Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides the preparation method of the D-psicose, which is used for improving the conversion rate of the D-fructose and reducing the production cost.
In order to solve the technical problems, the technical scheme of the invention is as follows:
A method for preparing D-psicose, the method comprising the steps of:
(1) D-fructose is used as a substrate, a solvent and a first whole-cell catalyst are added, the pH of a reaction system is adjusted to be slightly acidic, the temperature of the reaction system is controlled, and after the reaction is finished, the supernatant fluid centrifugally collected is the D-allitol solution;
(2) And (3) taking the D-allitol solution in the step (1), regulating the pH to be neutral, adding a second whole-cell catalyst under the ventilation and stirring states, and obtaining the psicose solution after the reaction is finished.
As an improved technical scheme, the first whole-cell catalyst is named as AO1, the first whole-cell catalyst is a whole-cell catalyst capable of simultaneously expressing an allose epimerase (DPE) gene, a allitol dehydrogenase (RDH) gene and a Formate Dehydrogenase (FDH) gene, the second whole-cell catalyst is named as PO3, and the second whole-cell catalyst is a whole-cell catalyst capable of simultaneously expressing the allitol dehydrogenase (RDH) gene and an NADH Oxidase (NOX) gene.
As an improved technical scheme, the first whole-cell catalyst-expressible allose epimerase gene-derived strain is selected from Clostridiales, the allitol dehydrogenase gene-derived strain is selected from Providencia alcalifaciens, the formate dehydrogenase gene-derived strain is selected from Starkeya, the second whole-cell catalyst-expressible allitol dehydrogenase (RDH) gene-derived strain is selected from Providencia alcalifaciens, and the NADH Oxidase (NOX) gene-derived strain is selected from Streptococcus pyogenes.
As an improved technical scheme, the preparation of the first whole-cell catalyst comprises the following steps of connecting psicose epimerase gene (DPE), allitol dehydrogenase gene (RDH) and formate dehydrogenase group (FDH) to pYB s plasmid, and transforming the obtained recombinant plasmid pYB s-DPE-RDH-FDH into escherichia coli BW25113 to obtain the first whole-cell catalyst;
The preparation of the second whole-cell catalyst comprises the following operation of connecting a allitol dehydrogenase (RDH) gene and an NADH oxidase gene (NOX) to pYB s plasmid, and converting the obtained recombinant plasmid pYB s-RDH-NOX into escherichia coli BW25113 to obtain the second whole-cell catalyst.
As an improved technical scheme, D-fructose and a first whole-cell catalyst in the step (1) are respectively added according to the proportion of 50-200g/L and the wet weight concentration of 5-30g/L, wherein the solvent is sodium formate solution or potassium formate solution, the concentration of the sodium formate solution or the potassium formate solution is 0.5-2.0mol/L, the pH of a reaction system is 5.8-6.8, the reaction temperature is 35-40 ℃, and the reaction time is 11-13h.
As a preferable technical scheme, in the step (1), D-fructose and the first whole-cell catalyst are added according to the proportion of 100g/L and the wet weight concentration of 20g/L in the step (1), the pH of a reaction system is 6.5, the reaction temperature is 40 ℃, and the reaction time is 12 hours.
As an improved technical scheme, in the step (2), the D-allitol solution and the second whole-cell catalyst are respectively added according to the proportion of 50-200g/L and the wet weight concentration of 5-30g/L, air is introduced according to the flow rate of 1.5-2.5L/min, the dissolved oxygen in the reaction system is maintained at 48-53% v/v, the stirring speed is gradually increased from 500rpm at the beginning to 1000rpm, the reaction time is controlled at 13-15h, and the reaction temperature is controlled at 35-40 ℃.
As a preferable technical scheme, the D-allitol solution and the second whole-cell catalyst in the step (2) are respectively added according to the proportion of 100g/L and 10g/L of wet weight concentration, air is introduced according to the flow rate of 1.8L/min, the dissolved oxygen amount in the reaction system is maintained at 50% v/v, the pH of the reaction system is 7.0, the reaction temperature is 40 ℃, and the reaction time is 14.5h.
In the preparation method of the invention, the reaction equation in the step (1) and the step (2) is as follows:
reaction of step (1):
Reaction of step (2):
After the technical scheme is adopted, the invention has the beneficial effects that:
the invention takes D-fructose as a substrate, adjusts the pH value of a reaction system to be weak acid, adopts a first whole-cell catalyst capable of simultaneously expressing psicose epimerase (DPE) gene, allitol dehydrogenase (RDH) gene and Formate Dehydrogenase (FDH) gene to obtain an NADH regeneration system, catalyzes D-fructose into D-allitol, and then adopts a second whole-cell catalyst capable of simultaneously expressing allitol dehydrogenase (RDH) gene and NADH Oxidase (NOX) gene, wherein the D-allitol is oxidized into D-psicose through the NADH regeneration system driven by NOX. In this synthetic route, two NADH regeneration systems are accomplished by FDH driven reduction and NOx driven oxidation, respectively. With this two-step multiple enzyme tandem bioconversion system, the maximum theoretical product yield of D-fructose to D-psicose was 100%.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1
A method for preparing D-psicose, comprising the following steps:
(1) 200g/L of substrate D-fructose and 2.0mol/L of sodium formate solution, wherein the addition amount of a first whole-cell catalyst is 30g (wet weight)/L, the first whole-cell catalyst (simultaneously expressing psicose epimerase gene, allitol dehydrogenase gene and formate dehydrogenase gene) is put into a bioreactor, the pH value of a reaction system is regulated to 5.8, the temperature of the reaction system is controlled to 35 ℃, the reaction is finished after 11 hours, and the supernatant collected by centrifugation is the D-allitol solution;
(2) Taking 0.5L of the D-allitol solution in the step (1), wherein the concentration of the D-allitol is 200g/L, placing the D-allitol solution in a 1.0L bioreactor, introducing air according to the flow rate of 2.5L/min, maintaining the dissolved oxygen in the reaction system at 53% v/v, regulating the pH value to be neutral, adding 30g (wet weight)/L of a second whole-cell catalyst (simultaneously expressing allitol dehydrogenase gene and NADH oxidase gene) in a stirring state, gradually increasing the temperature of the reaction system to 1000rpm according to 500rpm when stirring the reaction system, controlling the temperature of the reaction system to be 35 ℃, and finishing the reaction after 14 hours to obtain the D-allose solution.
Example 2
A method for preparing D-psicose, comprising the following steps:
(1) 180g/L of substrate D-fructose and 1.5mol/L of sodium formate solution, wherein the addition amount of a first whole-cell catalyst is 25g (wet weight)/L, the first whole-cell catalyst (simultaneously expressing psicose epimerase gene, allitol dehydrogenase gene and formate dehydrogenase gene) is put into a bioreactor, the pH value of a reaction system is regulated to 6.0, the temperature of the reaction system is controlled to 37 ℃, the reaction is finished after 11.5 hours, and the supernatant collected by centrifugation is the D-allitol solution;
(2) Taking 0.5L of D-allitol solution in the step (1), placing the solution with the concentration of 180g/L into a bioreactor with the volume of 1L, introducing air according to the flow rate of 2.3L/min, maintaining the dissolved oxygen content in a reaction system at 52% v/v, adding 25g (wet weight)/L of a second whole-cell catalyst (capable of simultaneously expressing allitol dehydrogenase gene and NADH oxidase gene), regulating pH to be neutral, stirring the reaction system, gradually increasing the temperature of the reaction system to be 1000rpm according to 500rpm, stirring the feed liquid in the reaction system, controlling the temperature of the reaction system to be 37 ℃, and finishing the reaction after 14.5h to obtain the D-psicose solution.
Example 3
A method for preparing D-psicose, comprising the following steps:
(1) 150g/L of substrate D-fructose and 1.0mol/L of sodium formate solution, wherein the addition amount of a first whole-cell catalyst is 20g (wet weight)/L, the first whole-cell catalyst (simultaneously expressing psicose epimerase gene, allitol dehydrogenase gene and formate dehydrogenase gene) is put into a bioreactor, the pH value of a reaction system is regulated to 6.3, the temperature of the reaction system is controlled to 38 ℃, the reaction is finished after 12 hours, and the supernatant collected by centrifugation is the D-allitol solution;
(2) Taking 0.5L of D-allitol solution in the step (1), placing the solution in a bioreactor with the volume of 1L, introducing air according to the flow rate of 2L/min, maintaining the dissolved oxygen in the reaction system at 51% v/v, adding 20g (wet weight)/L of a second whole-cell catalyst (capable of simultaneously expressing allitol dehydrogenase gene and NADH oxidase gene), regulating the pH to be neutral, stirring the reaction system, gradually increasing the temperature to 1000rpm according to 500rpm, stirring the feed liquid in the reaction system, controlling the temperature of the reaction system at 38 ℃, and finishing the reaction after 15 hours to obtain the D-psicose solution.
Example 4
A method for preparing D-psicose mannose, comprising the following steps:
(1) The substrate D-fructose is 100g/L, sodium formate solution is 1.5mol/L, the adding amount of the first whole-cell catalyst is 10g (wet weight)/L, the first whole-cell catalyst (simultaneously expressing psicose epimerase gene, allitol dehydrogenase gene and formate dehydrogenase gene) is put into a bioreactor, the pH value of a reaction system is regulated to 6.5, the temperature of the reaction system is controlled to 40 ℃, the reaction is finished after 12 hours, and the supernatant collected by centrifugation is the D-allitol solution;
(2) Taking 0.5L of D-allitol solution in the step (1), placing the solution with the concentration of 100g/L into a bioreactor with the volume of 1L, introducing air according to the flow rate of 1.8L/min, maintaining the dissolved oxygen content in a reaction system at 50% v/v, adding 10g (wet weight)/L of a second whole-cell catalyst (capable of simultaneously expressing allitol dehydrogenase gene and NADH oxidase gene), regulating the pH to be neutral, stirring the reaction system, gradually increasing the temperature of the reaction system to be 1000rpm according to 500rpm, stirring the feed liquid in the reaction system, controlling the temperature of the reaction system to be 40 ℃, and finishing the reaction after 14.5h to obtain the D-psicose solution.
Example 5
A method for preparing D-psicose, comprising the following steps:
(1) Substrate D-fructose 50g/L, sodium formate solution 1.0mol/L, first whole cell catalyst addition was 5g (wet weight)/L. Placing a first whole-cell catalyst (simultaneously expressing psicose epimerase gene, allitol dehydrogenase gene and formate dehydrogenase gene) into a bioreactor, adjusting the pH of a reaction system to 6.8, controlling the temperature of the reaction system to 40 ℃, finishing the reaction after 13 hours, and centrifuging the collected supernatant to obtain a D-allitol solution;
(2) Taking 0.5L of D-allitol solution in the step (1), placing the solution with the concentration of 100g/L into a bioreactor with the volume of 1L, introducing air according to the flow rate of 1.5L/min, maintaining the dissolved oxygen content in a reaction system at 49% v/v, adding 5g (wet weight)/L of a second whole-cell catalyst (capable of simultaneously expressing allitol dehydrogenase gene and NADH oxidase gene), regulating pH to be neutral, stirring the reaction system, gradually increasing the temperature of the reaction system to be 1000rpm according to 500rpm, stirring the feed liquid in the reaction system, controlling the temperature of the reaction system to be 39 ℃, and finishing the reaction after 13h to obtain the D-allose sugar solution.
Example 6
A method for preparing D-psicose, comprising the following steps:
(1) The substrate D-fructose 120g/L, potassium formate solution 2.0mol/L, the first whole-cell catalyst addition amount is 15g (wet weight)/L, the first whole-cell catalyst (simultaneously expressing psicose epimerase gene, allitol dehydrogenase gene and formate dehydrogenase gene) is put into a bioreactor, the pH value of a reaction system is regulated to 6.8, the temperature of the reaction system is controlled to 39 ℃, the reaction is finished after 13 hours, and the supernatant collected by centrifugation is the D-allitol solution;
(2) Taking 0.5L of D-allitol solution in the step (1), placing the solution with the concentration of 120g/L into a bioreactor with the volume of 1L, introducing air according to the flow rate of 1.6L/min, maintaining the dissolved oxygen content in a reaction system at 48% v/v, adding 15g (wet weight)/L of a second whole-cell catalyst (capable of simultaneously expressing allitol dehydrogenase gene and NADH oxidase gene), regulating pH to be neutral, stirring the reaction system, gradually increasing the temperature of the reaction system to be 1000rpm according to 500rpm, stirring the feed liquid in the reaction system, controlling the temperature of the reaction system to be 40 ℃, and finishing the reaction after 13h to obtain the D-allose sugar solution.
Example 7
A method for preparing D-psicose, comprising the following steps:
(1) The substrate D-fructose 80g/L, potassium formate solution 0.5mol/L, the first whole-cell catalyst addition is 5g (wet weight)/L, the first whole-cell catalyst (simultaneously expressing psicose epimerase gene, allitol dehydrogenase gene and formate dehydrogenase gene) is put into a bioreactor, the pH value of a reaction system is regulated to 6.8, the temperature of the reaction system is controlled to 40 ℃, the reaction is finished after 13 hours, and the supernatant collected by centrifugation is the D-allitol solution;
(2) Taking 0.5L of D-allitol solution in the step (1), placing the solution in a bioreactor with the volume of 1L, introducing air according to the flow rate of 2L/min, maintaining the dissolved oxygen in the reaction system at 50% v/v, adding 5g (wet weight)/L of a second whole-cell catalyst (capable of simultaneously expressing allitol dehydrogenase gene and NADH oxidase gene), regulating pH to be neutral, stirring the reaction system, gradually increasing to feed liquid in the stirring reaction system at 1000rpm according to 500rpm, controlling the temperature of the reaction system at 40 ℃, and finishing the reaction after 13h to obtain the D-allose sugar solution.
Wherein the first whole cell catalyst preparation method in examples 1 to 7 comprises the steps of:
(1) Construction of recombinant plasmid pYB s-DPE-RDH-FDH A construction of pYB s vector was performed by double digestion with XhoI and SpeI, and then vector fragments were recovered, and artificially synthesized DPE gene fragments, RDH gene fragments and FDH gene fragments (synthesized by Nanjin St Biotechnology Co., ltd.) were ligated with the recovered vector fragments by Gibson method (Gibson DG,Young L,Chuang RY,Venter JC,Hutchison CA,3rd,Smith HO:Enzymatic assembly of DNA molecules up toseveral hundred kilobases.Nat Methods 2009,6:343-345.) to obtain ligation products. The ligation products were transformed into DH 5. Alpha. Competent cells and coated with LB solid plates containing streptomycin. The monoclonal extracted plasmid was picked overnight at 37℃and sequenced for verification, and the correctly sequenced vector was designated pYB s-DPE-RDH-FDH.
(2) The induction expression of recombinant protein DPE, RDH and FDH includes chemical conversion of carrier pYB s-DPE-RDH-FDH to colibacillus BW25113 to obtain the first whole cell catalyst. Recombinant bacteria (first whole cell catalyst) were streaked onto LB plates containing agar (containing 50. Mu.g/mL of streptomycin) at a mass percent concentration of 1.5g/100mL, and cultured at 37℃for 12 hours. Selecting monoclonal, inoculating to liquid LB medium containing 50 μg/mL streptomycin, shake culturing at 37deg.C for 10h at 220rpm, inoculating to 500mL LB medium with 1% by volume, shake culturing at 37deg.C for 3h, adding arabinose with final concentration of 2g/L, and shake culturing at 30deg.C for 12h.
The preparation method of the second whole-cell catalyst in examples 1 to 7 comprises the following steps:
(1) Construction of recombinant plasmid pYB s-RDH-NOX, namely, after pYB s vector is digested with XhoI and SpeI, vector fragments are recovered, and artificially synthesized RDH gene fragments (source strain is selected from Providencia alcalifaciens) and NOX gene fragments (synthesized by Nanjing Jinsri Biotechnology Co., ltd.) are connected with the recovered vector fragments by using Gibson method to obtain a connection product. The ligation products were transformed into DH 5. Alpha. Competent cells and coated with LB solid plates containing streptomycin. The monoclonal extracted plasmid was picked overnight at 37℃and sequenced to verify that the correct vector was designated pYB s-RDH-NOX.
(2) And (3) carrying out induction expression on the recombinant protein RDH and NOX, namely converting the carrier pYB s-RDH-NOX into escherichia coli BW25113 by a chemical conversion method to obtain a second whole-cell catalyst. Recombinant bacteria (second whole cell catalyst) were streaked onto LB plates containing 1.5g/100mL of agar (containing 50. Mu.g/mL of streptomycin) at a mass percent concentration, and cultured at 37℃for 12 hours. Selecting monoclonal, inoculating to liquid LB medium containing 50 μg/mL streptomycin, shake culturing at 37deg.C for 10h at 220rpm, inoculating to 500mLLB medium with 1% by volume, shake culturing at 37deg.C for 3h, adding arabinose with final concentration of 2g/L, and shake culturing at 30deg.C for 12h.
In order to better prove that the preparation process has better technical effect, 3 comparative examples are given by taking example 4 as a reference, and the experimental results are shown in Table 1 in detail.
Comparative example 1
The difference from example 4 is that the recombinant plasmid constructed at the time of preparation of the first whole cell catalyst was pYB s-PRDH-SFDH-CBDPE, and the rest of the operations were exactly the same.
Comparative example 2
The difference from example 4 is that the recombinant plasmid constructed at the time of preparation of the first whole cell catalyst was pYB s-CBDPE-SFDH-PRDH, and the rest of the operations were exactly the same.
Comparative example 3
In contrast to example 4, the second whole-cell catalyst was prepared in which the RDH gene-derived strain was selected from Klebsiella oxytoca and the rest of the procedure was identical.
Comparative example 4
In contrast to example 4, the second whole-cell catalyst was prepared in which the RDH gene-derived strain was selected from Rubrivivax sp.
Comparative example 5
In contrast to example 4, the second whole-cell catalyst was prepared in which the RDH gene-derived strain was selected from Pantoea agglomerans and the rest of the procedure was identical.
TABLE 1
As can be seen from the data in Table 1, the preparation method of the invention is suitable for industrial production, improves the conversion rate of D-fructose and reduces the production cost. The concentration of D-fructose, allitol and D-psicose was measured by high performance liquid chromatography (AminexHPX-87C column (7.8X300 mm), detector (RID), mobile phase: double distilled water, column temperature 80 deg.C, flow rate 0.6 ml/min), and specific detection is shown in figure 1. The sequence of allose epimerase protein, allitol dehydrogenase protein, formate dehydrogenase protein, allitol dehydrogenase protein and NADH oxidase protein are shown in the sequence table.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.