Enzyme catalysis production method of D-psicose

Abstract

The invention belongs to the technical field of biological catalysis, and particularly relates to an enzyme catalysis production method of D-psicose, which comprises the following steps: by taking starch or dextrin as a substrate, adding a compound enzyme into the substrate, and carrying out enzyme catalysis reaction to synthesize the D-psicose, and the compound enzyme is alpha-glucan phosphorylase, glucose phosphate mutase, glucose phosphate isomerase, psicose 6-phosphoric acid-3-epimerase, and psicose 6-phosphate phosphatase. The invention further discloses a preparation method of the compound enzyme. The method for preparing the D-psicose has the advantages that the cost is low, the conversion rate can reach 66.63% and the like, cost reduction and efficiency improvement are realized, and the method is suitable for large-scale production of the D-psicose.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalysis, and particularly relates to an enzyme-catalyzed production method of D-psicose. Background Art

[0002] D-psicose is a diastereomer of fructose and a safe sweetener. Although its sweetness is only 70% of that of sucrose, the energy it produces is only 0.3% of that of sucrose, making it a truly low-energy sugar. In addition to its low energy content, D-psicose also has other physiological functions, such as being able to effectively prevent and treat obesity, diabetes, hyperlipidemia and other diseases. Therefore, it has great application value in the fields of food and medicine.

[0003] At present, the production of D-psicose in the market is mainly based on the single enzyme method, in which fructose is catalyzed by D-psicose-3-epimerase to produce D-psicose. Although the reaction process is simple and the product D-psicose can be obtained in one step, the price of fructose is relatively high, and there is a reaction equilibrium between fructose (i.e., substrate) and psicose (i.e., product), that is, the product / substrate ratio is about 20% to 35%, resulting in a limited conversion rate. Therefore, when using a single enzyme reaction to produce high-purity psicose, it is necessary to separate and remove high-concentration fructose from the reaction product. This process not only causes substrate waste, but also indirectly increases production costs due to its separation. In addition, although the prior art involves the use of multi-enzyme catalysis to produce D-psicose, the conversion rate of D-psicose is generally lower than 40% due to the different types and dosages of the composite enzymes used. At the same time, the generation of by-products other than the known glucose and fructose, and how to reduce the generation of by-products while ensuring the improvement of the conversion rate of D-psicose, have become constraints affecting the industrial promotion and application of D-psicose. Summary of the invention

[0004] The object of the present invention is to provide an enzymatic production method of D-psicose, thereby overcoming the shortcomings of the prior art and preparing D-psicose by a catalytic pathway of a multi-stage enzyme cascade reaction, which has the advantages of low cost, high conversion rate and few by-products, thereby achieving cost reduction and efficiency improvement, and is suitable for large-scale production of D-psicose. In order to achieve the above object, the technical solution of the present invention is: The present invention provides an enzymatic production method of D-psicose, which uses starch or dextrin as a substrate, adds a composite enzyme to the substrate to carry out an enzymatic reaction to synthesize D-psicose; The complex enzyme consists of alpha-glucan phosphorylase, glucose phosphomutase, glucose phosphomutase, psicose 6-phosphate-3-epimerase and psicose 6-phosphate phosphatase.

[0005] In some other embodiments, the conditions of the enzyme-catalyzed reaction are: temperature of 50-80° C., pH adjusted to 5-8 by adding a buffer solution, and reaction time of 12-20 h; Alternatively, the buffer solution is a phosphate buffer solution; the concentration of the buffer solution is 5-60 mM.

[0006] In some other embodiments, the conditions of the enzyme catalyzed reaction are: temperature of 65-80°C, pH adjusted to 7 by adding buffer solution, reaction time of 18h; Alternatively, the concentration of the buffer solution is 10-30 mM.

[0007] In some other embodiments, the method further includes pretreating the substrate, wherein the pretreatment method is: adding a pretreatment enzyme to enzymolyze the substrate; the pretreatment enzyme is isoamylase or pullulanase; the conditions for the enzymolysis are: temperature of 60-80°C, time of 3-5h; the amount of the pretreatment enzyme added is 0.1-3wt%.

[0008] In some other embodiments, the pretreatment enzyme is pullulanase; the conditions of the enzymolysis are: temperature is 80° C., time is 5 h; the addition amount of the pretreatment enzyme is 0.2-0.5 wt %; Or, the pullulanase is pullulanase Uniprot ID: P36905.

[0009] In some other embodiments, the method further comprises adding a metal ion to the substrate, wherein the metal ion is Mn 2+ Mg 2 + 、Co 2+ , Ca 2+ Cu 2+ ; The concentration of the metal ion is 0.5-6 mM.

[0010] In some other embodiments, the metal ion is Mn 2+ , the concentration of the metal ions is 1-3 mM.

[0011] In some other embodiments, the concentration of the substrate is 50-300 g / L; the amount of the complex enzyme added is 0.3-0.6 wt %; The mass percentages of the α-glucan phosphorylase, phosphoglucomutase, phosphoglucoside isomerase, psicose 6-phosphate-3-epimerase and psicose 6-phosphate phosphatase are (0.1-0.15): (0.05-0.08): (0.05-0.08): (0.07-0.15): (0.07-0.15); The concentration of the α-glucan phosphorylase is 0.01-5 wt %; The concentration of the glucose phosphomutase is 0.01-5 wt %; The concentration of the glucose phosphotisomerase is 0.01-5 wt%; The concentration of the psicose 6-phosphate 3-epimerase is 0.01-5wt%; The concentration of the psicose 6-phosphate phosphatase is 0.01-5wt%.

[0012] In some other embodiments, the concentration of the substrate is 200-300 g / L; The concentration of the α-glucan phosphorylase is 1-3wt%; The concentration of the glucose phosphomutase is 0.5-1 wt%; The concentration of the glucose phosphotisomerase is 0.5-1 wt%; The concentration of the psicose 6-phosphate 3-epimerase is 1-2wt%; The concentration of the psicose 6-phosphate phosphatase is 1-2 wt %.

[0013] In some other embodiments, the α-glucan phosphorylase is α-glucan phosphorylase Uniprot ID: Q9SD76; The glucose phosphomutase is glucose phosphomutase Uniprot ID: Q68BJ6; The glucose phosphotisomerase is glucose phosphotisomerase Uniprot ID: P0A0T1; The psicose 6-phosphate 3-epimerase is psicose 6-phosphate 3-epimerase Uniprot ID: C4L7F4; The psicose 6-phosphate phosphatase is psicose 6-phosphate phosphatase Uniprot ID: Q9I433; Preferably, α-glucan phosphorylase UniprotID: Q9SD76, glucose phosphotyrosinase UniprotID: Q68BJ6, glucose phosphotyrosinase UniprotID: P0A0T1, psicose 6-phosphate 3-epimerase UniprotID: C4L7F4, psicose 6-phosphate phosphatase UniprotID: Q9I433 and pullulanase UniprotID: P36905 can be prepared by recombinant Escherichia coli monoclonal or derived from thermophilic bacteria, and these thermostable enzymes can withstand the heating step of the sugar production process, which inactivates harmful activities contained in the cell lysate subjected to the conversion process. This heat inactivation step reduces the chance of microbial contamination negatively impacting production operations; for example, the α-glucan phosphorylase UniprotID: Q9SD76 is derived from Arabidopsis thaliana; the glucose phosphotyrosinase UniprotID: Q68BJ6 is derived from Thermococcus kodakarensis; the glucose phosphotyrosinase UniprotID: P0A0T1 is derived from Xanthomonas citri; the allulose 6-phosphate 3-epimerase UniprotID: C4L7F4 is derived from Tolumonas auensis; the allulose 6-phosphate phosphatase UniprotID: Q9I433 is derived from Pseudomonas aeruginosa; the pullulanase UniprotID: P36905 is derived from Thermoanaerobacterium pseudoethanolic thermoanaerobic bacteria saccharolyticum).

[0014] The inventors discovered during the research process that pullulanase (Pul) can decompose the long chains of starch or dextrin when used to treat starch or dextrin. Unlike isoamylase, pullulanase can decompose the smallest unit of branched chains to maximize the utilization of starch or dextrin. Although isoamylase can also hydrolyze the α-1,6 glycosidic bonds at branch points, it cannot hydrolyze side branches composed of 2 to 3 glucose residues. Pullulanase can process the substrate more thoroughly, so that subsequent reactions can make more full use of the substrate.

[0015] α-glucan phosphorylase (α-GP) is used to convert starch or dextrin into glucose-1-phosphate; Phosphoglucomutase (PGM) is used to convert glucose-1-phosphate into glucose-6-phosphate; Glucose-6-phosphate isomerase (PGI) is used to convert glucose-6-phosphate into fructose-6-phosphate; Allulose-6-phosphate 3-epimerase (A6PE) is used to convert fructose-6-phosphate into psicose-6-phosphate. During the experiment, the inventors also found that in addition to converting fructose-6-phosphate into psicose-6-phosphate, psicose-3-epimerase can also convert the product psicose into fructose; if the reverse reaction enzyme activity of psicose-6-phosphate-3-epimerase is very high, it will make it difficult to increase the conversion rate of the product. Therefore, it is necessary to screen a psicose-6-phosphate-3-epimerase with a specific UniProt number and strong substrate specificity for subsequent reactions; Allulose-6-phosphate phosphatase (A6PP) is used to dephosphorylate the phosphate groups of psicose-6-phosphate and convert it into psicose. During the experiment, the inventors also found that the selectivity of psicose-6-phosphate phosphatase for psicose-6-phosphate was different. This is because psicose-6-phosphate phosphatase can simultaneously dephosphorylate the phosphate groups of glucose-6-phosphate, fructose-6-phosphate and even glucose-1-phosphate, and convert them into byproducts glucose and fructose; it will also make it difficult to improve the conversion rate of the product. Therefore, it is necessary to screen a psicose-6-phosphate phosphatase with a specific Uniprot number and strong substrate specificity for subsequent reactions.

[0016] The addition of metal ions can promote the reaction because metal ions have the following functions: stabilizing the molecular conformation required for the catalytic activity of the enzyme protein; as a component of the active center of the enzyme, participating in the construction of the active center of the enzyme; acting as a bridge, chelating the substrate molecule with the enzyme protein.

[0017] Beneficial effects of the present invention: The present invention utilizes a multi-enzyme catalytic system and a one-pot method to produce D-psicose using starch or dextrin as a substrate. The method has low cost and a conversion rate of up to 66.63%, and is suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0019] Figure 1This is a high performance liquid phase diagram of the production of D-psicose by the reaction of dextrin in an example of the present invention. DETAILED DESCRIPTION

[0020] Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0021] The strains and expression vectors involved in the following examples: Escherichia coli strain BL21-Trxb (DE3) was purchased from Novagen, and the plasmids used were synthesized by Suzhou Genewise Biotechnology Co., Ltd. The enzymes used in the following examples were expressed by the above strains and vectors.

[0022] The culture medium involved in the following examples: LB liquid culture medium, whose composition is: yeast powder 5.0g·L -1 , Peptone 10.0 g·L -1 and NaCl 10.0 g·L -1 .

[0023] The detection methods involved in the following examples are as follows: High performance liquid chromatography (HPLC) detection: Ca 2+ Chromatographic column; the mobile phase is pure water, and the mobile phase flow rate is 0.6 mL / min; the chromatographic column temperature is 80°C; the detector is a differential refractometer.

[0024] Example 1 Construction of recombinant strains The plasmids pET32a-GP (Uniprot: Q9SD76), pET32a-PGI (Uniprot: Q68BJ6), pET32a- PGM (Uniprot: P0A0T1), pET32a-A6PE (Uniprot: C4L7F4, Uniprot: P32719, Uniprot: B7MJV9, Uniprot: A0A286BT41, Uniprot: F4T7D9, Uniprot: A0A222WNN1, Uniprot: G7LT81 or Uniprot: A0A0A2XT86), pET32a-A6PP (Uniprot: Q9I433, Uniprot: P64636, Uniprot: Q9NPB1 or Uniprot: P78330), and pET32a- IA (Uniprot: P10342), pET32a-Pul (Uniprot: P38939, Uniprot: Q9F930 or Uniprot: P36905) were transformed into Escherichia coli BL21-Trxb (DE3) competent cells, and the transformation products were spread on ampicillin-resistant LB solid medium plates and cultured at 37°C overnight (about 16 h).

[0025] Transformants were picked on LB solid medium to obtain recombinant Escherichia coli BL21-Trxb(DE3) / pET32a-GP (Uniprot: Q9SD76), BL21-Trxb(DE3) / pET32a-PGI (Uniprot: Q68BJ6), BL21-Trxb(DE3) / pET32a-PGM (Uniprot: P0A0T1), BL21-Trxb(DE3) / pET32a-A6PE (Uniprot: C4L7F4, Uniprot: P32719, Uniprot: B7MJV9, Uniprot: A0A286BT41, Uniprot: F4T7D9, Uniprot: A0A222WNN1, Uniprot: G7LT81 or Uniprot: A0A0A2XT86), BL21-Trxb(DE3) / pET32a-A6PP (Uniprot: Q9I433, Uniprot: P64636, Uniprot: Q9NPB1 or Uniprot: P78330), BL21-Trxb(DE3) / pET32a-IA (Uniprot: P10342), BL21-Trxb(DE3) / pET32a-Pul (Uniprot:P38939, Uniprot:Q9F930 or Uniprot:P36905).

[0026] Example 2 Enzyme preparation Recombinant E. coli BL21-Trxb(DE3) / pET32a- GP (Uniprot: Q9SD76), BL21-Trxb(DE3) / pET32a-PGI (Uniprot: Q68BJ6), BL21-Trxb(DE3) / pET32a-PGM (Uniprot: P0A0T1), BL21-Trxb(DE3) / pET32a-A6PE (Uniprot: C4L7F4, Uniprot: P32719, Uniprot: B7MJV9, Uniprot: A0A286BT41, Uniprot: F4T7D9, Uniprot: A0A222WNN1, Uniprot: G7LT81 or Uniprot: A0A0A2XT86) were picked from the plate. pET32a-A6PP (Uniprot: Q9I433, Uniprot: P64636, Uniprot: Q9NPB1 or Uniprot: P78330), BL21-Trxb(DE3) / pET32a-IA (Uniprot: P10342), BL21-Trxb(DE3) / pET32a-Pul (Uniprot: P38939, Uniprot: Q9F930 or Uniprot: P36905) monoclones were inoculated (directly insert the pipette tip into the test tube) into a test tube containing 5 mL of LB liquid medium with resistance, and cultured at 37°C, 200 rpm overnight (about 16 h).

[0027] Take 5 mL of overnight cultured seed culture and transfer it to a shake flask containing 500 ml of LB liquid medium with resistance. Cultivate at 37°C and 200 rpm until OD600 reaches about 0.4-0.6 (about 3 h). Add 5 μL of 1 M IPTG solution to the test tube to make the final IPTG concentration reach 0.1 mM. Induce overnight at 25°C and 180 rpm.

[0028] The overnight cultured cells were centrifuged and collected. The bacterial sludge was weighed and resuspended in a ratio of 0.2 g of cells to 1 mL of reaction buffer. The bacterial suspension was broken with an ultrasonic disruptor, ultrasonicated for 3 s, paused for 2 s, and lasted for 3 min (the time can be adjusted according to the size of the ultrasonic volume). After centrifugation of the ultrasonic system, the supernatant obtained was the enzyme solution of the recombinant strain, namely, α-glucan phosphorylase GP (Uniprot: Q9SD76), glucose phosphomutase PGI (Uniprot: Q68BJ6), glucose phosphotisomerase PGM (Uniprot: P0A0T1), allulose-6-phosphate-3-epimerase A6PE (Uniprot: C4L7F4, Uniprot: P32719, Uniprot: B7MJV9, Uniprot: A0A286BT41, Uniprot: prot: F4T7D9, Uniprot: A0A222WNN1, Uniprot: G7LT81 or Uniprot: A0A0A2XT86), psicose-6-phosphate phosphatase A6PP (Uniprot: Q9I433, Uniprot: P64636, Uniprot: Q9NPB1 or Uniprot: P78330), amylase IA (Uniprot: P10342) and pullulanase Pul (Uniprot: P38939, Uniprot: Q9F930 or Uniprot: P36905).

[0029] Example 3 Selection of Psicose 6-phosphate phosphatase (A6PP) During the research, it was found that the selectivity of allulose-6-phosphate phosphatase (A6PP) for allulose-6-phosphate varies greatly. Allulose-6-phosphate phosphatase can simultaneously dephosphorylate the phosphate groups of glucose-6-phosphate, fructose-6-phosphate and even glucose-1-phosphate, converting them into byproducts glucose and fructose. Therefore, it is necessary to screen out a allulose-6-phosphate phosphatase that has high selectivity for allulose-6-phosphate and poor selectivity for glucose-6-phosphate, fructose-6-phosphate and even glucose-1-phosphate.

[0030] The reaction system involved in the embodiment is a dextrin final concentration of 300 g / L, Mn 2+The final concentration was 2 mM, and the α-glucan phosphorylase GP (Uniprot: Q9SD76), glucose phosphoglucomutase PGI (Uniprot: Q68BJ6), glucose phosphophisomerase PGM (Uniprot: P0A0T1), allulose-6-phosphate-3-epimerase A6PE (Uniprot: C4L7F4), and allulose-6-phosphate phosphatase A6PP (Uniprot: Q9I433, Uniprot: P64636, Uniprot: Q9NPB1 or Uniprot: P78330) used were all prepared in Example 2 above.

[0031] Based on the determination of α-glucan phosphorylase GP (Uniprot: Q9SD76), glucose phosphomutase PGI (Uniprot: Q68BJ6), glucose phosphomimerase PGM (Uniprot: P0A0T1), and psicose-6-phosphate-3-epimerase A6PE (Uniprot: C4L7F4), the selectivity of four psicose-6-phosphate phosphatases A6PP (Uniprot: Q9I433, Uniprot: P64636, Uniprot: Q9NPB1, or Uniprot: P78330) for psicose-6-phosphate was screened, that is, 100 μl of the four psicose 6-phosphate phosphatases A6PP (Uniprot: Q9I433, Uniprot: P64636, Uniprot: Q9NPB1, or Uniprot: P78330) were taken, respectively, and KPB 7.0 buffer was added to make up the reaction system to 1. ml.

[0032] The reaction system described in the above example was placed in a constant temperature reactor at 60°C for reaction, and the rotation speed was set to 700 rpm. After 18 h of reaction, samples were taken and HPLC detection was performed. The results are shown in Table 1. Uniprot: Q9I433 was selected as the allulose 6-phosphate phosphatase (A6PP) in the reaction process.

[0033] Table 1 Reaction results of different psicose 6-phosphate phosphatases

[0034] Example 4 Selection of Psicose 6-phosphate 3-epimerase (A6PE) enzyme During the research, it was found that in addition to converting fructose-6-phosphate into allulose-6-phosphate, allulose 6-phosphate 3-epimerase can also convert the product allulose into fructose. At this time, it is necessary to screen for allulose 6-phosphate 3-epimerase (A6PE) that has a strong effect on converting fructose-6-phosphate into allulose-6-phosphate, but a weak effect on converting allulose into fructose.

[0035] The reaction system involved in the embodiment is a dextrin final concentration of 300 g / L, Mn 2+ The final concentration was 2 mM, and the α-glucan phosphorylase GP (Uniprot: Q9SD76), glucose phosphomutase PGI (Uniprot: Q68BJ6), glucose phosphomutase PGM (Uniprot: P0A0T1), allulose-6-phosphate phosphatase A6PP (Uniprot: Q9I433), and allulose-6-phosphate-3-epimerase A6PE (Uniprot: C4L7F4, Uniprot: P32719, Uniprot: B7MJV9, Uniprot: A0A286BT41, Uniprot: F4T7D9, Uniprot: A0A222WNN1, Uniprot: G7LT81 or Uniprot: A0A0A2XT86) used were all prepared in Example 2 above.

[0036] Based on the determination of α-glucan phosphorylase GP (Uniprot: Q9SD76), glucose phosphomutase PGI (Uniprot: Q68BJ6), glucose phosphomonomerase PGM (Uniprot: P0A0T1), and psicose-6-phosphate phosphatase A6PP (Uniprot: Q9I433), eight psicose-6-phosphate-3-epimerase A6PE (Uniprot: C4L7F4, Uniprot: P32719, Uniprot: B7MJV9, Uniprot: A0A286BT41, Uniprot: F4T7D9, Uniprot: A0A222WNN1, Uniprot: G7LT81, or Uniprot: A0A0A2XT86) were screened for their selectivity for psicose-6-phosphate, that is, 100% of each sample was taken. μl of eight kinds of psicose-6-phosphate-3-epimerase A6PE (Uniprot: C4L7F4, Uniprot: P32719, Uniprot: B7MJV9, Uniprot: A0A286BT41, Uniprot: F4T7D9, Uniprot: A0A222WNN1, Uniprot: G7LT81 or Uniprot: A0A0A2XT86), and KPB 7.0 buffer was added to make up the reaction system to 1 ml.

[0037] The reaction system described in the above embodiment was placed in a constant temperature reactor at 60°C for reaction, and the rotation speed was set to 700 rpm. After the reaction for 18 h, samples were taken and HPLC detection was performed. The results are shown in Table 2. Table 2 Reaction results of different psicose 6-phosphate 3-epimerase

[0038] The reaction system described in the above example was placed in a constant temperature reactor at 60° C. for reaction, and the rotation speed was set to 700 rpm. After 18 h of reaction, samples were taken and HPLC detection was performed. The results are shown in Table 3. The effects of psicose 6-phosphate 3-epimerase on the conversion of fructose-6-phosphate to psicose-6-phosphate and the conversion of the product psicose to fructose were different. The above experiment was intended to screen out an enzyme that has a strong effect on the conversion of fructose-6-phosphate to psicose-6-phosphate but a weak effect on the conversion of the product psicose to fructose. Based on the results of Tables 1 and 2, Uniprot:C4L7F4 was selected as the psicose 6-phosphate 3-epimerase in the reaction process.

[0039] Table 3 Reaction results of different psicose 6-phosphate 3-epimerase with psicose as substrate

[0040] Example 5 Reaction substrate pretreatment During the reaction, it was found that after a certain degree of reaction, the utilization rate of starch or dextrin would decrease. The reason may be that polar dextrin and other substrates that cannot be reused will appear at the end of the reaction. At this time, the substrate needs to be pretreated, and isoamylase or pullulanase needs to be added to enzymatically hydrolyze the substrate, especially when the amount of substrate is relatively high.

[0041] The reaction system involved in the embodiment is that the final concentration of dextrin is 300 g / L, amylase IA (Uniprot: P10342) or pullulanase Pul (Uniprot: P38939, Uniprot: Q9F930 or Uniprot: P36905) prepared in the above Example 2, 100 μl of each is added, and the mixture is treated at 80°C for 5 h, and Mn 2+ The final concentration was 2 mM, and the complex enzymes were α-glucan phosphorylase GP (Uniprot: Q9SD76), glucose phosphomutase PGI (Uniprot: Q68BJ6), glucose phosphomitomerase PGM (Uniprot: P0A0T1), allulose-6-phosphate phosphatase A6PP (Uniprot: Q9I433) and allulose-6-phosphate-3-epimerase A6PE (Uniprot: C4L7F4), 100 μl was added respectively, and KPB 7.0 buffer was added to make the reaction system up to 1 ml. HPLC detection was performed, and the results are shown in Table 4.

[0042] Table 4 Reaction results of different amylases

[0043] The reaction system described in the above embodiment was placed in a constant temperature reactor at 60°C for reaction, and the rotation speed was set to 700 rpm. After 18 hours of reaction, samples were taken and HPLC detection was performed. The results can be seen from Table 4 that the effect of isoamylase is weaker than that of pullulanase, among which Uniprot: P36905 had the best treatment effect of 45.33%.

[0044] Example 6 Optimization of reaction temperature In this reaction process, it was found that the higher the reaction temperature, the more conducive it is to the dissolution of the substrate. The solubilizing effect of high temperature is conducive to the forward progress of the reaction, but high temperature can easily lead to enzyme inactivation, so the reaction needs to find the most suitable reaction temperature.

[0045] The reaction system involved in the embodiment is that the final concentration of dextrin is 300 g / L, Pul (Uniprot: P36905) prepared in the above Example 5 is added with 100 μl, and the mixture is treated at 80°C for 5 h. 2+ The final concentration was 2 mM. The final composite enzyme screened based on Examples 3 and 4 was α-glucan phosphorylase GP (Uniprot: Q9SD76), glucose phosphomutase PGI (Uniprot: Q68BJ6), glucose phosphomutase PGM (Uniprot: P0A0T1), allulose-6-phosphate phosphatase A6PP (Uniprot: Q9I433) and allulose-6-phosphate-3-epimerase A6PE (Uniprot: C4L7F4), 100 μl of each was added, and KPB 7.0 buffer was added to make up the reaction system to 1 ml.

[0046] The reaction system described in the above embodiment was placed in a constant temperature reactor at 60°C, 65°C, and 70°C for reaction, and the rotation speed was set to 700 rpm. After 18 h of reaction, samples were taken and HPLC detection was performed. The results are shown in Table 5. Table 5 Reaction results at different reaction temperatures

[0047] The results showed that the highest conversion rate of D-psicose was 40.64% at a reaction temperature of 65°C.

[0048] Example 7 Optimization of reaction buffer concentration In this reaction process, phosphate ions will participate in the reaction process and are essential for the reaction. However, excessive phosphate ions will inhibit the forward progress of the reaction, so the concentration of the buffer needs to be optimized. The reaction system involved in the embodiment is a final concentration of dextrin of 300 g / L, 100 μl of Pul (Uniprot: P36905) is added, and the reaction is carried out at 80°C for 5 hours. 2+The final concentration was 2 mM, and the complex enzymes were α-glucan phosphorylase GP (Uniprot: Q9SD76), phosphoglucomutase PGI (Uniprot: Q68BJ6), phosphoglucoside isomerase PGM (Uniprot: P0A0T1), psicose-6-phosphate phosphatase A6PP (Uniprot: Q9I433) and psicose-6-phosphate-3-epimerase A6PE (Uniprot: C4L7F4), 100 μl each, and KPB 7.0 buffer was added at 0 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, and 60 mM, respectively. The reaction system described in the above example was placed in a constant temperature reactor at 65° C. and the rotation speed was set to 700 rpm. After 18 h of reaction, samples were taken and HPLC detection was performed. The results showed that the conversion rate of D-psicose could reach up to 41.54% when KPB 7.0 was between 10 and 30 mM.

[0049] Table 6 Reaction results at different reaction temperatures

[0050] Example 8 Mn 2+ Concentration Optimization The reaction system involved in the embodiment is that the final concentration of dextrin is 300 g / L, P36905 is 100 μl of the crude enzyme solution prepared in the above Example 2, and the mixture is treated at 80°C for 5 h. 2+ The final concentrations were 0.5 mM, 0.75 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, and 6 mM. The complex enzymes were α-glucan phosphorylase GP (Uniprot: Q9SD76), glucose phosphomutase PGI (Uniprot: Q68BJ6), glucose phosphomutase PGM (Uniprot: P0A0T1), allulose-6-phosphate phosphatase A6PP (Uniprot: Q9I433), and allulose-6-phosphate-3-epimerase A6PE (Uniprot: C4L7F4). 100 μl was added to each of them, and 10 mM KPB 7.0 buffer was added for HPLC detection. The results are shown in Table 7.

[0051] Table 7 Different Mn 2+ The final concentration of the reaction results

[0052] The reaction system described in the above embodiment was placed in a constant temperature reactor at 65°C for reaction, and the rotation speed was set to 700 rpm. After 18 h of reaction, samples were taken and HPLC was performed. The results are shown in Table 7. 2+ When the final concentration was 1 mM, the conversion rate of the reaction was as high as 44.72%, which was the optimal concentration for ion addition.

[0053] Example 9 Optimization of enzyme amounts between different enzymes The reaction system involved in the embodiment is that the final concentration of dextrin is 300 g / L, P36905 is 100 μl of the crude enzyme solution prepared in the above Example 2, and the mixture is treated at 80°C for 5 h. 2+ The final concentration was 2 mM. The complex enzymes were α-glucan phosphorylase GP (Uniprot: Q9SD76), phosphoglucomutase PGI (Uniprot: Q68BJ6), phosphoglucoside isomerase PGM (Uniprot: P0A0T1), psicose-6-phosphate phosphatase A6PP (Uniprot: Q9I433) and psicose-6-phosphate-3-epimerase A6PE (Uniprot: C4L7F4). The amounts shown in the table below were added respectively, and KPB 7.0 buffer was added to make up the reaction system to 1 ml.

[0054] The reaction system described in the above example was placed in a constant temperature reactor at 65° C. for reaction, and the rotation speed was set to 700 rpm. After 18 h of reaction, samples were taken and HPLC detection was performed. The results are shown in Table 8. The results showed that the formation of D-psicose varied greatly with the different enzyme dosage ratios. The conversion rate of D-psicose prepared with the enzyme addition amount in sequence number 11 was the highest at 60.46%.

[0055] Table 8 Reaction results corresponding to different enzyme amounts

[0056] Example 10 The inventors found that the impurities produced during the reaction included not only the known glucose and fructose, but also the impurities with a peak at 13.58 min in the HPLC liquid phase diagram, such as Figure 1As shown, the substance at 12.067min is glucose, the peak at 13.583min is an impurity, the peak at 14.9min is fructose, and the peak at 22.1min is allulose. The impurity at the peak at 13.583min not only affects the conversion rate, but also affects the subsequent separation and extraction. Therefore, it is necessary to consider controlling the generation of this impurity during the reaction. In addition, during the study, it was found that the impurity at 13.58 min would change with the amount of glucose phosphotisomerase PGM (Uniprot: P0A0T1) enzyme: when the amount of glucose phosphotisomerase PGM (Uniprot: P0A0T1) enzyme is low, the peak of this impurity is lower, but when the amount of glucose phosphotisomerase PGM (Uniprot: P0A0T1) enzyme is too low, it will affect the conversion rate of D-allulose, so the amount of glucose phosphotisomerase PGM (Uniprot: P0A0T1) enzyme needs to be further optimized.

[0057] The reaction system involved in the embodiment is that the final concentration of dextrin is 300 g / L, P36905 is 100 μl of the crude enzyme solution prepared in the above Example 2, and the mixture is treated at 80°C for 5 h. 2+ The final concentration was 2 mM. The complex enzymes were α-glucan phosphorylase GP (Uniprot: Q9SD76), phosphoglucomutase PGI (Uniprot: Q68BJ6), phosphoglucoside isomerase PGM (Uniprot: P0A0T1), psicose-6-phosphate phosphatase A6PP (Uniprot: Q9I433) and psicose-6-phosphate-3-epimerase A6PE (Uniprot: C4L7F4). The amounts shown in the table below were added respectively, and KPB 7.0 buffer was added to make up the reaction system to 1 ml.

[0058] The reaction system described in the above embodiment was placed in a constant temperature reactor at 65°C for reaction, and the rotation speed was set to 700 rpm. After 18 hours of reaction, samples were taken and HPLC detection was performed. The results are shown in Tables 9 and 10.

[0059] Table 9 Reaction results corresponding to different Uniprot:P0A0T1 enzyme amounts

[0060] Table 10 Impurity peak areas corresponding to different Uniprot: P0A0T1 enzyme amounts

[0061] It can be seen that when the glucose phosphotisomerase PGM (Uniprot: P0A0T1) is reduced to 0.01wt%, the impurity peak area can be reduced by more than 3 times, and the conversion rate of allulose is increased to 66.63%.

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for producing D-psicose by enzyme catalysis, It is characterized in that Starch or dextrin is used as a substrate, and a complex enzyme is added to the substrate to carry out an enzyme-catalyzed reaction to synthesize D-psicose; The complex enzyme consists of alpha-glucan phosphorylase, glucose phosphomutase, glucose phosphomutase, psicose 6-phosphate-3-epimerase and psicose 6-phosphate phosphatase.

2. The method for producing D-psicose by enzyme catalysis according to claim 1, It is characterized in that The conditions of the enzyme catalytic reaction are: temperature of 50-80°C, pH value adjusted to 5-8 by adding buffer solution, reaction time of 12-20h; Alternatively, the buffer solution is a phosphate buffer solution; the concentration of the buffer solution is 5-60 mM.

3. The method for producing D-psicose by enzyme catalysis according to claim 2, It is characterized in that The conditions of the enzyme catalytic reaction are: temperature of 65-80°C, pH value adjusted to 7 by adding buffer solution, reaction time of 18h; Alternatively, the concentration of the buffer solution is 10-30 mM.

4. The method for producing D-psicose by enzyme catalysis according to claim 1, It is characterized in that The method also includes pretreating the substrate, wherein the pretreating method is: adding a pretreating enzyme to enzymolyze the substrate; the pretreating enzyme is isoamylase or pullulanase; the enzymolysis conditions are: temperature of 60-80°C, time of 3-5h; the addition amount of the pretreating enzyme is 0.1-3wt%.

5. The method for producing D-psicose by enzyme catalysis according to claim 4, It is characterized in that The pretreatment enzyme is pullulanase; the enzymatic hydrolysis conditions are: temperature 80°C, time 5h; the addition amount of the pretreatment enzyme is 0.2-0.5wt%; Or, the pullulanase is pullulanase Uniprot ID: P36905.

6. The method for producing D-psicose by enzyme catalysis according to claim 1, It is characterized in that The method further comprises adding a metal ion to the substrate, wherein the metal ion is Mn 2+ Mg 2+ 、Co 2+ , Ca 2+ Cu 2+ ; The concentration of the metal ion is 0.5-6 mM.

7. The method for producing D-psicose by enzyme catalysis according to claim 6, It is characterized in that The metal ion is Mn 2 + , the concentration of the metal ions is 1-3 mM.

8. The method for producing D-psicose by enzyme catalysis according to claim 1, It is characterized in that The concentration of the substrate is 50-300 g / L; the amount of the complex enzyme added is 0.3-0.6 wt%; The mass percentages of the α-glucan phosphorylase, phosphoglucomutase, phosphoglucoside isomerase, psicose 6-phosphate-3-epimerase and psicose 6-phosphate phosphatase are (0.1-0.15): (0.05-0.08): (0.05-0.08): (0.07-0.15): (0.07-0.15); The concentration of the α-glucan phosphorylase is 0.01-5 wt %; The concentration of the glucose phosphomutase is 0.01-5 wt %; The concentration of the glucose phosphotisomerase is 0.01-5 wt%; The concentration of the psicose 6-phosphate 3-epimerase is 0.01-5wt%; The concentration of the psicose 6-phosphate phosphatase is 0.01-5wt%.

9. The method for producing D-psicose by enzyme catalysis according to claim 8, It is characterized in that The concentration of the substrate is 200-300 g / L; The concentration of the α-glucan phosphorylase is 1-3wt%; The concentration of the glucose phosphomutase is 0.5-1 wt%; The concentration of the glucose phosphotisomerase is 0.5-1 wt%; The concentration of the psicose 6-phosphate 3-epimerase is 1-2wt%; The concentration of the psicose 6-phosphate phosphatase is 1-2 wt %.

10. The method for producing D-psicose by enzyme catalysis according to claim 9, It is characterized in that The α-glucan phosphorylase is α-glucan phosphorylase Uniprot ID: Q9SD76; The glucose phosphomutase is glucose phosphomutase Uniprot ID: Q68BJ6; The glucose phosphotisomerase is glucose phosphotisomerase Uniprot ID: P0A0T1; The psicose 6-phosphate 3-epimerase is psicose 6-phosphate 3-epimerase Uniprot ID: C4L7F4; The psicose 6-phosphate phosphatase is psicose 6-phosphate phosphatase Uniprot ID: Q9I433.

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