A method for producing D-allulose and sodium gluconate as a byproduct

Abstract

The present application relates to the technical field of biotechnology, and particularly relates to a preparation method of co-production of D-allulose and sodium gluconate, which comprises the following steps: (1) taking D-fructose and D-glucose as substrates, adding a solvent and a whole-cell catalyst I, adjusting pH, controlling temperature, and obtaining a mixed solution of D-taurulose and sodium gluconate; (2) taking supernatant, adjusting pH to neutral, adding a whole-cell catalyst II under the conditions of ventilation and stirring, and obtaining a mixed solution after reaction; (3) taking the mixed solution, passing through a cation resin column first, collecting effluent, passing through an anion resin column again, and obtaining an allulose product through treatment of the collected effluent; and washing the anion resin column with a sodium hydroxide solution, collecting effluent, and obtaining a sodium gluconate product through treatment of the collected effluent. By using the two-step multi-enzyme cascade biotransformation system, D-fructose and D-glucose are catalyzed to co-produce D-allulose and sodium gluconate, and the production cost is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, in particular to a preparation method of D-psicose and sodium gluconate. BACKGROUND

[0002] D-psicose is a very important rare ketohexose, with a sweetness of 70% of sucrose, but its energy is almost 0, and its solvent properties and taste are similar to sucrose. In the food and medicine industry, D-psicose is an ideal substitute for sucrose. In addition, D-psicose has the functions of inhibiting obesity, reducing blood sugar, reducing blood lipids, etc., and is very suitable for consumption by diabetic patients and obese people, so D-psicose has significant efficacy in medical and health care. At present, the production of D-psicose is mainly based on the isomerization reaction of D-fructose catalyzed by isomerase. This reaction is a reversible reaction, and there is a problem of low conversion rate, with a conversion rate of about 30%.

[0003] Sodium gluconate, also known as sodium pentahydroxyhexanoate, is a white or light yellow crystalline powder, easily soluble in water and slightly soluble in alcohol, insoluble in ether. As an organic acid sodium salt with wide application, it is mainly used in construction, chemical industry, food industry, pharmaceutical industry and other industries. At present, the production method of sodium gluconate is mainly biological fermentation by using strains such as Aspergillus niger or double enzyme oxidation by using glucose oxidase and catalase. The biological fermentation method has the characteristics of stable and mild production, easy scale industrial production, etc., but also has some defects, such as difficult control of product color, high degree of sterilization requirement, etc. The double enzyme oxidation method has the characteristics of fast speed, simple process, high product purity, low energy consumption, etc., but also has some shortcomings: the current enzyme preparation price is high, which increases the production cost. Therefore, in view of the above problems, a preparation method for co-production of D-psicose and sodium gluconate is established. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a preparation method for co-production of D-psicose and sodium gluconate, which simultaneously produces D-psicose and sodium gluconate by using the preparation method, thereby reducing the production cost.

[0005] To solve the above technical problems, the technical scheme of the present application is as follows:

[0006] A preparation method for co-production of D-psicose and sodium gluconate, the preparation method comprising the following steps:

[0007] (1) taking D-fructose and D-glucose as substrates, adding a solvent and a whole-cell catalyst Ⅰ co-expressing three enzymes, adjusting the pH of the reaction system to be weakly acidic, controlling the temperature of the reaction system, and centrifuging the supernatant collected after the reaction to obtain a mixed solution of D-allulose and sodium gluconate;

[0008] (2) taking the supernatant in step (1), adjusting the pH to be neutral, adding a whole-cell catalyst Ⅱ co-expressing two enzymes under ventilation and stirring, and obtaining a mixed solution of D-allulose and sodium gluconate after the reaction;

[0009] (3) taking the mixed solution in step (2), passing through a cation resin column first, collecting the effluent, then passing through an anion resin column, evaporating, concentrating and crystallizing the collected effluent to obtain an allulose product, and using sodium hydroxide solution to flush the anion resin column, evaporating, concentrating and crystallizing the collected effluent to obtain a sodium gluconate product.

[0010] As an improved technical solution, the whole-cell catalyst Ⅰ is a whole-cell catalyst simultaneously expressing an allulose epimerase (DPE) gene, a trehalose dehydrogenase (RDH) gene and a glucose dehydrogenase (GDH) gene; and the whole-cell catalyst Ⅱ is a whole-cell catalyst simultaneously expressing a trehalose dehydrogenase (RDH) gene and a NADH oxidase (NOX) gene.

[0011] As an improved technical solution, the allulose epimerase gene expressed by the whole-cell catalyst Ⅰ is derived from a Clostridiales strain, the trehalose dehydrogenase gene is derived from a Providencia alcalifaciens strain, and the glucose dehydrogenase gene is derived from a Bacillaceae strain; the trehalose dehydrogenase (RDH) gene expressed by the whole-cell catalyst Ⅱ is derived from a Rubrivivax sp. strain, and the NADH oxidase (NOX) gene is derived from a Streptococcus pyogenes strain.

[0012] As an improved technical solution, the preparation of the whole-cell catalyst Ⅰ includes the following operations: linking an allulose epimerase gene (DPE), a trehalose dehydrogenase gene (RDH) and a glucose dehydrogenase gene (GDH) to a pYB1s plasmid, transforming the obtained recombinant plasmid pYB1s-DPE-RDH-GDH into Escherichia coli BW25113 to obtain the whole-cell catalyst Ⅰ;

[0013] The preparation of the whole cell catalyst II comprises the following steps: connecting a gene of a mannitol dehydrogenase (RDH) and a gene of an NADH oxidase (NOX) to a pYB1s plasmid, transforming the obtained recombinant plasmid pYB1s-RDH-NOX into Escherichia coli BW25113 to obtain the whole cell catalyst II.

[0014] As an improved technical solution, in step (1), D-fructose and D-glucose are added in a ratio of 50-100 g / L respectively, the whole cell catalyst I is added in a ratio of 10-30 g / L of wet weight concentration, and the solvent is a 0.05-0.1 mol / L sodium phosphate solution; the pH of the reaction system is 5.5-6.5, the reaction temperature is 38-45℃, and the reaction time is 10-12 h.

[0015] As a preferred technical solution, in step (1), D-fructose and D-glucose are added in a ratio of 100 g / L respectively, the whole cell catalyst I is added in a ratio of 10 g / L of wet weight concentration, the pH of the reaction system is 6.0, the reaction temperature is 40℃, and the reaction time is 12 h.

[0016] As an improved technical solution, in step (2), the mixed solution of D-mannitol and sodium gluconate is added in a ratio of 50-100 g / L of D-mannitol and sodium gluconate respectively, and the whole cell catalyst II is added in a ratio of 10-30 g / L of wet weight concentration; air is introduced at a flow rate of 1.5-2.5 L / min, and the dissolved oxygen in the reaction system is maintained at 30-40% v / v, the stirring speed is gradually increased from 500 rpm at the beginning to 1000 rpm, the reaction time is controlled at 20-24 h, and the reaction temperature is controlled at 40-45℃.

[0017] As a preferred technical solution, in step (2), the mixed solution of D-mannitol and sodium gluconate is added in a ratio of 50 g / L, the whole cell catalyst II is added in a ratio of 30 g / L of wet weight concentration, air is introduced at a flow rate of 1.8 L / min, the amount of dissolved oxygen in the reaction system is maintained at 35% v / v, the pH of the reaction system is 7.0, the reaction temperature is 40℃, and the reaction time is 24 h.

[0018] The reaction equations in steps (1) and (2) in the preparation method of the present application are as follows:

[0019] The reaction of step (1) is as follows:

[0020]

[0021] The reaction of step (2) is as follows:

[0022]

[0023] The application has the following beneficial effects:

[0024] The application uses D-fructose and D-glucose as substrates, adjusts the pH of the reaction system to be weakly acidic, uses whole-cell catalyst I which simultaneously expresses allulose epimerase (DPE) gene, tetritol dehydrogenase (RDH) gene and glucose dehydrogenase (GDH) gene to obtain an NADH regeneration system, and catalyzes D-fructose and D-glucose into D-tetritol and sodium gluconate respectively; then, whole-cell catalyst II which simultaneously expresses tetritol dehydrogenase (RDH) gene and NADH oxidase (NOX) gene is used to oxidize D-tetritol into D-allulose, and sodium gluconate remains in the reaction system. In this synthesis route, two NADH regeneration systems are completed through reduction driven by GDH and oxidation driven by NOX respectively. By using this two-step multi-enzyme cascade biotransformation system, D-fructose and D-glucose are catalyzed to co-produce D-allulose and sodium gluconate, and the production cost is reduced. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The high-phase liquid chromatogram of the supernatant collected for the reaction of step (1) in Example 4;

[0026] Figure 2 The high-phase liquid chromatogram of the mixed solution collected for the reaction of step (2) in Example 4. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical scheme and advantages of the application clearer, the application is further described in detail in combination with the following examples. It should be understood that the specific examples described herein are only used to explain the application, and are not used to limit the application.

[0028] Example 1

[0029] A preparation method for co-producing D-allulose and sodium gluconate, comprising the following steps:

[0030] (1) The substrates D-fructose and D-glucose are 100 g / L and 0.05 mol / L sodium phosphate solution respectively, the whole-cell catalyst I (simultaneously expressing allulose epimerase gene, tetritol dehydrogenase gene and glucose dehydrogenase gene) is added in an amount of 30 g (wet weight) / L, the pH of the reaction system is adjusted to 5.5, the temperature of the reaction system is controlled to 38℃, and after 12 h of reaction, the reaction is ended, and the supernatant collected by centrifugation is a mixed solution of D-tetritol and sodium gluconate;

[0031] (2) Take the mixed solution from step (1) and dilute it to 0.5L. The concentration of D-aluronate is 90g / L and the concentration of sodium gluconate is 97g / L. Place it in a 1.0L bioreactor and introduce air at a flow rate of 2.5L / min to maintain the dissolved oxygen content in the reaction system at 40% v / v. Adjust the pH to neutral and add 30g (wet weight) / L of whole cell catalyst II (which simultaneously expresses the alliin dehydrogenase gene and the NADH oxidase gene) while stirring. When stirring the reaction system, gradually increase the stirring speed of the mixed solution of sodium gluconate to 1000rpm at 500rpm. Control the temperature of the reaction system at 45℃. After 24h of reaction, the reaction ends and a mixed solution of D-aluronate and sodium gluconate is obtained.

[0032] (3) Take the mixture from step (2) and feed it into a cation exchange resin column (resin column packing is 001*16) at a flow rate of 0.5 bv / h. The collected effluent is then fed into an anion exchange resin column (resin column packing is D941) at a flow rate of 0.5 bv / h. The collected effluent is evaporated and concentrated to a solid content of 70-85 wt%. 95 wt% ethanol is added, and the temperature is lowered to 45°C. 0.5% of the concentrated solution mass of seed crystals is added. The mixture is then stirred and slowly cooled to room temperature before centrifugation to obtain allulose product. The anion exchange resin column is rinsed with sodium hydroxide solution. The collected effluent is evaporated and concentrated to a solid content of 70-80 wt%. The concentrated solution is cooled to 45°C, and 0.5% of the concentrated solution mass of seed crystals is added. The mixture is kept at 45°C and a vacuum of -0.05 MPa for isothermal evaporation and crystallization. The mixture is then centrifuged to obtain sodium gluconate product.

[0033] Example 2

[0034] A method for preparing D-allulose and sodium gluconate co-products includes the following steps:

[0035] (1) The substrates D-fructose and D-glucose were 100 g / L and sodium phosphate solution was 0.05 mol / L. The amount of whole-cell catalyst I (which simultaneously expresses allulose epimerase gene, allitol dehydrogenase gene and glucose dehydrogenase gene) added was 20 g (wet weight) / L. The pH of the reaction system was adjusted to 6.0 and the temperature of the reaction system was controlled at 38℃. The reaction ended after 12 h. The supernatant collected by centrifugation was the mixed solution of D-allitol solution and sodium gluconate.

[0036] (2) Take the mixed solution from step (1) and dilute it to 0.5L. The concentration of D-aluronate is 80g / L and the concentration of sodium gluconate is 86g / L. Place it in a bioreactor with a volume of 1L. Introduce air at a flow rate of 2.3L / min to maintain the dissolved oxygen content in the reaction system at 38% v / v. Add 20g (wet weight) / L of whole cell catalyst II (which simultaneously expresses the alliin dehydrogenase gene and the NADH oxidase gene). Adjust the pH to neutral. Stir the reaction system at a stirring speed of 500rpm to 1000rpm. Control the temperature of the reaction system at 40℃. After 20h, the reaction ends and a mixed solution of D-aluronate and sodium gluconate is obtained.

[0037] (3) Take the mixture from step (2) and feed it into a cation exchange resin column (resin column packing is 001*16) at a flow rate of 0.5 bv / h. The collected effluent is then fed into an anion exchange resin column (resin column packing is D941) at a flow rate of 0.5 bv / h. The collected effluent is evaporated and concentrated to a solid content of 70-85 wt%. 95 wt% ethanol is added, and the temperature is lowered to 45°C. 0.5% of the concentrated solution mass of seed crystals is added. The mixture is then stirred and slowly cooled to room temperature before centrifugation to obtain allulose product. The anion exchange resin column is rinsed with sodium hydroxide solution. The collected effluent is evaporated and concentrated to a solid content of 70-80 wt%. The concentrated solution is cooled to 45°C, and 0.5% of the concentrated solution mass of seed crystals is added. The mixture is kept at 45°C and a vacuum of -0.05 MPa for isothermal evaporation and crystallization. The mixture is then centrifuged to obtain sodium gluconate product.

[0038] Example 3

[0039] A method for preparing D-allulose and sodium gluconate co-products includes the following steps:

[0040] (1) The substrates D-fructose and D-glucose were 60 g / L and sodium phosphate solution was 0.05 mol / L. The amount of whole-cell catalyst I (which simultaneously expresses allulose epimerase gene, allitol dehydrogenase gene and glucose dehydrogenase gene) added was 20 g (wet weight) / L. The pH of the reaction system was adjusted to 6.0 and the temperature of the reaction system was controlled at 40℃. The reaction was completed after 10 h. The supernatant collected by centrifugation was the mixed solution of D-allitol solution and sodium gluconate.

[0041] (2) Take 0.5L of the mixed solution from step (1) (D-aluronate concentration is 60g / L, sodium gluconate concentration is 65g / L), place it in a 1L bioreactor, introduce air at a flow rate of 2.0L / min to maintain the dissolved oxygen content in the reaction system at 35% v / v, add 20g (wet weight) / L of whole-cell catalyst II (which simultaneously expresses alliin dehydrogenase gene and NADH oxidase gene), adjust the pH to neutral, and stir the reaction system at a stirring speed of 500rpm to 1000rpm, control the temperature of the reaction system at 42℃, and end the reaction after 22h to obtain a mixed solution of D-aluronate and sodium gluconate.

[0042] (3) Take the mixture from step (2) and feed it into a cation exchange resin column (resin column packing is 001*16) at a rate of 0.5 bv / h. The collected effluent is then fed into an anion exchange resin column (resin column packing is D941) at a rate of 0.5 bv / h. The collected effluent is evaporated and concentrated to a solid content of 70-85 wt%. 95 wt% ethanol is added, and the temperature is lowered to 45°C. 0.5% of the concentrated solution mass of seed crystals is added. The mixture is then stirred and slowly cooled to room temperature before centrifugation to obtain allulose product. The anion exchange resin column is rinsed with sodium hydroxide solution. The collected effluent is evaporated and concentrated to a solid content of 70-80 wt%. The concentrated solution is cooled to 45°C, and 0.5% of the concentrated solution mass of seed crystals is added. The mixture is kept at 45°C and a vacuum of -0.05 MPa for isothermal evaporation and crystallization. The mixture is then centrifuged to obtain sodium gluconate product.

[0043] Example 4

[0044] A method for preparing D-allulose and sodium gluconate co-products includes the following steps:

[0045] (1) The substrates D-fructose and D-glucose were 100 g / L, sodium phosphate solution was 0.1 mol / L, whole cell catalyst I (which simultaneously expresses allulose epimerase gene, allitol dehydrogenase gene and glucose dehydrogenase gene) was added at 10 g (wet weight) / L, the pH of the reaction system was adjusted to 6.0, the temperature of the reaction system was controlled at 40℃, the reaction was completed after 12 h, and the supernatant collected by centrifugation was the mixed solution of D-allitol solution and sodium gluconate;

[0046] (2) Take the mixed solution from step (1) and dilute it to 0.5L. The concentration of D-aluronate is 50g / L and the concentration of sodium gluconate is 55g / L. Place it in a bioreactor with a volume of 1L. Introduce air at a flow rate of 1.8L / min to maintain the dissolved oxygen content in the reaction system at 35% v / v. Add 30g (wet weight) / L of whole cell catalyst II (which simultaneously expresses the alliin dehydrogenase gene and the NADH oxidase gene). Adjust the pH to neutral. Stir the reaction system at a stirring speed of 500rpm to 1000rpm. Control the temperature of the reaction system at 40℃. After 24h of reaction, the reaction ends and a mixed solution of D-aluronate and sodium gluconate is obtained.

[0047] (3) Take the mixture from step (2) and feed it into a cation exchange resin column (resin column packing is 001*16) at a flow rate of 0.5 bv / h. The collected effluent is then fed into an anion exchange resin column (resin column packing is D941) at a flow rate of 0.5 bv / h. The collected effluent is evaporated and concentrated to a solid content of 70-85 wt%. 95 wt% ethanol is added, and the temperature is lowered to 45°C. 0.5% of the concentrated solution mass of seed crystals is added. The mixture is then stirred and slowly cooled to room temperature before centrifugation to obtain allulose product. The anion exchange resin column is rinsed with sodium hydroxide solution. The collected effluent is evaporated and concentrated to a solid content of 70-80 wt%. The concentrated solution is cooled to 45°C, and 0.5% of the concentrated solution mass of seed crystals is added. The mixture is kept at 45°C and a vacuum of -0.05 MPa for isothermal evaporation and crystallization. The mixture is then centrifuged to obtain sodium gluconate product.

[0048] Example 5

[0049] A method for preparing D-allulose and sodium gluconate co-products includes the following steps:

[0050] (1) The substrates D-fructose and D-glucose were 60 g / L and sodium phosphate solution was 0.08 mol / L. The amount of whole-cell catalyst I (which simultaneously expresses allulose epimerase gene, allitol dehydrogenase gene and glucose dehydrogenase gene) added was 25 g (wet weight) / L. The pH of the reaction system was adjusted to 6.3 and the temperature of the reaction system was controlled at 45℃. The reaction ended after 11 h. The supernatant collected by centrifugation was the mixed solution of D-allitol solution and sodium gluconate.

[0051] (2) Take 0.5L of the mixed solution from step (1) (D-aluronate concentration is 50g / L, sodium gluconate concentration is 55g / L), place it in a 1L bioreactor, introduce air at a flow rate of 1.5L / min to maintain the dissolved oxygen content in the reaction system at 30% v / v, add 20g (wet weight) / L of whole cell catalyst II (which simultaneously expresses alliin dehydrogenase gene and NADH oxidase gene), adjust the pH to neutral, and stir the reaction system at a stirring speed of 500rpm to 1000rpm, control the temperature of the reaction system at 43℃, and end the reaction after 24h to obtain a mixed solution of D-aluronate and sodium gluconate.

[0052] (3) Take the mixture from step (2) and feed it into a cation exchange resin column (resin column packing is 001*16) at a flow rate of 0.5 bv / h. The collected effluent is then fed into an anion exchange resin column (resin column packing is D941) at a flow rate of 0.5 bv / h. The collected effluent is evaporated and concentrated to a solid content of 70-85 wt%. 95 wt% ethanol is added, and the temperature is lowered to 45°C. 0.5% of the concentrated solution mass of seed crystals is added. The mixture is then stirred and slowly cooled to room temperature before centrifugation to obtain allulose product. The anion exchange resin column is rinsed with sodium hydroxide solution. The collected effluent is evaporated and concentrated to a solid content of 70-80 wt%. The concentrated solution is cooled to 45°C, and 0.5% of the concentrated solution mass of seed crystals is added. The mixture is kept at 45°C and a vacuum of -0.05 MPa for isothermal evaporation and crystallization. The mixture is then centrifuged to obtain sodium gluconate product.

[0053] Example 6

[0054] A method for preparing D-allulose and sodium gluconate co-products includes the following steps:

[0055] (1) The substrates D-fructose and D-glucose were 80 g / L and sodium phosphate solution was 0.1 mol / L. The amount of whole-cell catalyst I (which simultaneously expresses allulose epimerase gene, allitol dehydrogenase gene and glucose dehydrogenase gene) added was 15 g (wet weight) / L. The pH of the reaction system was adjusted to 6.5 and the temperature of the reaction system was controlled at 42℃. The reaction ended after 12 h. The supernatant collected by centrifugation was the mixed solution of D-allitol solution and sodium gluconate.

[0056] (2) Take the mixed solution in step (1) to dilute to 0.5 L, the concentration of D-sugar alcohol is 70 g / L, and the concentration of sodium gluconate is 76 g / L, and place in a bioreactor with a volume of 1 L, and air is passed at a flow rate of 2.5 L / min to maintain the amount of dissolved oxygen in the reaction system at 35% v / v, and 30 g (wet weight) of whole cell catalyst II (expressing both the D-sugar alcohol dehydrogenase gene and the NADH oxidase gene) is added, the pH is adjusted to neutral, and the reaction system is stirred at a speed of 500 rpm to gradually increase to 1000 rpm in the liquid in the stirred reaction system, the temperature of the reaction system is controlled at 45°C, and after 24 h of reaction, the reaction is completed, and a mixed solution of D-allulose and sodium gluconate is obtained;

[0057] (3) Take the mixed solution of step (2) and pass it into a cation resin column (resin column filler is 001*16) at a flow rate of 0.5 bv / h, collect the effluent and pass it into an anion resin column (resin column filler is D941) at a flow rate of 0.5 bv / h, evaporate and concentrate the collected effluent to a solid content of 70-85 wt%, add 95 wt% ethanol, cool to 45°C, add 0.5% of the concentrated solution by mass of seed crystals, then stir slowly and cool to room temperature, and centrifuge to obtain the allulose product; wash the anion resin column with sodium hydroxide solution, collect the effluent, evaporate and concentrate to a solid content of 70-80 wt%, cool the concentrated solution to 45°C, add 0.5% of the concentrated solution by mass of seed crystals, maintain 45°C, perform constant temperature evaporation crystallization at a vacuum of -0.05 MPa, and centrifuge to obtain the sodium gluconate product.

[0058] The preparation method of the whole cell catalyst I in examples 1-6 includes the following steps:

[0059] (1) Construction of recombinant plasmid pYB1s-DPE-RDH-GDH: The pYB1s vector was double-digested with Xho I and Spe I, and the vector fragment was recovered. The DPE gene fragment, the RDH gene fragment and the GDH gene fragment (synthesized by Nanjing Jin Sui Biological Technology Co., Ltd.) were ligated with the recovered vector fragment by Gibson method (Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, 3rd, Smith HO: Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 2009, 6:343-345.) to obtain a ligation product. The ligation product was transformed into DH5a competent cells, and spread on a LB solid plate containing streptomycin. After 37°C overnight, a single colony was picked for plasmid extraction, sequencing verification, and the correct vector was named pYB1s-DPE-RDH-GDH.

[0060] (2) Induced expression of recombinant proteins DPE, RDH and GDH: The vector pYB1s-DPE-RDH-GDH was transformed into E. coli BW25113 by chemical transformation to obtain whole-cell catalyst I. The recombinant bacteria (whole-cell catalyst I) were streaked on a LB plate containing 1.5 g / 100 mL of agar (containing 50 μg / mL of streptomycin), and cultured at 37°C for 12 h. A single colony was picked and inoculated in liquid LB medium containing 50 μg / mL of streptomycin, and cultured at 37°C for 10 h with shaking at 220 rpm. The culture was inoculated into 500 mL of LB medium at a volume percentage of 1%, and cultured at 37°C for 3 h with shaking, and then 2 g / L of arabinose was added for induced expression, and the culture was incubated at 30°C for 12 h with shaking.

[0061] The preparation method of the whole-cell catalyst II in Examples 1-6 includes the following steps:

[0062] (1) Construction of recombinant plasmid pYB1s-RDH-NOX: The pYB1s vector was double-digested with Xho I and Spe I, and the vector fragment was recovered. The RDH gene fragment and the NOX gene fragment (synthesized by Nanjing Jin Sui Biological Technology Co., Ltd.) were ligated with the recovered vector fragment by Gibson method to obtain a ligation product. The ligation product was transformed into DH5a competent cells, and spread on a LB solid plate containing streptomycin. After 37°C overnight, a single colony was picked for plasmid extraction, sequencing verification, and the correct vector was named pYB1s-RDH-NOX.

[0063] (2) Induced expression of recombinant proteins RDH and NOX: The vector pYB1s-RDH-NOX was transformed into E. coli BW25113 by chemical transformation method to obtain whole-cell catalyst II. The recombinant bacteria (whole-cell catalyst II) were streaked onto LB plates containing 1.5 g / 100 mL of agar (containing 50 μg / mL of streptomycin) and incubated at 37 °C for 12 h. Single colonies were picked and inoculated into liquid LB medium containing 50 μg / mL of streptomycin and incubated at 37 °C for 10 h at a shaking speed of 220 rpm. The culture was inoculated into 500 mL of LB medium at a volume percentage of 1% and incubated at 37 °C for 3 h. Then, 2 g / L of arabinose was added to induce expression, and the culture was incubated at 30 °C for 12 h.

[0064] To better prove that the method of the present application can simultaneously obtain high yield of D-psicose product and sodium gluconate product, four comparative examples are given, and the yield of the substrate and the yield of the product are shown in Table 1.

[0065] Comparative Example 1

[0066] Different from Example 4: in step (1), whole-cell catalyst I was added at a wet weight concentration of 40 g / L, and the rest of the operations were the same.

[0067] Comparative Example 2

[0068] Different from Example 4: in step (1), the reaction temperature was 35 °C, and the rest of the operations were the same.

[0069] Comparative Example 3

[0070] Different from Example 4: in step (2), the dissolved oxygen in the reaction system was maintained at 40% v / v, and the rest of the operations were the same.

[0071] Table 1

[0072]

[0073] From the data in Table 1, it can be found that the preparation method of the present application can be suitable for industrial production. D-psicose and sodium gluconate are co-produced by a multi-enzyme system catalyzing D-fructose and D-glucose, which reduces the production cost. In Example 4, the supernatant after step (1) reaction was determined by high performance liquid chromatography (Aminex HPX-87C column (7.8*300 mm), detector (RID), mobile phase: double distilled water, column temperature: 80 °C, flow rate: 0.6 ml / min) to determine the concentrations of dulcitol and sodium gluconate. The specific detection is shown in the following table. Figure 1 .

[0074] The mixture after the reaction of step (2) in Example 4 was subjected to high performance liquid chromatography (Aminex HPX-87H column (7.8*300 mm), detector (DAD, 210 nm), mobile phase: 5 mM sulfuric acid, column temperature 50°C, flow rate: 0.5 ml / min) to determine the concentrations of sodium gluconate and D-allulose. The detailed detection is shown in the following Table 1. Figure 2 .

[0075] The allulose epimerase protein sequence, the mannosonate dehydrogenase protein sequence, the glucose dehydrogenase protein sequence, the mannosonate dehydrogenase protein sequence, and the NADH oxidase protein sequence are shown in the sequence listing.

[0076] The above merely provides the preferred embodiments of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present application shall fall within the protection scope of the present application.

Claims

1. A method for preparing D-psicose and sodium gluconate in combination, the method comprising the following steps: (1) taking D-fructose and D-glucose as substrates, adding a solvent and a whole-cell catalyst I co-expressing three enzymes, adjusting the pH of the reaction system to be weakly acidic, controlling the temperature of the reaction system, and collecting the supernatant after centrifugation to obtain a mixed solution of D-threitol and sodium gluconate after the reaction; the whole-cell catalyst I is a whole-cell catalyst co-expressing a D-psicose epimerase (DPE) gene, a threitol dehydrogenase (RDH) gene and a glucose dehydrogenase (GDH) gene; the D-psicose epimerase gene expressed by the whole-cell catalyst I is derived from a strain selected from Clostridiales, the threitol dehydrogenase gene is derived from a strain selected from Providencia alcalifaciens, and the glucose dehydrogenase gene is derived from a strain selected from Bacillaceae; (2) taking the supernatant in step (1), adjusting the pH to be neutral, and adding a whole-cell catalyst II co-expressing two enzymes under the conditions of ventilation and stirring to obtain a mixed solution of D-psicose and sodium gluconate after the reaction; the whole-cell catalyst II is a whole-cell catalyst co-expressing a threitol dehydrogenase (RDH) gene and a NADH oxidase (NOX) gene; the threitol dehydrogenase (RDH) gene expressed by the whole-cell catalyst II is derived from a strain selected from Rubrivivax sp., and the NADH oxidase (NOX) gene is derived from a strain selected from Streptococcus pyogenes; (3) taking the mixed solution in step (2), passing it through a cation resin column, collecting the effluent, passing the effluent through an anion resin column, and obtaining a D-psicose product after evaporation, concentration, crystallization and filtration; and (4) washing the anion resin column with a sodium hydroxide solution, collecting the effluent, and obtaining a sodium gluconate product after evaporation, concentration, crystallization and filtration. The preparation of the whole-cell catalyst I comprises the following operations: linking a D-psicose epimerase gene (DPE), a threitol dehydrogenase gene (RDH) and a glucose dehydrogenase gene (GDH) to a pYB1s plasmid, transforming the obtained recombinant plasmid pYB1s-DPE-RDH-GDH into Escherichia coli BW25113 to obtain the whole-cell catalyst I. The preparation of the whole-cell catalyst II comprises the following operations: linking a threitol dehydrogenase gene (RDH) and a NADH oxidase gene (NOX) to a pYB1s plasmid, transforming the obtained recombinant plasmid pYB1s-RDH-NOX into Escherichia coli BW25113 to obtain the whole-cell catalyst II. In step (1), D-fructose and D-glucose are added in a ratio of 50-100 g / L, the whole-cell catalyst I is added in a wet weight concentration of 10-30 g / L, and the solvent is a 0.05-0.1 mol / L sodium phosphate solution; the pH of the reaction system is 5.5-6.5, the reaction temperature is 38-45℃, and the reaction time is 10-12 h. ​ 2. The method of claim 1, wherein the method is characterized by: ​ ​ 3. The method of claim 1, wherein the method is characterized by: ​ 4. The method of claim 3, wherein the method is characterized by: In step (1), D-fructose and D-glucose were added at a ratio of 100 g / L, respectively, and whole cell catalyst I was added at a ratio of 10 g / L of wet weight concentration. The pH of the reaction system was 6.0, the reaction temperature was 40°C, and the reaction time was 12 h.

5. The method of claim 1, wherein the method is characterized by: In step (2), the mixed solution of D-allitol and sodium gluconate was added at a ratio of 50-100 g / L of D-allitol and sodium gluconate, respectively, and whole cell catalyst II was added at a ratio of 10-30 g / L of wet weight concentration. Air was introduced at a flow rate of 1.5-2.5 L / min, and the dissolved oxygen in the reaction system was maintained at 30-40% v / v. The stirring speed was gradually increased from 500 rpm at the beginning to 1000 rpm, the reaction time was controlled at 20-24 h, and the reaction temperature was controlled at 40-45°C.

6. The method of claim 5, wherein the method is characterized by: In step (2), the mixed solution of D-allitol and sodium gluconate was added at a ratio of 50 g / L, and whole cell catalyst II was added at a ratio of 30 g / L of wet weight concentration. Air was introduced at a flow rate of 1.8 L / min, the dissolved oxygen in the reaction system was maintained at 35% v / v, the pH of the reaction system was 7.0, the reaction temperature was 40°C, and the reaction time was 24 h.

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