D-psicose 3-epimerase mutant and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a D-psicose 3-epimerase mutant and application thereof.The epimerase mutant is mutated at at least one key site in an amino acid sequence shown in SEQ ID NO: 20, and the mutation is beneficial to improving the catalytic activity of D-psicose formed by D-fructose C3 epimerization; through the mode, the epimerase mutant shows excellent catalytic activity of forming D-psicose through epimerization of D-fructose C3, can efficiently convert D-fructose into D-psicose, and is beneficial to improving the synthesis efficiency of D-psicose.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, and in particular to a D-psicose 3-epimerase mutant and application thereof. BACKGROUND

[0002] At present, the industrial production of D-psicose is by chemical synthesis and enzymatic synthesis. Chemical synthesis involves the use of strong acid, strong base and metal catalysts, which has high catalytic cost, complex post-processing and great environmental pressure. Enzymatic synthesis usually uses D-psicose 3-epimerase (D-psicose 3-epimerase, EC: 5.1.3.30), referred to as DPE, to produce D-psicose. Enzymatic synthesis of D-psicose has the advantages of environmental protection and sustainability compared with chemical synthesis. However, due to the defects of existing DPE such as low specific enzyme activity, poor thermal stability and low conversion rate, the application range is limited, and the expression amount of DPE in host cells directly affects the production cost of D-psicose. The above factors are not conducive to improving the synthesis efficiency of D-psicose. SUMMARY

[0003] In view of the above problems, the embodiments of the present application provide a D-psicose 3-epimerase mutant and application thereof to solve the technical problem of not conducive to improving the synthesis efficiency of D-psicose.

[0004] In a first aspect, the embodiments of the present application provide a D-psicose 3-epimerase mutant, which has a mutation at position 68 in the amino acid sequence shown in SEQ ID NO: 20, and has an amino acid sequence shown in any one of SEQ ID NO: 1-5.

[0005] In a second aspect, the embodiments of the present application provide a polynucleotide encoding the above-mentioned D-psicose 3-epimerase mutant.

[0006] In a third aspect, the embodiments of the present application provide a recombinant vector comprising the above-mentioned polynucleotide.

[0007] Optionally, the recombinant vector comprises a first promoter having a nucleotide sequence shown in SEQ ID NO: 21.

[0008] Optionally, the recombinant vector comprises, in sequence, a nucleotide sequence shown in SEQ ID NO: 22, a nucleotide sequence encoding any of the D-psicose 3-epimerase mutants, and a nucleotide sequence shown in SEQ ID NO: 23.

[0009] In a fourth aspect, the embodiments of the present application provide a host cell, which comprises the recombinant vector described above.

[0010] In a fifth aspect, the embodiments of the present application provide a method for synthesizing D-psicose, which comprises:

[0011] The epimerase mutant described above or the transformed host cell containing the epimerase mutant or the culture of the transformed host cell containing the epimerase mutant is contacted with D-fructose, and D-fructose is converted into D-psicose.

[0012] Optionally, the epimerase mutant or the transformed host cell containing the epimerase mutant or the culture of the transformed host cell containing the epimerase mutant is added into a reaction solution containing D-fructose for catalysis, and the reaction solution comprises a PBS buffer or a Tris-HCl buffer.

[0013] Optionally, the reaction solution comprises at least one of cobalt ions, potassium ions and manganese ions.

[0014] Optionally, the catalysis temperature is 55-70°C.

[0015] The D-psicose 3-epimerase mutant and the application thereof provided by the embodiments of the present application, the epimerase mutant is mutated at least one key site in the amino acid sequence shown in SEQ ID NO: 20, and the mutation is beneficial to improve the catalytic activity of D-fructose C3 epimerization to form D-psicose; in the above manner, the epimerase mutant shows excellent catalytic activity of D-fructose C3 epimerization to form D-psicose, and can efficiently convert D-fructose into D-psicose, which is beneficial to improve the synthesis efficiency of D-psicose.

[0016] These aspects or other aspects of the present application will be more apparent in the following description of the embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 A protein three-dimensional structure prediction diagram of a wild-type D-psicose 3-epimerase amino acid sequence is shown.

[0018] Figure 2 A structure schematic diagram of a pMA5-DPE plasmid in the embodiments of the present application is shown.

[0019] Figure 3 A protein SDS-PAGE diagram of an epimerase mutant in the embodiments of the present application is shown.

[0020] Figure 4 A liquid chromatogram standard curve of D-psicose in the embodiments of the present application is shown.

[0021] Figure 5 A liquid chromatography standard curve of D-fructose in the embodiment of the present application is shown.

[0022] Figure 6 A liquid chromatography result chart catalyzed by the epimerase mutant in the embodiment of the present application is shown.

[0023] Figure 7 A comparison chart of protein amounts of transcription expression of plasmids constructed by different promoters in the embodiment of the present application is shown.

[0024] Figure 8 A comparison chart of catalysis results of different concentrations of B. subtilis in the embodiment of the present application is shown.

[0025] Figure 9 A comparison chart of catalysis results of different pH values in the embodiment of the present application is shown.

[0026] Figure 10 A comparison chart of catalysis results of different catalysis temperatures in the embodiment of the present application is shown.

[0027] Figure 11 A comparison chart of catalysis results of different catalysis times in the embodiment of the present application is shown.

[0028] Figure 12 A comparison chart of catalysis results of different metal salts in the embodiment of the present application is shown. DETAILED DESCRIPTION

[0029] The embodiments of the present application will be described in detail below, examples of the embodiments are shown in the accompanying drawings, wherein the same or similar notations represent the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by referring to the accompanying drawings are exemplary, only for explaining the present application, and cannot be understood as limiting the present application.

[0030] In order to make the person skilled in the art better understand the scheme of the present application, the technical scheme in the embodiments of the present application will be described clearly and completely below by combining the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by the person skilled in the art without making creative efforts are within the scope of protection of the present application.

[0031] In the embodiments of the present application, it should be noted that, in this paper, relationship terms such as first and second are only used 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 the entities or operations.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0037] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0038] In this article, the terms "epimerase mutant for the synthesis of D-allulose", "epimerase mutant", "D-allulose 3-epimerase" and "D-allulose 3-epimerase mutant" all refer to enzymes that exhibit D-fructose C3 epimerization activity, which can convert D-fructose into D-allulose.

[0039] The reaction principle for converting D-fructose to D-allulose using the aforementioned epimerase mutants or D-allulose 3-epimerase mutants for the synthesis of D-allulose is shown below:

[0040]

[0041] 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.

[0042] One embodiment of this application provides a D-allulose 3-epimerase mutant, which produces a mutation at position 68 in the amino acid sequence shown in SEQ ID NO: 20, and the epimerase mutant has any of the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 5.

[0043] 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.

[0044] right Figure 1The 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.

[0045] 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.

[0046] Among the mutants, the D-allulose 3-epimerase mutant 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.

[0047] This application also provides a polynucleotide encoding a D-allulose 3-epimerase mutant, the polynucleotide comprising the nucleotide sequence corresponding to the amino acid sequence of the above-mentioned D-allulose 3-epimerase mutant.

[0048] The polynucleotide is the nucleotide sequence corresponding to SEQ ID NO: 1 to SEQ ID NO: 5.

[0049] The polynucleotide is a DNA or RNA chain formed by the polymerization of several nucleotides.

[0050] The polynucleotide only needs to encode the D-allulose 3-epimerase mutant mentioned above, and any nucleotide in the polynucleotide can be chemically modified.

[0051] This application also provides a recombinant vector comprising the polynucleotide encoding the D-allulose 3-epimerase mutant described above.

[0052] In this context, a recombinant vector refers to a DNA preparation containing a nucleic acid sequence encoding a polynucleotide of a D-allulose 3-epimerase mutant. It may also contain a control sequence. Within the recombinant vector, the nucleic acid sequence encoding the D-allulose 3-epimerase mutant is operatively linked to a suitable control sequence, allowing the D-allulose 3-epimerase mutant to be expressed in a suitable host. Specifically, the control sequence may include, but is not limited to, promoters capable of initiating transcription, arbitrary operon sequences for regulating transcription, suitable mRNA ribosome binding sites, and sequences for controlling transcription and translation termination. After transformation into a suitable host cell, the recombinant vector can replicate or function independently of the host genome, or it can integrate into the genome itself for replication or function.

[0053] There are no particular restrictions on the type of recombinant vector; any vector known in the art can be used as long as it can replicate in the host cell. Exemplarily, commonly used vectors in the art can include plasmids, granules, viruses, and bacteriophages in their natural or recombinant states. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or granule vectors, and the pBR system, pUC system, pBluescript II system, pGEM system, pTZ system, pCL system, and pET system can be used as plasmid vectors. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0054] In one embodiment, the recombinant vector includes a first promoter (promoter Pyblp) having a nucleotide sequence as shown in SEQ ID NO: 21.

[0055] In this embodiment, the use of the first promoter can improve the transcription and expression of polynucleotides encoding the D-allulose 3-epimerase mutant, and the amount of protein produced by the recombinant vector using the first promoter is greatly increased.

[0056] In some embodiments, the recombinant vector can be a pMA5 plasmid, the structure of which is as follows: Figure 2 As shown, the recombinant vector sequentially includes the nucleotide sequence shown in SEQ ID NO: 22, the nucleotide sequence encoding any D-allulose 3-epimerase isomer, and the nucleotide sequence shown in SEQ ID NO: 23.

[0057] This application also provides a host cell, including the recombinant vector described above.

[0058] In this embodiment, the recombinant vector is transformed into host cells, enabling the synthesis of the D-allulose 3-epimerase mutant in the host cells. The recombinant vector, when introduced into the host cells, allows the polynucleotide encoding the D-allulose 3-epimerase mutant in the recombinant vector to be expressed in the host cells, thereby enabling the host cells to synthesize the aforementioned D-allulose 3-epimerase mutant. This polynucleotide can be inserted into the host cell's chromosome, located outside the host cell's chromosome, or simultaneously inserted into the host cell's chromosome and located outside the chromosome. The polynucleotide can be DNA or RNA, as long as it can be expressed in the host cells. Exemplarily, the recombinant vector can be an expression cassette, including a promoter, transcription termination element, ribosomal domain, and translation termination element operatively linked to the polynucleotide.

[0059] In one implementation, the host cell may be a species of Escherichia, Erwinia, Serratia, Providencia, Corynebacterium, or Brevibacterium; exemplary, the host cell may be Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, or Aspergillus oryzae.

[0060] In one implementation, when the recombinant vector can be a pMA5 plasmid, the host cell is Bacillus subtilis.

[0061] One embodiment of this application provides a method for preparing a D-allulose 3-epimerase mutant, comprising the following steps: culturing and transforming host cells with a recombinant vector, so that the D-allulose 3-epimerase mutant accumulates in the bacterial cells of the transformed host cells, wherein the recombinant vector contains a nucleotide sequence encoding the above-mentioned D-allulose 3-epimerase mutant.

[0062] The process involves growing transformed host cells containing the recombinant vector under appropriate environmental conditions. The methods of culturing host cells can include, but are not limited to, batch culture, continuous culture, or fed-batch culture. To achieve appropriate environmental conditions, the pH can be adjusted using alkaline or acidic compounds to a suitable value, such as 5–9, 6–8, or 6.5–7.0. For example, the alkaline compound can be sodium hydroxide, potassium hydroxide, or ammonia, and the acidic compound can be hydrochloric acid, phosphoric acid, or sulfuric acid. To achieve appropriate environmental conditions, the temperature can be controlled at 25–60°C, or at 30–45°C. Alternatively, oxygen or an oxygen-containing gas mixture can be injected to maintain an aerobic environment.

[0063] The carbon source in the host cell culture medium may include, but is not limited to, at least one of carbohydrates, oils or fats, fatty acids, alcohols, or organic acids. Carbohydrates include, for example, glucose, sucrose, lactose, fructose, maltose, molasses, starch, and cellulose; oils or fats include, for example, soybean oil, sunflower oil, peanut oil, and coconut oil; fatty acids include, for example, palmitic acid, stearic acid, and linoleic acid; alcohols include, for example, glycerol and ethanol; and organic acids include, for example, acetic acid. The phosphorus source in the host cell culture medium may include, but is not limited to, at least one of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, and disodium hydrogen phosphate.

[0064] The culture medium for the host cells may further include metal salts, such as magnesium sulfate or ferric sulfate, and / or the culture medium for the host cells may further include other substances required for growth, such as amino acids or vitamins.

[0065] In one implementation method, the host cell is Escherichia coli.

[0066] In one implementation method, the host cell is Bacillus subtilis.

[0067] One embodiment of this application provides a method for synthesizing D-allulose, comprising:

[0068] The above-mentioned D-allulose 3-epimerase mutant, or a culture of a host cell containing the above-mentioned D-allulose 3-epimerase mutant, or a culture of a host cell containing the above-mentioned D-allulose 3-epimerase mutant, is brought into contact with D-fructose to convert D-fructose into D-allulose.

[0069] Among them, using the D-allulose 3-epimerase mutant mentioned above to catalyze the conversion of D-fructose to D-allulose can improve the conversion efficiency of D-allulose.

[0070] The catalytic system for D-fructose catalyzed by the epimerase mutant 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 D-fructose catalyzed by the epimerase mutant 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.

[0071] The catalytic system for D-fructose catalyzed by the D-allulose 3-epimerase mutant 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.

[0072] For example, a D-allulose 3-epimerase mutant or a transformed host cell containing the D-allulose 3-epimerase mutant or a culture of a transformed host cell containing the D-allulose 3-epimerase mutant is added to a reaction solution containing D-fructose for catalysis. The reaction solution includes PBS buffer (Phosphate-Buffered Saline) or Tris-HCl buffer (Tris(hydroxymethyl)aminomethane-Hydrochloride).

[0073] For example, the concentration of the D-allulose 3-epimerase mutant in the reaction solution is 1 mg / mL to 10 mg / mL.

[0074] The contact between the D-allulose 3-epimerase mutant and D-fructose can be carried out at pH 4.0 to 10.0. For example, the contact between the D-allulose 3-epimerase mutant and D-fructose can be carried out at pH 4.0, pH 5.0, pH 6.0, pH 7.0, pH 8.0, pH 9.0 or pH 10.0.

[0075] The contact between the D-allulose 3-epimerase mutant and D-fructose can be carried out at temperatures ranging from 50°C to 85°C, i.e., the catalytic temperature range is 50°C to 85°C. For example, the contact between the D-allulose 3-epimerase mutant and D-fructose can be carried out at 50°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, or 85°C.

[0076] The contact time between the D-allulose 3-epimerase mutant and D-fructose can be 0.5 hours to 12 hours. For example, the contact time between the D-allulose 3-epimerase mutant and D-fructose can be 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours or 11 hours.

[0077] In one embodiment, the transformed host cell containing the above-mentioned epimerase mutant is contacted with D-fructose to generate D-allulose. The concentration of the host cell is 10 g / L to 200 g / L. For example, the concentration of the host cell can be 10 g / L, 25 g / L, 50 g / L, 75 g / L, 100 g / L, 150 g / L or 200 g / L.

[0078] As one implementation method, the concentration of D-fructose is 50 g / L to 500 g / L. For example, the concentration of D-fructose can be 50 g / L, 100 g / L, 200 g / L, 300 g / L, 400 g / L or 500 g / L.

[0079] As one implementation method, the concentration of the D-allulose 3-epimerase mutant is 1 mg / mL to 10 mg / mL, for example, the concentration of the D-allulose 3-epimerase mutant is 1 mg / mL, 3 mg / mL, 5 mg / mL or 10 mg / mL.

[0080] As one implementation method, the total reaction system can be 0.5 mL, 1 mL, 10 mL, 1 L, 3.5 L, or 35 L.

[0081] Example

[0082] The coding genes for the D-allulose 3-epimerase mutants in Examples 1 to 19 of this application, and the coding gene for the D-allulose 3-epimerase in Comparative Example 1, can be referred to as DPE genes. These DPE genes contain the nucleotide sequences corresponding to the amino acid sequences of the aforementioned epimerase isomers or epimerases. For example, the DPE genes in Examples 1 to 19 contain the nucleotide sequences corresponding to any of the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 19. Constructing this DPE gene into the pMA5 plasmid yields the following results: Figure 2 The recombinant vector shown.

[0083] For example, Figure 2An 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) mentioned above. 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.

[0084] 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.

[0085] The steps in the embodiments and Comparative Example 1 of this application are as follows:

[0086] Transformation of Bacillus subtilis

[0087] The transformation of Bacillus subtilis specifically includes the following steps:

[0088] 1. Add 0.1-5 μg of plasmid DNA to 150 μL of melted competent cells and mix by inverting.

[0089] 2. Transfer to a pre-cooled 1mm shock cup, ice bath for 5 minutes, then place in the electric shock tank, 2.0KV, and shock once.

[0090] 3. Transfer to a 1.5ml EP sterile tube and add 1ml of hay fever recovery solution, then mix gently.

[0091] Incubate at 4.37℃ and 200rpm for 3 hours.

[0092] 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.

[0093] Expression and purification of protein

[0094] 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 release the expressed target protein, which was then dissolved 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. The supernatant was then purified by nickel affinity chromatography, as detailed below:

[0095] (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.

[0096] (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.

[0097] (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.

[0098] Finally, the protein eluent containing the target protein was collected and concentrated by centrifugation (4°C, 3400 rpm) 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.

[0099] Protein concentration determination

[0100] Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).

[0101] 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.

[0102] Enzymatic reaction

[0103] In vitro enzyme catalytic reaction conditions:

[0104] The reaction buffer solution is selected from at least one of Tris-HCl, sodium phosphate buffer, and potassium phosphate buffer.

[0105] Reaction pH: pH 4, pH 5, pH 6, pH 7, pH 8, pH 9, or pH 10;

[0106] The final fructose concentrations are 50 g / L, 100 g / L, 200 g / L, 300 g / L, 400 g / L, or 500 g / L;

[0107] Metal salt types: ferrous sulfate, ferric sulfate, calcium chloride, ammonium chloride, cobalt chloride, manganese sulfate, calcium chloride, magnesium chloride, magnesium sulfate, or nickel sulfate;

[0108] The final concentrations of the metal salts are 0.1 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, or 10 mM;

[0109] Amount of protease: 1 mg / mL, 3 mg / mL, 5 mg / mL, or 10 mg / mL;

[0110] Reaction temperature: 50℃, 60℃, 70℃, 80℃, or 90℃;

[0111] Reaction time: 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, or 24h;

[0112] The total reaction volume is 0.5 mL, 1 mL, 5 mL, or 10 mL.

[0113] The embodiments and comparative examples can be prepared by adding cobalt chloride metal salt, enzyme (epimerase mutant or 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.

[0114] D-allulose detection

[0115] Liquid chromatography detection conditions:

[0116] Mobile phase: 80% acetonitrile, 20% water;

[0117] Liquid column Shimpack GIST, NH2;

[0118] Temperature: 30℃;

[0119] Flow rate: 0.8 mL / min;

[0120] Injection volume: 10 μL.

[0121] 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 [reference needed]. 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.

[0122] The results of Examples 1 to 19 and Comparative Example 1 are shown in Table 1. In Comparative Example 1, the relative activity of the epimerase is 1. The relative activities of the epimerase isomers in Examples 1 to 19 are shown based on the relative activity of the epimerase in Comparative Example 1.

[0123] Table 1. Parameters of Examples 1-6 and Comparative Example 1

[0124]

[0125] Enzyme expression amount experiment of pMA5-DPE plasmid constructed by different promoters

[0126] The promoters of the pMA5-DPE plasmids in Examples 20 to 23 are different, but other parameters are the same.

[0127] Table 2 Parameters of Examples 20-23

[0128]

[0129] In Examples 20-23, protein concentration was determined after protein expression and purification, and the results are as follows: Figure 7 As shown. In Example 21, the amount of protein transcribed by promoter Hpa II and in Example 22 by promoter P43 was similar; in Example 23, the amount of protein transcribed by promoter Pyblp was significantly increased, and compared with the amount of protein transcribed by promoter T7 in Example 20, the amount of protein transcribed by promoter Pyblp in Example 23 was increased by 5.8 times.

[0130] Different cell amount experiment

[0131] In Examples 24 to 26, the concentration of Bacillus subtilis was different, the initial concentration of substrate (D-fructose) was 700 g / L, and other parameters were the same.

[0132] Table 3 Parameters of Examples 24-26

[0133]

[0134] After the catalytic reaction, the conversion rate of the substrate was detected, and the results are as follows: Figure 8 As shown. When confirming the initial concentration of the substrate, appropriately increasing the amount of Bacillus subtilis can accelerate the attainment of reaction equilibrium.

[0135] Different pH value influence on catalytic reaction experiment

[0136] The pH values ​​differed in Examples 27 to 35, but all other parameters remained the same.

[0137] Table 4 Parameters of Examples 27 to 35

[0138]

[0139]

[0140] After the catalytic reaction, the yield of the product was measured, and the results are as follows: Figure 9 As shown, the pH ranges corresponding to the Tris-HCl buffer in Examples 30-32 and the pH ranges corresponding to the PBS buffer in Examples 33-35 resulted in relatively high product yields.

[0141] Different catalytic temperature influence on catalytic reaction experiment

[0142] The catalytic temperature was different in Examples 36 to 43, but all other parameters were the same.

[0143] Table 5 Parameters of Examples 36 to 43

[0144]

[0145] After the catalytic reaction, the yield of the product was measured, and the results are as follows: Figure 10 As shown, the catalytic temperatures of Examples 39 to 42 resulted in relatively high product yields.

[0146] Different catalytic reaction time experiment

[0147] The catalytic time differed in Examples 44 to 50, while other parameters remained the same.

[0148] Table 6 Parameters of Examples 44 to 50

[0149]

[0150]

[0151] After the catalytic reaction, the yield of the product was measured, and the results are as follows: Figure 11 As shown in the examples 46 to 50, the catalytic time resulted in relatively high product yields.

[0152] Different metal salt influence on catalytic reaction experiment

[0153] The metal salts in Examples 51 to 62 are different, but all other parameters are the same.

[0154] Table 7 Parameters of Examples 51 to 62

[0155]

[0156] After the catalytic reaction, the yield of the product was measured, and the results are as follows: Figure 12 As shown. The cobalt ions in Example 54, the potassium ions in Example 58, the magnesium ions in Example 59, and the manganese ions in Example 60 yielded relatively high product yields.

[0157] 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. A mutant of D-allulose 3-epimerase, characterized in that, The epimerase mutant has a mutation at position 68 in the amino acid sequence set forth in SEQ ID NO: 20, and has an amino acid sequence set forth in any one of SEQ ID NO: 1 to SEQ ID NO:

5.

2. A polynucleotide, comprising, The polynucleotide encodes the D-allulose 3-epimerase mutant according to claim 1.

3. A recombinant vector, characterized in that, The recombinant vector comprises the polynucleotide according to claim 2.

4. The recombinant vector of claim 3, wherein, The recombinant vector comprises a first promoter having a nucleotide sequence set forth in SEQ ID NO:

21.

5. The recombinant vector of claim 4, wherein, The recombinant vector comprises, in order, a nucleotide sequence set forth in SEQ ID NO: 22, a nucleotide sequence encoding any one of the D-allulose 3-epimerase isomers, and a nucleotide sequence set forth in SEQ ID NO:

23.

6. A host cell, characterized in that, The host cell comprises the recombinant vector according to claim 3.

7. A method of synthesizing D-allulose, characterized by, Comprising: contacting the epimerase mutant according to claim 1 or a transformed host cell containing the epimerase mutant according to claim 1 or a culture of the transformed host cell containing the epimerase mutant according to claim 1 with D-fructose to convert the D-fructose into D-allulose.

8. The method of synthesizing D-psicose according to claim 7, wherein, The epimerase mutant according to claim 1 or a transformed host cell containing the epimerase mutant according to claim 1 or a culture of the transformed host cell containing the epimerase mutant according to claim 1 is added to a reaction solution containing D-fructose to catalyze the reaction, wherein the reaction solution comprises a PBS buffer or a Tris-HCl buffer.

9. The method of synthesizing D-psicose according to claim 8, wherein, The reaction solution comprises at least one of cobalt ions, potassium ions, and manganese ions. 10.The method of synthesizing D-psicose according to claim 8, characterized by, The catalytic temperature is 55°C to 70°C.

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