured at 37°C 220 r / min until OD 600 =0.6-0.8, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 1 mmol / L, and induce gene expression for 8 hours at 30°C and 200 r / min. After the induction culture, the cells were collected by centrifugation at 4°C and 8000 r / min, and the cells were washed twice with physiological saline.

[0061] SDS-PAGE electrophoresis verification: After the artificial polypeptide fermentation broth is induced, take an appropriate amount of cells for SDS-PAGE electrophoresis detection and observe the electrophoresis bands. The theoretical size of the four artificial polypeptides GRA, GFA, ARG and AFG is 79kDa. The electrophoresis results are shown in the attached Figure 4 As shown, after the induction of the recombinant cells, a new obvious protein band appeared at about 79 kDa, indicating that the artificial polypeptide was successfully expressed.

[0062] Example 4: Verification of the catalytic function of artificial peptides

[0063] Recombinant E. coli cells expressing the four artificial polypeptides were used as catalysts to react with D-glucose and D-fructose, respectively, to test the production of the products D-fructose and D-psicose. The reaction conditions are shown in Table 2.

[0064] Table 1 Reaction conditions for verification of artificial peptide catalytic function

[0065]

[0066]

[0067] The reaction was carried out for 30 min and then heated to boil for 10 min.

[0068] The production of D-fructose and D-psicose was analyzed by high performance liquid chromatography (HPLC). Figure 5 , Attachment Figure 6 The HPLC analysis conditions are as follows: the chromatographic column is Waters Sugar-Pak I, the detector is a differential detector (RID), the mobile phase is ultrapure water, the flow rate is 0.6 mL / min, the injection volume is 10 μL, the detector temperature is 45°C, and the column oven temperature is 80°C. Under these conditions, the elution times of D-glucose, D-fructose, and D-psicose are 8.5 min, 10.3 min, and 14.6 min, respectively.

[0069] The above experimental results ( Figure 5 and Figure 6 ) showed that the blank control group (empty plasmid E. coli, ck) had no reaction; the four artificial peptides GRA, GFA, ARG and AFG expressed in E. coli all successfully catalyzed the partial conversion of D-glucose into D-fructose ( Figure 6 a), among which GFA had the highest conversion rate of 28.92%, ARG was 21.5%, GRA was 17.14%, and AFG was 16.85%; E. coli expressing ARG and AFG, two artificial peptides, could catalyze the conversion of D-fructose into D-psicose, with conversion rates of 31.17% and 30.6%, respectively, while E. coli expressing GRA and GFA, two artificial peptides, could not catalyze the production of D-psicose. This shows that different linking methods have a significant effect on the catalytic activity of the functional domain.

[0070] Specific Example 5: Changes in the amount of product at different reaction times when D-glucose is used as a substrate

[0071] Substrate concentration: 100 g / L D-glucose, reaction temperature: 55 °C, 50 mmol / L PBS, pH 7.0, 1 mmol / L Co 2+ , cell addition amount 40g / L. Three experimental groups were set up in parallel. Samples were taken at 0, 0.5, 1, 2, 4, 8, and 12h of reaction. The samples were boiled for 10min, diluted 5 times, filtered with a 0.22μm filter membrane, and then tested by HPLC. The results are shown in the attached Figure 7As shown. The results showed that only two artificial peptides, ARG and AFG, successfully catalyzed D-glucose to D-psicose, with yields of 4.38 g / L and 2.72 g / L, respectively; ARG had the best catalytic effect under the same conditions. Compared with the flexible connecting peptide, the rigid α-helical connecting peptide was more conducive to the improvement of glucose isomerase activity, thereby improving the overall conversion rate of D-glucose to D-psicose.

[0072] Example 6: Comparison of catalytic effects of Escherichia coli and Bacillus subtilis expressing ARG

[0073] The recombinant Escherichia coli (BL21-ARG) and recombinant Bacillus subtilis (WB600-ARG) expressing ARG constructed in Example 2 were cultured separately as follows:

[0074] When the host cell is Escherichia coli, the culture method is the same as that in Example 3 "Cultivation of recombinant microorganisms and expression of artificial polypeptides".

[0075] When the host cell is Bacillus subtilis, the culture method is as follows: the recombinant Bacillus subtilis is inoculated into LB-LB-Kana liquid culture medium, and cultured overnight at 37°C 220r / min as the primary seed liquid. The primary seed liquid is taken and inoculated into LB liquid culture medium containing kanamycin sulfate (50μg / mL) at a 2% (v / v) inoculation amount, and the culture time at 37°C 220r / min is the same as that of Escherichia coli. The cells are collected by centrifugation at 4°C and 8000r / min, and the cells are washed twice with ultrapure water.

[0076] The specific reaction conditions were the same as those in Example 5. The curves of the changes in the production of D-psicose from D-glucose catalyzed by different ARG expression hosts over time are shown in the attached figure. Figure 8 The results showed that both hosts were able to catalyze the conversion of D-glucose into D-psicose, and the recombinant E. coli produced more D-psicose per unit time, so it was the preferred host.

[0077] Example 7: Experiment on the production of D-psicose from fructose syrup by ARG and comparison of different catalytic conditions

[0078] The optimal catalytic reaction conditions for ARG to catalyze fructose syrup to produce D-psicose were investigated. Different temperatures, pH values, metal ions, cell addition amounts and substrate concentrations were used for the reactions. The reaction temperature range was 45-75°C, the reaction pH range was sodium acetate buffer (50mmol / L, pH 4.5-6.0), sodium phosphate buffer (50mmol / L, pH 6.0-7.0) and Tris-HCl buffer (50mmol / L, pH7.0-9.0), and the metal ion range was 1mmol / L Co 2+, Mn 2 + 、Co 2+ / Mn 2+ (0.5mmol / L CO 2+ and 0.5mmol / L Mn 2+ ),Mg 2+ , Ca 2+ , Cu 2+ , Fe 2+ 、Ni 2+ or Zn 2+ Or ethylenediaminetetraacetic acid (EDTA), the range of cell addition was 10-100 g / L. The catalytic condition optimization test method was to conduct the experiment under standard conditions (55°C, 50 mmol / L PBS buffer, pH 7.0, 1 mmol / L Co 2+ , 40g / L cells, reaction time 1h, take out after the reaction, boil for 10min. Centrifuge at 10,000r / min for 10min, take 200μL supernatant, add to 800μL ultrapure water, mix well, dilute 5 times. Filter with 0.22μm filter membrane, HPLC detection). The experimental results are shown in the attached Fig. 9 , Attachment Fig.10 , Attachment Fig.11 , Attachment Fig.12 , Attachment Fig.13 ARG can catalyze the reaction of fructose syrup to produce D-psicose under different reaction conditions. The reaction conditions have a great influence on the amount of D-psicose produced. The preferred reaction conditions are: 65℃, 50mmol / L Tris-HCl pH7.5, 0.5mmol / L Co 2+ / Mn 2+ , cell concentration 75g / L. Under the optimal conditions, when the volume ratio of substrate F55 fructose syrup added was 70% (v / v), 92.7g / L of D-psicose was produced. The final ratio of D-glucose, D-fructose and D-psicose was 2.5:2.3:1, and the proportion of D-psicose in the generated mixed syrup was 17.2%.

[0079] Example 8: Comparison of different catalytic conditions for ARG to produce D-psicose from D-glucose

[0080] The reaction conditions were similar to those in Example 6, except that the reaction substrate was changed to glucose. The reaction was carried out at different temperatures, pH values, metal ions, cell addition amounts, and substrate concentrations. The amount of D-psicose produced was as shown in the attached table. Fig.14 , Attachment Fig.15 , Attachment Fig.16 , Attachment Fig.17 and attached Fig.18The results show that the optimal conditions for producing D-psicose using D-glucose as substrate are: 65℃, 50mmol / L Tris-HCl pH 7.5, 0.5mmol / L Co 2+ / Mn 2+ , cell concentration 75g / L. Under this condition, when the concentration of substrate glucose was 600g / L, 103.2g / L g / L of D-psicose was produced, the ratio of D-glucose, D-fructose and D-psicose in the final product was 2.5:2.3:1, and the proportion of D-psicose in the generated mixed syrup was 17.2%.

[0081] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for converting glucose or fructose syrup into D-psicose, characterized in that: A microorganism expressing an artificial polypeptide is added to the reaction system, wherein the amino acid sequence of the artificial polypeptide is selected from SEQ ID NO: 3 and SEQ ID NO:

4.

2. The microorganism according to claim 1, characterized in that It is a type of Escherichia coli or Bacillus subtilis.

3. The microorganism according to claim 2, characterized in that A polynucleotide encoding an artificial polypeptide, the sequence of which is selected from SEQ ID NO: 9 and SEQ ID NO: 10, or a plasmid encoding an artificial polypeptide, the sequence of which is selected from SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO:

18.

4. The reaction system according to claim 1, characterized in that: Contains D-glucose or F55 fructose syrup as a substrate, wherein the amount of D-glucose added is 50-600 g / L, and the volume ratio of F55 fructose syrup added is 5%-60%; the pH value is 5.0-9.0; contains Co2+, Mn 2+ Mg 2+ , Ca 2+ , Cu 2+ , Fe 2+ 、Ni 2+ 、Zn 2+ One or more of the following: the amount of microorganism added is 10 to 100 g / L, the reaction temperature is 45 to 75°C, and the reaction time is 0.5 to 12 hours.

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