Immobilized enzyme for synthesizing d-allulose and application thereof

Abstract

This application relates to the field of biotechnology, and more particularly to an immobilized enzyme for the synthesis of D-allulose and its application. The immobilized enzyme for the synthesis of D-allulose provided in this application has an epimerase in which at least one key site in the amino acid sequence shown in SEQ ID NO: 20 is mutated. This mutation is beneficial to improving the catalytic activity of D-fructose C3 epimerization to form D-allulose. Furthermore, the epimerase is immobilized on a resin support to form a highly stable immobilized enzyme. Through the above method, the immobilized enzyme exhibits excellent catalytic activity for the D-fructose C3 epimerization to form D-allulose, and can efficiently convert D-fructose to D-allulose, thus improving the efficiency of D-allulose synthesis.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to an immobilized enzyme for the synthesis of D-allulose and its applications. Background Technology

[0002] Currently, the industrial production of D-allulose involves chemical synthesis and enzymatic synthesis. Chemical synthesis involves the use of strong acids, strong bases, and metal catalysts, resulting in high catalytic costs, complex post-processing, and significant environmental impact. Enzymatic synthesis typically uses D-allulose 3-epimerase (D-psicose 3-epimerase, EC: 5.1.3.30), abbreviated as DPE, to produce D-allulose. Enzymatic synthesis of D-allulose has advantages over chemical synthesis, such as being environmentally friendly and sustainable. However, existing DPEs have limitations, such as low specific enzyme activity, poor thermal stability, and low conversion rates, which restrict their application. Furthermore, the expression level of DPE in the host cell directly affects the production cost of D-allulose. All of these factors are detrimental to improving the synthesis efficiency of D-allulose. Summary of the Invention

[0003] In view of the above problems, this application provides an immobilized enzyme for synthesizing D-allulose and its application, so as to solve the above-mentioned technical problems that are not conducive to improving the synthesis efficiency of D-allulose.

[0004] In a first aspect, embodiments of this application provide an immobilized enzyme for synthesizing D-allulose, comprising a D-allulose 3-epimerase and a resin carrier for immobilizing the D-allulose 3-epimerase, wherein the D-allulose 3-epimerase has an amino acid sequence as shown in any of SEQ ID NO: 1 to SEQ ID NO: 5.

[0005] Optionally, the resin carrier includes an amino resin or an IDA resin.

[0006] Optionally, the mass ratio of the D-allulose 3-epimerase to the resin carrier is 10-12:1000.

[0007] Secondly, embodiments of this application provide a method for preparing an immobilized enzyme for synthesizing D-allulose, comprising: The resin carrier is activated to obtain an activated resin carrier; D-allulose 3-epimerase was added to an activated resin support to immobilize the D-allulose 3-epimerase on the activated resin support, thus obtaining an immobilized enzyme for the synthesis of D-allulose. The D-allulose 3-epimerase has an amino acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 5.

[0008] Optionally, the resin carrier may be activated using a glutaraldehyde solution with a mass concentration of 0.05% to 1%.

[0009] Optionally, the cross-linking reaction between the D-allulose 3-epimerase and the activated resin carrier is carried out at a pH of 7.0 to 8.0; and the cross-linking reaction time between the D-allulose 3-epimerase and the activated resin carrier is 2.8 to 3.2 hours.

[0010] Optionally, the mass ratio of the D-allulose 3-epimerase to the resin carrier is 10-12:1000; the resin carrier includes amino resin or IDA resin.

[0011] Thirdly, embodiments of this application provide a method for synthesizing D-allulose, comprising: The immobilized enzyme used to synthesize D-allulose is brought into contact with D-fructose to convert D-fructose into D-allulose.

[0012] Optionally, the catalytic pH is 7.5 and the catalytic temperature is 60℃~65℃.

[0013] Optionally, the catalytic time is 6 to 8 hours.

[0014] The immobilized enzyme for synthesizing D-allulose and its application provided in this application have an epimerase in which at least one key site is mutated in the amino acid sequence shown in SEQ ID NO: 20. This mutation is beneficial to improving the catalytic activity of D-fructose C3 epimerization to form D-allulose. Furthermore, the epimerase is immobilized on a resin support to form a highly stable immobilized enzyme. Through the above method, the immobilized enzyme exhibits excellent catalytic activity of D-fructose C3 epimerization to form D-allulose, and can efficiently convert D-fructose to D-allulose, which is beneficial to improving the synthesis efficiency of D-allulose.

[0015] These or other aspects of this application will become more apparent in the following description of the embodiments. Attached Figure Description

[0016] Figure 1 The predicted three-dimensional structure of the protein based on the amino acid sequence of the wild-type D-allulose 3-epimerase is shown.

[0017] Figure 2 A schematic diagram of the structure of the pMA5-DPE plasmid in an embodiment of this application is shown.

[0018] Figure 3 The SDS-PAGE diagram of the epimerase mutant in the embodiments of this application is shown.

[0019] Figure 4 The liquid chromatography standard curve of D-allulose in the embodiments of this application is shown.

[0020] Figure 5 The liquid chromatography standard curve of D-fructose in the embodiments of this application is shown.

[0021] Figure 6 The figure shows the liquid chromatography results of protein purification catalysis by epimerase in the embodiments of this application.

[0022] Figure 7 The images show SDS-PAGE gel images of proteins before and after purification by adsorption of epimerases on different types of resin carriers in the embodiments of this application.

[0023] Figure 8 The accompanying diagram shows a comparison of the catalytic results of immobilized enzymes prepared using different types of resin supports in the embodiments of this application.

[0024] Figure 9 The diagram shows a comparison of the catalytic results of epimerases with different mass ratios and immobilized enzymes prepared on resin carriers in the embodiments of this application.

[0025] Figure 10 The diagram shows a comparison of the catalytic results of immobilized enzymes prepared with different concentrations of glutaraldehyde solution in the embodiments of this application.

[0026] Figure 11 The diagram shows a comparison of the catalytic results of immobilized enzymes prepared by epimerase and resin support at different cross-linking times in the embodiments of this application.

[0027] Figure 12 The diagram shows a comparison of the catalytic results of the epimeric enzyme and the immobilized enzyme prepared by the resin carrier at different cross-linking pH values ​​in the embodiments of this application.

[0028] Figure 13 The diagram shows a comparison of the catalytic results of immobilized enzymes prepared by epimerase and resin carrier at different crosslinking temperatures in the embodiments of this application.

[0029] Figure 14 The accompanying diagram shows a comparison of the catalytic results of the immobilized enzyme at different catalytic temperatures in the embodiments of this application.

[0030] Figure 15 The accompanying diagram shows a comparison of the catalytic results of the immobilized enzyme at different catalytic times in the embodiments of this application.

[0031] Figure 16The accompanying diagram shows a comparison of the catalytic results of the immobilized enzyme at different catalytic pH values ​​in the embodiments of this application.

[0032] Figure 17 The accompanying diagram shows a comparison of the catalytic results of immobilized enzymes at different catalytic cycles in the embodiments of this application. Detailed Implementation

[0033] The embodiments of this application are described in detail below. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0034] To enable those skilled in the art to better understand the solutions of this application, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0035] In the embodiments of this application, it should be noted that, in this document, relational terms such as first and second are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations.

[0036] Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0037] In the description of the embodiments of this application, the words "example" or "for example" are used to indicate exemplification, illustration, or description. Any embodiment or design described as "example" or "for example" in the embodiments of this application is not to be construed as being more preferred or having more advantages than another embodiment or design. The use of the words "example" or "for example" is intended to present relative concepts in a clear manner.

[0038] Furthermore, in the embodiments of this application, "multiple" refers to two or more. Therefore, in the embodiments of this application, "multiple" can also be understood as "at least two". "At least one" can be understood as one or more, such as one, two, or more. For example, including at least one means including one, two, or more, and is not limited to which ones are included. For example, including at least one of A, B, and C, then it could include A, B, C, A and B, A and C, B and C, or A and B and C.

[0039] It should be noted that in the embodiments of this application, "and / or" describes the relationship between associated objects, indicating that there can be three relationships. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. In addition, the character " / ", unless otherwise specified, generally indicates that the associated objects before and after it are in an "or" relationship.

[0040] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0041] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0042] In this article, the terms "epimerase for the synthesis of D-allulose", "epimerase" and "D-allulose 3-epimerase" all refer to enzymes that exhibit D-fructose C3 epimerization activity, which can convert D-fructose into D-allulose.

[0043] The reaction principle for converting D-fructose to D-allulose using the aforementioned D-allulose 3-epimerase and the immobilized enzyme for synthesizing D-allulose is shown below: .

[0044] The description herein refers to "a polypeptide, protein, mutant, or enzyme having the amino acid sequence shown in SEQ ID NO:". Obviously, polypeptides, proteins, mutants, or enzymes having the amino acid sequence shown in SEQ ID NO:, even with some sequence deletions, modifications, substitutions, conserved substitutions, or additions, can also be used in this application, as long as they exhibit the same or corresponding activity as the polypeptide, protein, mutant, or enzyme with the amino acid sequence shown in SEQ ID NO:. For example, it is not excluded to add sequences that do not alter protein function, naturally occurring mutations, their silent mutations, or conserved substitutions before or after "the polypeptide, protein, mutant, or enzyme with the amino acid sequence shown in SEQ ID NO:". Furthermore, polypeptides, proteins, mutants, or enzymes having the amino acid sequence shown in SEQ ID NO:, when subjected to the addition of the aforementioned sequences that do not alter protein function, naturally occurring mutations, their silent mutations, or conserved substitutions, also fall within the scope of this application, as long as they exhibit the same or corresponding activity as the amino acid sequence shown in SEQ ID NO: after the addition of the aforementioned sequences.

[0045] One embodiment of this application provides an immobilized enzyme for synthesizing D-allulose, comprising a D-allulose 3-epimerase and a resin carrier for immobilizing the D-allulose 3-epimerase, the D-allulose 3-epimerase having an amino acid sequence as shown in any of SEQ ID NO: 1 to SEQ ID NO: 5.

[0046] In this specification, the enzyme formed by the amino acid sequence shown in SEQ ID NO: 20 can be called wild-type D-allulose 3-epimerase, and the enzyme formed by the mutated amino acid sequences can be called mutant D-allulose 3-epimerase or D-allulose 3-epimerase mutant. Inputting the amino acid sequence shown in SEQ ID NO: 20 into the AlphaFold2 model yields the following results: Figure 1 The diagram shows the predicted three-dimensional structure of the protein and its docking with the substrate D-fructose.

[0047] right Figure 1 The three-dimensional structure of the protein was analyzed. The key site P68 (proline P at position 68) in the amino acid sequence shown in SEQ ID NO: 20, which is associated with the binding of the substrate D-fructose and the exercise of D-fructose C3 epimerization activity, was selected as the research object. Saturation mutation screening was performed on the key site P68 to obtain a D-allulose 3-epimerase mutant with improved D-fructose C3 epimerization activity. In the corresponding mutation, the proline residue at position P68 in the amino acid sequence shown in SEQ ID NO: 20 was replaced by other amino acids.

[0048] In this embodiment, the term "other amino acids" is not limited, as long as it differs from the amino acids corresponding to each position. Specifically, the other amino acids in this embodiment may be one or more amino acids selected from the following: nonpolar amino acids glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), phenylalanine (F), tryptophan (W), and proline (P); polar amino acids serine (S), threonine (T), cysteine ​​(C), tyrosine (Y), asparagine (N), and glutamine (Q); acidic amino acids aspartic acid (D) and glutamic acid (E); and basic amino acids lysine (K), arginine (R), and histidine (H), but are not limited thereto.

[0049] In the mutant, the D-allulose 3-epimerase with any of the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 5 exhibits excellent catalytic activity for the D-allulose C3 epimerization to form D-allulose, and can efficiently convert D-fructose to D-allulose, which is beneficial to improving the D-allulose synthesis efficiency.

[0050] To improve the thermal stability of D-allulose 3-epimerase, it is beneficial to immobilize D-allulose 3-epimerase on a resin support, which can improve the catalytic activity of the immobilized enzyme and increase the recycling rate of the immobilized enzyme.

[0051] In one embodiment, the resin carrier includes an amino resin or an IDA (iminodiacetic acid) resin, wherein the amino resin is a type of thermosetting resin containing an amino (-NH2) functional group, and the IDA resin is an iminodiacetic acid type chelating resin.

[0052] In one embodiment, the mass ratio of the D-allulose 3-epimerase to the resin carrier is 10–12:1000. Exemplarily, the mass ratio of the D-allulose 3-epimerase to the resin carrier can be 10 mg:1 g, 11 mg:1 g, or 12 mg:1 g.

[0053] One embodiment of this application provides a method for preparing an immobilized enzyme for synthesizing D-allulose, comprising the following steps: The resin carrier is activated to obtain an activated resin carrier; D-allulose 3-epimerase was added to an activated resin support to immobilize the D-allulose 3-epimerase on the activated resin support, thus obtaining an immobilized enzyme for the synthesis of D-allulose. The D-allulose 3-epimerase has an amino acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 5.

[0054] As one implementation method, the resin carrier can be an amino resin, EA (Epoxy Acrylate) resin, modified epoxy resin (EP resin), IDA resin, HA resin (using a copolymer system of styrene monomers and divinylbenzene), epoxy resin, D101 resin, or D301 resin. The above-mentioned epoxy resin molecules contain two or more epoxy groups, with the molecular formula (C... 11 H 12 03) n It is a polycondensation product of epichlorohydrin and bisphenol A or polyol. The above-mentioned modified epoxy resin (EP resin) is obtained by modifying epoxy resin, and toughening is achieved using liquid carboxyl-terminated nitrile butadiene rubber (CTBN). The amount of CTBN added is 10% (mass percentage), and 30% (mass concentration) of silica is added to avoid strength reduction after the addition of CTBN. The acrylonitrile content of CTBN is 18-30%. In some embodiments, the resin carrier can be an amino resin or an IDA resin.

[0055] As one implementation method, glutaraldehyde is used to activate the resin carrier.

[0056] For example, the resin carrier can be activated using a glutaraldehyde solution with a mass concentration of 0.05% to 1%, that is, a glutaraldehyde solution with a mass concentration of approximately 0.0005 g / mL to 0.01 g / mL can be used to activate the resin carrier, such as a 0.05% glutaraldehyde solution, a 0.1% glutaraldehyde solution, a 0.3% glutaraldehyde solution, a 0.5% glutaraldehyde solution, a 0.8% glutaraldehyde solution, or a 1.0% glutaraldehyde solution.

[0057] The activation time of glutaraldehyde on the resin carrier can be 20 min to 240 min. For example, the activation time can be 20 min, 40 min, 60 min, 90 min, 120 min, 150 min, 180 min or 240 min.

[0058] The activation temperature of the resin carrier by glutaraldehyde can be from 4°C to 60°C. For example, the activation temperature can be 4°C, 10°C, 20°C, 30°C, 40°C, 50°C or 60°C.

[0059] In one embodiment, the cross-linking reaction of the D-allulose 3-epimerase with the activated resin carrier is at a pH of 7.0 to 8.0; exemplaryly, the cross-linking reaction of the D-allulose 3-epimerase with the activated resin carrier is at a pH of 7.0, 7.5, or 8.0.

[0060] In one embodiment, the cross-linking reaction time between the D-allulose 3-epimerase and the activated resin carrier is 2.8 to 3.2 hours. Exemplarily, the cross-linking reaction time between the D-allulose 3-epimerase and the activated resin carrier is 3 hours.

[0061] In one embodiment, the mass ratio of the D-allulose 3-epimerase to the resin carrier is 10–12:1000. Exemplarily, the mass ratio of the D-allulose 3-epimerase to the resin carrier can be 10 mg:1 g, 11 mg:1 g, or 12 mg:1 g.

[0062] One embodiment of this application provides a method for synthesizing D-allulose, comprising the following steps: The immobilized enzyme used to synthesize D-allulose is brought into contact with D-fructose to convert D-fructose into D-allulose.

[0063] The catalytic system for immobilized enzyme catalyzing D-fructose may include suitable excipients, which may include, but are not limited to, at least one of preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents. For example, the catalytic system for immobilized enzyme catalyzing D-fructose may include a buffer solution, which may include, but is not limited to, at least one of Tris-HCl, sodium phosphate buffer, and potassium phosphate buffer.

[0064] The catalytic system for immobilized enzyme catalyzing D-fructose may further include a metal salt, such as an ammonium salt, nickel salt, iron salt, cobalt salt, magnesium salt, or manganese salt. Exemplarily, the metal salt may include at least one of ferrous sulfate, ferric sulfate, calcium chloride, ammonium chloride, cobalt chloride, manganese sulfate, calcium chloride, magnesium chloride, and nickel sulfate. Furthermore, the concentration of the metal salt may be from 0.1 mM to 10 mM, for example, 0.1 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, or 10 mM.

[0065] The contact between the immobilized enzyme and D-fructose can be carried out at pH 6.0 to 8.5. For example, the contact between the immobilized enzyme and D-fructose can be carried out at pH 6.0, pH 6.5, pH 7.0, pH 7.5, pH 8.0, or pH 8.5.

[0066] The contact between the immobilized enzyme and D-fructose can be carried out at a temperature of 60℃ to 70℃. For example, the contact between the immobilized enzyme and D-fructose can be carried out at 60℃, 65℃ or 70℃.

[0067] The contact time between the immobilized enzyme and D-fructose can be from 1 hour to 10 hours. For example, the contact time between the immobilized enzyme and D-fructose can be 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, or 10 hours.

[0068] The concentration of the immobilized enzyme can be from 1 mg / mL to 10 mg / mL, for example, it can be 1 mg / mL, 3 mg / mL, 5 mg / mL or 10 mg / mL.

[0069] The initial concentration of D-fructose can be 100 g / L to 500 g / L, for example, it can be 100 g / L, 200 g / L, 300 g / L, 400 g / L or 500 g / L.

[0070] Example The coding genes for the mutant D-allulose 3-epimerase in Examples 1 to 19 of this application and the coding gene for the wild-type D-allulose 3-epimerase in Comparative Example 1 can be referred to as DPE genes. The DPE gene contains the nucleotide sequence corresponding to the amino acid sequence of the mutant or wild-type epimerase described above. For example, in Examples 1 to 19, the DPE gene contains the nucleotide sequence corresponding to any of the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 19. Constructing the DPE gene into the pMA5 plasmid yields the following results: Figure 2 The recombinant vector shown.

[0071] For example, Figure 2 An example of the recombinant vector shown, the pMA5-DPE plasmid, comprises, in sequence, the nucleotide sequence shown in SEQ ID NO: 22, the nucleotide sequence corresponding to any of the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 20, and the nucleotide sequence shown in SEQ ID NO: 23. Figure 2 The promoter in the plasmid shown is the first promoter (promoter Pyblp). The nucleotide sequence of the first promoter is shown in SEQ ID NO: 21. The first promoter is located at positions 4700 to 4802 in the nucleotide sequence shown in SEQ ID NO: 22. In addition, the plasmid uses the kanamycin resistance gene, which is located at positions 3083 to 3844 in the nucleotide sequence shown in SEQ ID NO: 22.

[0072] The recombinant vector used in the various embodiments and Comparative Example 1 of this application is a pMA5 plasmid containing the DPE gene, hereinafter referred to as pMA5-DPE; the host cell used in the various embodiments and Comparative Example 1 of this application is Bacillus subtilis.

[0073] The steps in the embodiments and Comparative Example 1 of this application are as follows: Transformation of Bacillus subtilis The transformation of Bacillus subtilis specifically includes the following steps: 1. Add 0.1-5 μg of plasmid DNA to 150 μL of melted competent cells and mix by inverting.

[0074] 2. Transfer to a pre-cooled 1mm shock cup, ice bath for 5 minutes, then place in the electric shock tank and perform one shock at 2.0 KV.

[0075] 3. Transfer to a 1.5ml EP sterile tube and add 1ml of hay fever recovery solution, then mix gently.

[0076] 4. Incubate at 37℃ and 200rpm for 3 hours.

[0077] 5. Centrifuge at 4500 rpm for 5 min, discard the supernatant, and resuspend the bacterial cells in 100 μL of liquid. Spread the entire suspension onto solid agar plates containing the corresponding antibiotic and incubate at 37°C for 16-48 h until positive colonies appear on the plates.

[0078] Protein expression and purification After growth and induction culture of Bacillus subtilis, the bacterial cells were collected by centrifugation and lysed using a high-pressure cryo-lysis device at 800-1000 bar and 4°C for 3-5 minutes to fully lyse the cells, thereby releasing the expressed target protein and dissolving it in protein buffer (50 mM Tris-HCl, pH 7.5). The lysed bacterial culture was centrifuged at 8000 rpm for 60 minutes in a pre-cooled centrifuge at 4°C. The precipitate and supernatant were collected and prepared as a sample, and the supernatant was collected. The supernatant was purified by nickel affinity chromatography, with the specific steps as follows: (1) Column equilibration: First wash with dd H2O for 2 column volumes, then equilibrate the Ni affinity chromatography column with protein buffer for 1 column volume.

[0079] (2) Sample loading: Take 50 μL of the supernatant and slowly pass it through the Ni affinity chromatography column. Flow through (can be repeated once) and take the first few drops of the flow-through sample.

[0080] (3) Elution of target protein: Use 30 mL of protein buffer containing 20 mM, 50 mM, 100 mM, 200 mM and 300 mM imidazole respectively to elute the bound impurities. Take the first few drops of each sample to flow through the sample, prepare the sample, and detect it by 12% SDS-PAGE.

[0081] Finally, the protein eluent containing the target protein was concentrated by centrifugation (4 ℃, 3400 r / min) using a 50 mL Amicon ultrafiltration tube (10 kDa, Millipore) to a final volume of 1 mL. Then, 10 mL of protein buffer was added, and the concentration was repeated to 1 mL. This process was repeated once to ensure the removal of imidazole from the protein, resulting in purified protein.

[0082] Protein concentration determination Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).

[0083] First, the protein concentration was initially determined using the absorbance at 280 nm. Then, based on the initial measurement, the protein concentration was diluted to 0.5-1 mg / mL. Reagents A and B from the BCA Protein Assay Kit were prepared at a 50:1 ratio to form the reaction solution. 200 μL of the reaction solution was placed in an ELISA plate, and 25 μL of diluted protein was added. The mixture was then incubated at 37°C for 30 min. The plate was then placed in an ELISA reader to measure the absorbance at 562 nm, and the data was processed according to the protein standard curve to obtain the protein concentration. An SDS-PAGE gel image of one of the epimerase mutants is shown below. Figure 3 As shown.

[0084] Enzyme-catalyzed reactions In vitro enzyme catalytic reaction conditions: The reaction buffer is selected from at least one of Tris-HCl, sodium phosphate buffer, and potassium phosphate buffer. Reaction pH: pH 4, pH 5, pH 6, pH 7, pH 8, pH 9, or pH 10; The final fructose concentrations are 50 g / L, 100 g / L, 200 g / L, 300 g / L, 400 g / L, or 500 g / L; Metal salt types: ferrous sulfate, ferric sulfate, calcium chloride, ammonium chloride, cobalt chloride, manganese sulfate, calcium chloride, magnesium chloride, magnesium sulfate, or nickel sulfate; The final concentrations of the metal salts are 0.1 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, or 10 mM; Amount of protease: 1 mg / mL, 3 mg / mL, 5 mg / mL, or 10 mg / mL; Reaction temperature: 50℃, 60℃, 70℃, 80℃, or 90℃; Reaction time: 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, or 24h; The total reaction volume is 0.5 mL, 1 mL, 5 mL, or 10 mL.

[0085] The embodiments and comparative examples can be prepared by adding cobalt chloride metal salt, enzyme (mutant epimerase or wild-type epimerase), and D-fructose to Tris-HCl at pH 8 to form a catalytic solution. In this catalytic solution, the concentration of cobalt chloride is 2 mM, the enzyme concentration is 3 mg / mL, and the D-fructose concentration is 200 g / L. The catalytic solution is reacted at 60°C for 2 hours, and the conversion activity of each embodiment and comparative example is measured.

[0086] D-allulose detection Liquid chromatography detection conditions: Mobile phase: 80% acetonitrile, 20% water; Liquid column Shimpack GIST, NH2; Temperature: 30℃; Flow rate: 0.8 mL / min; Injection volume: 10 μL.

[0087] For the standard curve of D-allulose, please refer to [link / reference needed]. Figure 4 As shown, the standard curve for D-fructose can be found in [reference needed]. Figure 5 As shown, the liquid phase diagram of the reaction in the embodiment is referred to Figure 6 As shown, each epimerase in Examples 1 to 19 and Comparative Example 1 converts D-fructose into D-allulose and all exhibits D-fructose-C3-epimerization activity.

[0088] The results of Examples 1 to 19 and Comparative Example 1 are shown in Table 1. The relative activity of the wild-type epimerase in Comparative Example 1 is 1. The relative activities of the mutant epimerases in Examples 1 to 19 are shown based on the relative activities of the epimerase in Comparative Example 1.

[0089] Table 1 Parameters of Examples 1 to 19 and Comparative Example 1 Immobilized enzyme preparation The purified epimerase was immobilized on a resin support to obtain the immobilized enzyme.

[0090] The conditions for immobilization, including the concentration of epimerase, immobilization time, temperature, pH, glutaraldehyde solution concentration, activation temperature, and activation time, were optimized. Ultimately, the immobilized enzyme can be reused more than 80 times, with the remaining activity maintained at over 50%.

[0091] Enzyme concentration (epomerase mg / resin carrier g): 0.45, 0.9, 2.25, 4.5, 11.25, 22.5, 33.75, 45; Fixed time: 30min, 60min, 120min, 180min, 240min; Fixed temperatures: 4℃, 16℃, 25℃, 37℃; Fixed pH values: 6, 6.5, 7, 7.5, 8, 8.5; Glutaraldehyde concentrations: 0.05%, 0.1%, 0.25%, 0.5%, 0.75%, 1%; Activation temperatures: 10℃, 20℃, 30℃, 40℃, 50℃, 60℃; Activation time: 20 min, 40 min, 60 min, 90 min, 120 min, 150 min, 180 min, 240 min.

[0092] Immobilized enzyme catalytic reaction In vitro enzyme catalytic reaction conditions: Reaction buffers: Tris-HCl buffer, PBS buffer; Reaction pH: 6.5, 7.0, 7.5, 8.0, 8.5; The final concentrations of D-fructose are 50 g / L, 100 g / L, 200 g / L, 300 g / L, 400 g / L, and 500 g / L. Metal salt types: ferrous sulfate, ferric sulfate, calcium chloride, ammonium chloride, cobalt chloride, manganese sulfate, calcium chloride, magnesium chloride, nickel sulfate; The final concentrations of the metal salts were 0.1 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, and 10 mM. The amount of immobilized enzyme: 1 mg / mL, 3 mg / mL, 5 mg / mL, 10 mg / mL; Reaction temperatures: 60℃, 65℃, 70℃; Reaction time: 1h, 2h, 3h, 4h, 6h, 8h, 10h; The total reaction volume was 0.5 mL, 1 mL, 5 mL, and 10 mL.

[0093] Experiment on the effect of resin support type on the catalytic activity of immobilized enzymes In Examples 20 to 27, the types of resin carriers were different, but other parameters were the same.

[0094] In preparing the immobilized enzyme, the resin carrier was activated with a 1% glutaraldehyde solution for 60 min at a temperature of 37°C. The purified D-allulose 3-epimerase protein was then immobilized and cross-linked with the activated resin carrier. The mass ratio of D-allulose 3-epimerase to resin carrier was 10 mg: 1 g. The immobilization and cross-linking temperature was 37°C, the immobilization and cross-linking time was 3 hours, and the pH value for immobilization and cross-linking was 7.5.

[0095] For immobilized enzyme catalysis, cobalt chloride metal salt, immobilized enzyme, and D-fructose were added to a Tris-HCl buffer at pH 7.5 to form a catalytic solution. In this solution, the concentration of cobalt chloride was 2 mM, the concentration of the immobilized enzyme was 3 mg / mL, and the concentration of D-fructose was 500 g / L. The catalytic solution was reacted at 60°C for 6 hours. The relative activities of the immobilized enzyme in each embodiment were measured. The embodiment with the highest relative activity was represented as 100%, and the relative activities of the other embodiments were based on the embodiment with the highest relative activity.

[0096] Table 2 Parameters of Examples 20 to 27 The results are as follows Figure 7 and Figure 8 As shown, Figure 7 The images show SDS-PAGE gel images of the immobilized enzyme before and after protein purification following the adsorption of epimerase onto the resin carrier. Figure 8 As shown, the amino resin in Example 20 and the IDA resin in Example 23 have relatively high activity.

[0097] Experiment on the effect of the amount of purified epimerase added on the catalytic activity of immobilized enzyme In Examples 28 to 35, the mass ratio of epimerase-purified protein to resin carrier was different, while other parameters were the same.

[0098] When preparing the immobilized enzyme, the amino resin was activated with a 1% glutaraldehyde solution for 60 min at a temperature of 37°C. The purified D-allulose 3-epimerase protein was then immobilized and cross-linked with the activated amino resin at a temperature of 37°C for 3 hours at a pH of 7.5.

[0099] For immobilized enzyme catalysis, cobalt chloride metal salt, immobilized enzyme, and D-fructose were added to a Tris-HCl buffer solution at pH 7.5 to form a catalytic solution. In this solution, the concentration of cobalt chloride was 2 mM, the concentration of the immobilized enzyme was 3 mg / mL, and the concentration of D-fructose was 500 g / L. The catalytic solution was reacted at 60°C for 6 hours, and the product concentration of each example was measured.

[0100] Table 3 Parameters of Examples 28 to 35 The results are as follows Figure 9 As shown, in Example 31, the mass ratio of D-allulose 3-epimerase to resin carrier was 11.25 mg: 1 g, resulting in the highest product concentration.

[0101] Experiment on the effect of cross-linking agent concentration on the catalytic activity of immobilized enzymes In Examples 36 to 41, the concentration of the crosslinking agent glutaraldehyde was different, while other parameters were the same.

[0102] In preparing the immobilized enzyme, the amino resin was activated with glutaraldehyde solution for 60 min at 37℃. The purified D-allulose 3-epimerase protein was then immobilized and cross-linked with the activated amino resin. The mass ratio of D-allulose 3-epimerase to the resin carrier was 10 mg: 1 g. The immobilization and cross-linking temperature was 37℃, the immobilization and cross-linking time was 3 hours, and the pH value for immobilization and cross-linking was 7.5.

[0103] For immobilized enzyme catalysis, cobalt chloride metal salt, immobilized enzyme, and D-fructose were added to a Tris-HCl buffer solution at pH 7.5 to form a catalytic solution. In this solution, the concentration of cobalt chloride was 2 mM, the concentration of the immobilized enzyme was 3 mg / mL, and the concentration of D-fructose was 500 g / L. The catalytic solution was reacted at 60°C for 6 hours, and the product concentration of each example was measured.

[0104] Table 4 Parameters of Examples 36 to 41 The results are as follows Figure 10 As shown, in Example 41, a glutaraldehyde solution with a mass concentration of 1.0% was used for activation, resulting in the highest product concentration.

[0105] The effect of cross-linking time between epimerase purified protein and resin support on the catalytic activity of immobilized enzyme In Examples 42 to 46, the cross-linking time between the epimerase-purified protein and the resin carrier was different, while other parameters were the same.

[0106] In preparing the immobilized enzyme, the amino resin was activated with a 1% glutaraldehyde solution for 60 min at a temperature of 37°C. The purified D-allulose 3-epimerase protein was then immobilized and cross-linked with the activated amino resin. The mass ratio of D-allulose 3-epimerase to the resin carrier was 10 mg: 1 g. The immobilization and cross-linking temperature was 37°C, and the pH value was 7.5.

[0107] For immobilized enzyme catalysis, cobalt chloride metal salt, immobilized enzyme, and D-fructose were added to a Tris-HCl buffer solution at pH 7.5 to form a catalytic solution. In this solution, the concentration of cobalt chloride was 2 mM, the concentration of the immobilized enzyme was 3 mg / mL, and the concentration of D-fructose was 500 g / L. The catalytic solution was reacted at 60°C for 6 hours, and the product concentration of each example was measured.

[0108] Table 5 Parameters of Examples 42 to 46 The results are as follows Figure 11 As shown, in Example 45, the cross-linking time between the epimerase-purified protein and the resin carrier was 3 hours, resulting in the highest product concentration.

[0109] Experiment on the effect of pH value on the catalytic activity of epimerase purified protein crosslinking with resin support In Examples 47 to 52, the cross-linking pH values ​​between the epimerase-purified protein and the resin carrier were different, while other parameters were the same.

[0110] In preparing the immobilized enzyme, the amino resin was activated with a 1% glutaraldehyde solution for 60 min at a temperature of 37°C. The purified D-allulose 3-epimerase protein was then immobilized and cross-linked with the activated amino resin. The mass ratio of D-allulose 3-epimerase to the resin carrier was 10 mg: 1 g. The immobilization and cross-linking temperature was 37°C, and the immobilization and cross-linking time was 3 hours.

[0111] For immobilized enzyme catalysis, cobalt chloride metal salt, immobilized enzyme, and D-fructose were added to a Tris-HCl buffer solution at pH 7.5 to form a catalytic solution. In this solution, the concentration of cobalt chloride was 2 mM, the concentration of the immobilized enzyme was 3 mg / mL, and the concentration of D-fructose was 500 g / L. The catalytic solution was reacted at 60°C for 6 hours, and the product concentration of each example was measured.

[0112] Table 6 Parameters of Examples 47 to 52 The results are as follows Figure 12As shown, in Example 50, the cross-linking pH of the epimerase-purified protein with the resin carrier was 7.5, resulting in the highest product concentration.

[0113] The effect of cross-linking temperature between epimerase purified protein and resin support on the catalytic activity of immobilized enzyme In Examples 53 to 56, the cross-linking temperatures of the epimerase-purified proteins and the resin carriers were different, while other parameters were the same.

[0114] In preparing the immobilized enzyme, the amino resin was activated with a 1% glutaraldehyde solution for 60 min at a temperature of 37°C. The purified D-allulose 3-epimerase protein was then immobilized and cross-linked with the activated amino resin. The mass ratio of D-allulose 3-epimerase to the resin carrier was 10 mg: 1 g. The immobilization and cross-linking time was 3 hours, and the pH value for immobilization and cross-linking was 7.5.

[0115] For immobilized enzyme catalysis, cobalt chloride metal salt, immobilized enzyme, and D-fructose were added to a Tris-HCl buffer solution at pH 7.5 to form a catalytic solution. In this solution, the concentration of cobalt chloride was 2 mM, the concentration of the immobilized enzyme was 3 mg / mL, and the concentration of D-fructose was 500 g / L. The catalytic solution was reacted at 60°C for 6 hours, and the product concentration of each example was measured.

[0116] Table 7 Parameters of Examples 53 to 56 The results are as follows Figure 13 As shown, different cross-linking temperatures in Examples 53 to 56 had little effect on the catalytic activity of the immobilized enzyme, with Example 56 having the highest product concentration.

[0117] Optimization experiment of immobilized enzyme catalytic temperature The catalytic temperature was different in Examples 57 to 59, but other parameters were the same.

[0118] In preparing the immobilized enzyme, the amino resin was activated with a 1% glutaraldehyde solution for 60 min at a temperature of 37°C. The purified D-allulose 3-epimerase protein was then immobilized and cross-linked with the activated amino resin. The mass ratio of D-allulose 3-epimerase to the resin carrier was 10 mg: 1 g. The immobilization and cross-linking temperature was 37°C, the immobilization and cross-linking time was 3 hours, and the pH value for immobilization and cross-linking was 7.5.

[0119] For immobilized enzyme catalysis, cobalt chloride metal salt, immobilized enzyme, and D-fructose were added to a Tris-HCl buffer at pH 8.5 to form a catalytic solution. In this solution, the concentration of cobalt chloride was 2 mM, the concentration of the immobilized enzyme was 3 mg / mL, and the concentration of D-fructose was 500 g / L. The catalytic solution was reacted for 6 hours, and the product yield of each example was measured.

[0120] Table 8 Parameters of Examples 57 to 59 The results are as follows Figure 14 As shown, different catalytic temperatures in Examples 57 to 59 had little effect on the catalytic activity of the immobilized enzyme, with Example 59 showing the highest product yield.

[0121] Experiment on the effect of immobilized enzyme catalytic time on immobilized enzyme catalytic activity In Examples 60 to 66, the immobilized enzyme catalytic time was different, while other parameters were the same.

[0122] In preparing the immobilized enzyme, the amino resin was activated with a 1% glutaraldehyde solution for 60 min at a temperature of 37°C. The purified D-allulose 3-epimerase protein was then immobilized and cross-linked with the activated amino resin. The mass ratio of D-allulose 3-epimerase to the resin carrier was 10 mg: 1 g. The immobilization and cross-linking time was 3 hours, the pH value was 7.5, and the temperature was 37°C.

[0123] For immobilized enzyme catalysis, cobalt chloride metal salt, immobilized enzyme, and D-fructose were added to a Tris-HCl buffer at pH 7.5 to form a catalytic solution. In this solution, the concentration of cobalt chloride was 2 mM, the concentration of the immobilized enzyme was 3 mg / mL, and the concentration of D-fructose was 500 g / L. The catalytic reaction was carried out at 60°C, and the product yields of each example were measured.

[0124] Table 9 Parameters of Examples 60 to 66 The results are as follows Figure 15 As shown, different catalytic times in Examples 60 to 66 have different effects on the catalytic activity of the immobilized enzyme, and the product yields in Examples 64 and 65 are higher.

[0125] Experiment on the effect of pH value on the catalytic activity of immobilized enzymes In Examples 67 to 71, the immobilized enzymes were catalyzed at different pH values, while other parameters remained the same.

[0126] In preparing the immobilized enzyme, the amino resin was activated with a 1% glutaraldehyde solution for 60 min at a temperature of 37°C. The purified D-allulose 3-epimerase protein was then immobilized and cross-linked with the activated amino resin. The mass ratio of D-allulose 3-epimerase to the resin carrier was 10 mg: 1 g. The immobilization and cross-linking time was 3 hours, the pH value was 7.5, and the temperature was 37°C.

[0127] For immobilized enzyme catalysis, cobalt chloride metal salt, immobilized enzyme, and D-fructose were added to Tris-HCl buffer to form a catalytic solution. In this solution, the concentration of cobalt chloride was 2 mM, the concentration of the immobilized enzyme was 3 mg / mL, and the concentration of D-fructose was 500 g / L. The catalytic solution was reacted at 60°C for 6 hours, and the product yield of each example was measured.

[0128] Table 10 Parameters of Examples 67 to 71 The results are as follows Figure 16 As shown, different catalytic pH values ​​have little effect on the catalytic activity of immobilized enzymes, and the product yields of Examples 68 and 71 are relatively high.

[0129] Immobilized enzyme repetition experiment In preparing the immobilized enzyme, the amino resin was activated with a 1% glutaraldehyde solution for 60 min at a temperature of 37°C. The purified D-allulose 3-epimerase protein was then immobilized and cross-linked with the activated amino resin. The mass ratio of D-allulose 3-epimerase to the resin carrier was 10 mg: 1 g. The immobilization and cross-linking time was 3 hours, the pH value was 7.5, and the temperature was 37°C.

[0130] For immobilized enzyme catalysis, cobalt chloride metal salt, immobilized enzyme, and D-fructose were added to a Tris-HCl buffer solution with a pH of 7.5 to form a catalytic solution. In this solution, the concentration of cobalt chloride was 2 mM, the concentration of the immobilized enzyme was 3 mg / mL, and the concentration of D-fructose was 500 g / L. The catalytic solution was reacted at 60°C for 6 hours, and the product concentration of each example was measured.

[0131] The results are as follows Figure 17 As shown, the immobilized enzyme exhibits higher catalytic activity after 5-20 catalytic cycles than the initially immobilized enzyme, and retains more than 50% of its activity after 80 catalytic cycles. It is necessary to refer to the instruction manual. Figure 17In the study, the immobilized enzymes with 5-20 catalytic cycles exhibited higher activity than the initially immobilized enzymes. The applicant speculates the following reasons: After the immobilized enzymes were prepared, a large number of epimerases were embedded in the resin support. Microscopically, epimerase molecules are stacked on the resin support. In the initial immobilized enzymes, a large number of epimerase molecules were embedded inside the resin support, and epimerase molecules were also present on the surface of the resin support. When the initial immobilized enzymes had fewer catalytic cycles, the epimerase molecules embedded inside could not participate in the reaction. As the number of catalytic cycles increased, the epimerase molecules on the surface of the resin support inevitably detached, and more epimerase molecules participated in the reaction. Therefore, the activity of the immobilized enzymes with 5-20 catalytic cycles increased slightly. Subsequently, as the epimerase molecules further detached, the activity gradually decreased.

[0132] The above description is merely an embodiment of this application. It should be noted that those skilled in the art can make improvements without departing from the inventive concept of this application, but these improvements all fall within the protection scope of this application.

Claims

1. An immobilized enzyme for the synthesis of D-allulose, characterized in that, The invention includes a D-allulose 3-epimerase and a resin carrier immobilizing the D-allulose 3-epimerase, the D-allulose 3-epimerase having an amino acid sequence as shown in any of SEQ ID NO: 1 to SEQ ID NO:

5.

2. The immobilized enzyme for synthesizing D-allulose according to claim 1, characterized in that, The resin carrier includes amino resin or IDA resin.

3. The immobilized enzyme for synthesizing D-allulose according to claim 1, characterized in that, The mass ratio of the D-allulose 3-epimerase to the resin carrier is 10-12:1000.

4. A method for preparing an immobilized enzyme for the synthesis of D-allulose, characterized in that, include: The resin carrier is activated to obtain an activated resin carrier; D-allulose 3-epimerase was added to an activated resin support to immobilize the D-allulose 3-epimerase on the activated resin support, thus obtaining an immobilized enzyme for the synthesis of D-allulose. The D-allulose 3-epimerase has an amino acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO:

5.

5. The method for preparing the immobilized enzyme for synthesizing D-allulose according to claim 4, characterized in that, The resin carrier was activated using a glutaraldehyde solution with a mass concentration of 0.05% to 1%.

6. The method for preparing the immobilized enzyme for synthesizing D-allulose according to claim 4, characterized in that, The cross-linking reaction between the D-allulose 3-epimerase and the activated resin carrier is carried out at a pH of 7.0–8.0; the cross-linking reaction time between the D-allulose 3-epimerase and the activated resin carrier is 2.8–3.2 hours.

7. The method for preparing the immobilized enzyme for synthesizing D-allulose according to claim 4, characterized in that, The mass ratio of the D-allulose 3-epimerase to the resin carrier is 10-12:1000; the resin carrier includes amino resin or IDA resin.

8. A method for synthesizing D-allulose, characterized in that, include: The immobilized enzyme for synthesizing D-allulose according to any one of claims 1 to 3 is contacted with D-fructose to convert D-fructose into D-allulose.

9. The method for synthesizing D-allulose according to claim 8, characterized in that, The catalytic pH value is 7.5, and the catalytic temperature is 60℃~65℃.

10. The method for synthesizing D-allulose according to claim 9, characterized in that, The catalytic time is 6 to 8 hours.

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