Hexaldehyde aldose-2-epimerase mutant and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a hexanose-2-epimerase mutant and application thereof. According to the hexaldehyde aldose-2-epimerase mutant and the application of the hexaldehyde aldose-2-epimerase mutant in the embodiment of the invention, the hexaldehyde aldose-2-epimerase mutant generates mutation beneficial to improvement of hexaldehyde aldose-2 hydroxyl isomerization catalytic activity at at least one key site in an amino acid sequence as shown in SEQ ID NO: 19; through the mode, the hexaldehyde sacchar-2-epimerase mutant shows excellent hexaldehyde sugar C-2 hydroxyl isomerization catalytic activity, can efficiently convert D-glucose into D-mannose, and is beneficial to improving the synthesis efficiency of the D-mannose.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a hexanoose-2-epimerase mutant and its applications. Background Technology

[0002] With the development of modern society and the improvement of living standards, people have begun to pay attention to functional sugars and their applications in health. As a sucrose substitute, functional sugars have broad prospects in the fields of food, pharmaceuticals, and health care due to their unique physiological functions.

[0003] D-mannose is a functional sugar with many physiological benefits: as a functional sweetener, it has prebiotic functions; it inhibits type 1 diabetes and lung airway inflammation by regulating T cell immunity; it enhances wound healing and prevents bacterial infection; and it is one of the important precursors for the synthesis of vitamins, anti-tumor drugs, and immunostimulatory drugs.

[0004] Currently, the main methods for synthesizing D-mannose include plant extraction, chemical synthesis, and enzymatic methods. While plant extraction is widely used, it is significantly affected by factors such as insufficient raw material supply, season, and climate, and is also detrimental to environmental protection. Chemical synthesis is complex, costly, and generates large amounts of acidic wastewater that pollute the environment; furthermore, the final separation and purification of D-mannose is difficult. Enzymatic methods, while offering mild reaction conditions, fewer byproducts, easy separation and purification, no environmental pollution, and low cost, suffer from low enzyme catalytic efficiency, hindering the improvement of D-mannose synthesis efficiency. Summary of the Invention

[0005] Based on this, this application provides a hexanoose-2-epimerase mutant and its application to solve the above-mentioned technical problems that are not conducive to improving the synthesis efficiency of D-mannose.

[0006] In a first aspect, embodiments of this application provide an aldose-2-epimerase mutant, the amino acid sequence of which is shown in any one of SEQ ID NO: 1 to SEQ ID NO: 5.

[0007] Secondly, embodiments of this application provide a polynucleotide that encodes the aforementioned aldose-2-epimerase mutant.

[0008] Thirdly, embodiments of this application provide a recombinant vector comprising the aforementioned polynucleotides.

[0009] Fourthly, embodiments of this application provide a host cell, the host cell comprising the recombinant vector described above.

[0010] Optionally, the host cell is a bacterial cell, specifically Escherichia coli or Bacillus subtilis.

[0011] Fifthly, embodiments of this application provide a whole-cell catalyst, the preparation steps of which include:

[0012] Transformed host cells containing a recombinant vector are cultured and transformed to allow the aldose-2-epimerase mutant to accumulate in the bacterial cells of the transformed host cells. The bacterial cells are then collected to obtain the whole-cell catalyst. The recombinant vector contains a nucleotide sequence encoding the aforementioned aldose-2-epimerase mutant.

[0013] Fifthly, embodiments of this application provide an immobilized cell, including a carrier and a host cell immobilized on the carrier, wherein the host cell synthesizes the aforementioned aldose-2-epimerase mutant.

[0014] Sixthly, embodiments of this application provide the use of the above-described aldose-2-epimerase mutant, the above-described polynucleotide, the above-described recombinant vector, or the above-described host cell in any of the following:

[0015] A1) Production of D-mannose;

[0016] A2) Prepare products for the production of D-mannose;

[0017] A3) catalyzes the conversion of D-glucose to D-mannose.

[0018] Seventhly, embodiments of this application provide a method for synthesizing D-mannose, comprising:

[0019] The above-mentioned aldose-2-epimerase mutant or the transformed host cell containing the above-mentioned aldose-2-epimerase mutant is brought into contact with D-glucose to convert D-glucose into D-mannose.

[0020] Optionally, the reaction temperature is 45℃~55℃, and the reaction pH is 7~10.

[0021] The aldose-2-epimerase mutant and its application in this application are described. The aldose-2-epimerase mutant produces a mutation at at least one key site in the amino acid sequence shown in SEQ ID NO: 19 that is beneficial to improving the catalytic activity of aldose C-2 hydroxyl isomerization. In this way, the aldose-2-epimerase mutant exhibits excellent aldose C-2 hydroxyl isomerization catalytic activity, which can efficiently convert D-glucose to D-mannose, thereby improving the synthesis efficiency of D-mannose.

[0022] These or other aspects of this application will become more apparent in the following description of the embodiments. Attached Figure Description

[0023] Figure 1 The predicted three-dimensional structure of the protein based on the amino acid sequence of the wild-type hexanoose-2-epimerase is shown.

[0024] Figure 2 The liquid chromatography standard curve of D-glucose in the embodiments of this application is shown.

[0025] Figure 3 The liquid chromatography standard curve of D-mannose in the embodiments of this application is shown.

[0026] Figure 4 The figure shows the liquid chromatography results catalyzed by the epimerase mutant in the embodiments of this application.

[0027] Figure 5 The diagram shows the change in catalytic activity of the epimerase mutant in the embodiments of this application as a function of temperature. Detailed Implementation

[0028] The technical solutions in 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. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0029] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0030] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0031] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0032] In this article, the terms "aldeoxy-2-epimerase" and "aldeoxy-2-epimerase mutant" refer to enzymes that exhibit catalytic activity of isomerization of the C-2 hydroxyl group of aldose, which can convert D-glucose into D-mannose.

[0033] The reaction principle for converting D-glucose to D-mannose using the aforementioned aldose-2-epomerase or aldose-2-epomerase mutant is shown below:

[0034]

[0035] 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.

[0036] One embodiment of this application provides a hexanoose-2-epimerase mutant, which generates at least one mutation in the amino acid sequence shown in SEQ ID NO: 19.

[0037] Among them, the amino acid sequence shown in SEQ ID NO: 19 is derived from the AGE family epimerase of Dyadobacter beijingensis.

[0038] In this specification, the enzyme formed by the amino acid sequence shown in SEQ ID NO: 19 can be called wild-type aldose-2-epimerase, and the enzyme formed by the mutated amino acid sequences can be called mutant aldose-2-epimerase or aldose-2-epimerase mutant.

[0039] The three-dimensional structure of the protein was predicted by performing an amino acid sequence as shown in SEQ ID NO: 19. Figure 1 The three-dimensional predicted structure of the protein is shown. Analysis of the three-dimensional predicted structure of the protein reveals two key sites in the amino acid sequence shown in SEQ ID NO: 19, namely I213 (isoleucine I at position 213) and T240 (threonine T at position 240).

[0040] Saturation mutation screening was performed on the two key sites mentioned above to obtain a hexanoose-2-epimerase mutant with improved catalytic activity of hexanoose C-2 hydroxyl isomerization.

[0041] In this embodiment, the amino acid sequence of the hexanoose-2-epimerase mutant is shown as any one of SEQ ID NO: 1 to SEQ ID NO: 5.

[0042] In the aldose-2-epimerase mutant that produces a mutation, the aldose-2-epimerase mutant corresponding to the amino acid sequence shown in SEQ ID NO: 1, which mutates isoleucine I at position 213 to valine V, exhibits a significant increase in the catalytic activity of aldose C-2 hydroxyl isomerization.

[0043] Among the two mutant aldose-2-epimerase mutants, the aldose-2-epimerase mutants corresponding to the amino acid sequences shown in SEQ ID NO: 2, which produce a mutation of isoleucine I at position 213 to valine V and threonine T at position 240 to alanine A, exhibit a significant increase in the catalytic activity of aldose C-2 hydroxyl isomerization.

[0044] Among the two mutant aldose-2-epimerase mutants, the aldose-2-epimerase mutants corresponding to the amino acid sequences shown in SEQ ID NO: 3, which produce the mutation of isoleucine I at position 213 to valine V and threonine T at position 240 to serine S, exhibit a significant increase in the catalytic activity of aldose C-2 hydroxyl isomerization.

[0045] Among the two mutant aldose-2-epimerase mutants, the aldose-2-epimerase mutants corresponding to the amino acid sequences shown in SEQ ID NO: 4, which produce the mutation of isoleucine I at position 213 to valine V and threonine T at position 240 to aspartic acid D, exhibit a significant increase in the catalytic activity of aldose C-2 hydroxyl isomerization.

[0046] Among the two mutant aldose-2-epimerase mutants, the aldose-2-epimerase mutants corresponding to the amino acid sequences shown in SEQ ID NO: 5, which produce the mutation of isoleucine I at position 213 to valine V and threonine T at position 240 to methionine M, exhibit a significant increase in the catalytic activity of aldose C-2 hydroxyl isomerization.

[0047] One embodiment of this application provides a polynucleotide encoding an aldose-2-epimerase mutant, the polynucleotide comprising the nucleotide sequence corresponding to the amino acid sequence of the aforementioned aldose-2-epimerase mutant.

[0048] The polynucleotide has the nucleotide sequences 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 be able to encode the above-mentioned aldose-2-epimerase mutant, and any nucleotide in the polynucleotide can be chemically modified.

[0051] This application also provides a recombinant vector comprising the polynucleotide encoding the above-described aldose-2-epimerase mutant.

[0052] The recombinant vector is any natural or artificially constructed expression vector that encodes the above-mentioned nucleic acid molecule of aldose-2-epimerase mutant, which can be catalyzed by cellular transcriptases and / or translatases.

[0053] Specifically, a recombinant vector is a DNA preparation containing a nucleic acid sequence encoding a polynucleotide of an aldose-2-epimerase mutant. It may also contain a control sequence. In the recombinant vector, the nucleic acid sequence encoding the polynucleotide of the aldose-2-epimerase mutant is operatively linked to a suitable control sequence, allowing the aldose-2-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 to replicate or function.

[0054] 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, bacteriophages, or transposons 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.

[0055] This application also provides a host cell, including the recombinant vector described above.

[0056] In this embodiment, the recombinant vector is transformed into host cells, enabling the synthesis of the aldose-2-epimerase mutant within the host cells. The recombinant vector, when introduced into host cells, allows the polynucleotide encoding the aldose-2-epimerase mutant in the recombinant vector to be expressed in the host cells, thereby enabling the host cells to synthesize the aforementioned aldose-2-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.

[0057] The host cell can be a eukaryotic cell or a prokaryotic cell, and further, the prokaryotic cell can be a bacterial cell.

[0058] 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.

[0059] One embodiment of this application provides a whole-cell catalyst, the preparation steps of which include:

[0060] Transformed host cells containing a recombinant vector are cultured and transformed to allow the aldose-2-epimerase mutant to accumulate in the bacterial cells of the transformed host cells. The bacterial cells are collected to obtain the whole-cell catalyst, wherein the recombinant vector contains a nucleotide sequence encoding the aforementioned aldose-2-epimerase mutant.

[0061] The recombinant vector can be the recombinant vector described in the above embodiments, and the transformed host cell transformed with the recombinant vector can be the host cell described in the above embodiments. For example, the host cell can be cultured in a growth medium, then induced, and the cells collected to obtain the whole-cell catalyst.

[0062] One embodiment of this application provides an immobilized cell comprising a carrier and a host cell immobilized on the carrier, wherein the host cell synthesizes the aforementioned aldose-2-epimerase mutant.

[0063] The host cell can be the host cell described in the above embodiment. For example, the host cell can be cultured in a growth medium, then induced, the bacterial cells collected, and then the bacterial cells immobilized on the carrier to obtain immobilized cells.

[0064] One embodiment of this application provides the use of the above-described aldose-2-epimerase mutant, the above-described polynucleotide, the above-described recombinant vector, or the above-described host cell in any of the following: producing D-mannose; preparing a product for producing D-mannose; catalyzing the conversion of D-glucose to D-mannose.

[0065] One embodiment of this application provides a method for synthesizing D-mannose, comprising:

[0066] The above-mentioned aldose-2-epimerase mutant or the transformed host cell containing the above-mentioned aldose-2-epimerase mutant is brought into contact with D-glucose to convert D-glucose into D-mannose.

[0067] The catalytic reaction between D-glucose and the hexanoose-2-epimerase mutant or host cells containing the hexanoose-2-epimerase mutant can be carried out at 45℃ to 55℃, for example, at 45℃, 50℃ or 55℃.

[0068] The catalytic reaction between D-glucose and auroral-2-epimerase mutant or host cells containing auroral-2-epimerase mutant can be carried out at pH 7–10, for example, at pH 7.0, pH 7.5, pH 8.0, pH 8.5, pH 9.0, pH 9.5 or pH 10.0.

[0069] The catalytic system containing a hexanoose-2-epimerase mutant or a host cell containing a hexanoose-2-epimerase mutant and D-glucose 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 described above 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.

[0070] The catalytic system of D-glucose with aldose-2-epimerase mutant or host cell containing aldose-2-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. Further, 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.

[0071] The catalytic time between D-glucose and the hexalose-2-epimerase mutant or host cells containing the hexalose-2-epimerase mutant can be 2 hours to 24 hours. For example, the catalytic time can be 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours or 24 hours.

[0072] In one implementation, when host cells containing the hexanoose-2-episomerase mutant undergo a catalytic reaction with D-glucose, the concentration of the host cells is 10 g / L to 50 g / L, for example, the concentration of the host cells can be 10 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, or 50 g / L; and the concentration of D-glucose is 50 g / L to 500 g / L, for example, the concentration of D-glucose can be 50 g / L, 100 g / L, 200 g / L, 300 g / L, 400 g / L, or 500 g / L.

[0073] Example

[0074] The gene encoding the aldose-2-epimerase mutant and aldose-2-epimerase can be called the UxaE gene. The UxaE gene contains the nucleotide sequence corresponding to the amino acid sequence of the aforementioned aldose-2-epimerase mutant or aldose-2-epimerase mutant. For example, in Examples 1 to 5, the gene contains the nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 5. The UxaE gene was constructed in the pET-28a plasmid to obtain a recombinant vector. In Comparative Examples 1 to 14, the gene contains the nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO: 6 to SEQ ID NO: 19. The UxaE gene was constructed in the pET-28a plasmid to obtain a recombinant vector.

[0075] The recombinant vector is a pET-28a plasmid containing the UxaE gene, hereinafter referred to as pET-28a-UxaE. The host cells used in all embodiments and comparative examples in this application are Escherichia coli. Specifically, the structure and sequence of the pET-28a plasmid can be found in CN202410706921.3. The nucleotide sequences corresponding to the amino acid sequences shown in Examples 1 to 5 and Comparative Examples 1 to 14 of this application are used as the target expression genes to construct the vector.

[0076] Expression of hexanoose-2-epimerase mutant

[0077] a. Transform pET-28a-UxaE into Escherichia coli (e.g., competent E. coli BL21(DE3) cells); pick single clones of pET-28a-UxaE or strains stored at -80℃ and inoculate them into 5 mL of LB liquid medium (Kan + In a small test tube (100 μg / mL), the culture was carried out overnight at 37°C and 220 rpm to obtain the seed culture.

[0078] b. Transfer the seed culture to 50 mL LB liquid medium (Kan + Activated again by incubation in a shaker at 37°C and 220 rpm (100 μg / mL).

[0079] c. Transfer the reactivated bacterial culture to 800 mL of 2YT liquid culture medium (Kan) at an inoculation rate of 1%. + In 100 μg / mL, cultured on a shaker at 37℃ and 220 rpm until the OD 600 is approximately 0.6-0.8.

[0080] d. Lower the temperature of the shaker to 16℃-18℃. After the temperature of the cultured bacterial solution has decreased, add isopropylthio-β-D-galactoside (IPTG) to a final concentration of 0.5mM and induce expression for 14-16h. The collected bacterial cells are the whole-cell catalyst.

[0081] Purification of hexanoose-2-epimerase mutant protein

[0082] a. Preparation of crude enzyme solution: The collected bacterial cells were resuspended in buffer and then subjected to high pressure low temperature disruption 3-4 times at 1200 bar and 4°C to fully lyse the cells, thereby releasing the expressed target protein and dissolving it in protein buffer.

[0083] b. Centrifugation: Centrifuge the broken bacterial culture at 8000 rpm for 50 min in a pre-cooled centrifuge at 4℃. Take the precipitate and supernatant after centrifugation, prepare the sample, and collect the supernatant.

[0084] c. Purification: The supernatant was filtered through a 0.45 μm microporous membrane and purified by nickel affinity chromatography. The specific steps are as follows:

[0085] (1) Column equilibration: First wash with dd H2O for 2 column volumes, then equilibrate the Ni affinity chromatography column with 20mM imidazole protein buffer for 1 column volume.

[0086] (2) Sample loading: Pass the supernatant slowly through the nickel affinity chromatography column at a flow rate of 0.5 mL / min, and repeat once more to flow through.

[0087] (3) Elution of contaminating proteins: Wash with protein buffer for one column volume, then use 50 mL of protein buffer containing 50 mM imidazole to elute the contaminating proteins with strong binding. Take the first few drops to flow through the sample and prepare the sample.

[0088] (4) Elution of target protein: Elute the target protein with 20 mL of imidazole protein buffer containing 100 mM, 200 mM and 300 mM respectively. Take the first few drops of the sample to flow through, prepare the sample and detect it with 12% SDS-PAGE.

[0089] d. Concentration and buffer replacement: The collected target protein was concentrated by centrifugation (4℃, 3400 rpm) using a 50 mL Amicon ultrafiltration tube (30 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, yielding purified protein UxaE.

[0090] Electrophoretic analysis of hexanoose-2-epimerase mutant protein

[0091] a. Protein sample preparation: Add the purified protein solution and 5× loading buffer at a ratio of 1:4 (v / v), heat in boiling water for 10 min, and set aside for later use.

[0092] b. Sample loading and electrophoresis: Place the precast protein gel (Genscript, SurePAGE, 4%–20%) in the electrophoresis tank, and add the protein sample and marker to the sample wells of the protein gel using a pipette.

[0093] c. Staining and destaining: Remove the outer shell of the pre-cast gel after electrophoresis, and automatically destain and stain using a protein staining and destaining instrument for 15 minutes.

[0094] d. Gel image analysis: The stained and destained protein gels were photographed and saved using a gel imaging system.

[0095] Enzyme-catalyzed reaction system

[0096] In vitro enzyme catalytic reaction conditions:

[0097] The reaction buffers were Tris-HCl, sodium phosphate buffer, and potassium phosphate buffer.

[0098] Reaction pH: 4, 5, 6, 7, 8, 9, 10;

[0099] The final concentrations of D-glucose are 50 g / L, 100 g / L, 200 g / L, 300 g / L, 400 g / L, and 500 g / L.

[0100] Metal salt types: ferrous sulfate, ferric sulfate, calcium chloride, ammonium chloride, cobalt chloride, manganese sulfate, calcium chloride, magnesium chloride, nickel sulfate;

[0101] The final concentrations of the metal salts were 0.1 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, and 10 mM.

[0102] Amount of wet bacterial cells (whole-cell catalyst): 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L;

[0103] Reaction temperatures: 40℃, 45℃, 50℃, 55℃, 60℃;

[0104] Reaction time: 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h;

[0105] The total reaction volume was 0.5 mL, 1 mL, 5 mL, and 10 mL.

[0106] In Examples 1 to 5 and Comparative Examples 1 to 14, all parameters were the same except for the amino acid sequence. For example, the reaction buffer could be Tris-HCl, the reaction pH was 7, the reaction temperature was 50°C, the concentration of magnesium sulfate was 2 mM, the concentration of D-glucose was 200 g / L, and the concentration of wet bacterial cells (whole-cell catalyst) was 30 g / L.

[0107] D-mannose detection

[0108] Liquid chromatography detection conditions:

[0109] Mobile phase: 80% acetonitrile, 20% water;

[0110] Liquid column Shimpack GIST, NH2;

[0111] Temperature: 40℃;

[0112] Flow rate: 0.5 mL / min;

[0113] Injection volume: 10 μL;

[0114] For the standard curve of D-mannose, please refer to [link / reference needed]. Figure 2 As shown, the standard curve for D-glucose can be found in [reference needed]. Figure 3 As shown, the liquid phase diagram of the reaction in the embodiment is referred to [reference needed]. Figure 4 As shown, each epimerase (mutant) in Examples 1 to 5 and Comparative Examples 1 to 14 converts D-glucose to D-mannose and all have catalytic activity for the C-2 hydroxyl isomerization of hexanoose.

[0115] The results of Examples 1 to 5 and Comparative Examples 1 to 14 are shown in Table 1. The relative activity of the epimerase in Comparative Example 14 is 1. The relative activities of the epimerase mutants in Examples 1 to 5 and Comparative Examples 1 to 13 are shown as a basis.

[0116] Table 1. Parameters of Examples 1-5 and Comparative Examples 1-14

[0117] Mutation site 1 Mutation site 2 enzyme amino acid sequence relative enzyme activity Example 1 213: I→V / SEQ ID NO: 1 186％ Example 2 213: I→V 240: T→A SEQ ID NO: 2 241％ Example 3 213: I→V 240: T→S SEQ ID NO: 3 264％ Example 4 213: I→V 240: T→D SEQ ID NO: 4 131％ Example 5 213: I→V 240: T→M SEQ ID NO: 5 173％ Comparative Example 1 213: I→A / SEQ ID NO: 6 92％ Comparative Example 2 213: I→L / SEQ ID NO: 7 75％ Comparative Example 3 213: I→N / SEQ ID NO: 8 122％ Comparative Example 4 213: I→C / SEQ ID NO: 9 54％ Comparative Example 5 213: I→Y / SEQ ID NO: 10 30％ Comparative Example 6 213: I→T / SEQ ID NO: 11 47％ Comparative Example 7 213: I→H / SEQ ID NO: 12 118％ Comparative Example 8 213: I→K / SEQ ID NO: 13 67％ Comparative Example 9 213: I→V 240: T→C SEQ ID NO: 14 91％ Comparative Example 10 213: I→V 240: T→F SEQ ID NO: 15 107％ Comparative Example 11 213: I→V 240: T→Y SEQ ID NO: 16 84％ Comparative Example 12 213: I→V 240: T→L SEQ ID NO: 17 121％ Comparative Example 13 213: I→V 240: T→I SEQ ID NO: 18 102％ Comparative Example 14 / / SEQ ID NO: 19 100％

[0118] Optimization of catalytic reaction temperature example

[0119] The catalytic temperature was different in Examples 6 to 10, but all other parameters were the same.

[0120] Table 3 Parameters of Examples 6 to 10

[0121]

[0122]

[0123] The results are as follows Figure 5 As shown, the relative activities of the catalytic enzymes in Examples 6 to 10 are based on the relative activities of the epimerase mutant in Example 8, with Example 8 representing 100%. Good catalytic activity is observed at catalytic temperatures of 45°C to 55°C.

[0124] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0125] The above embodiments merely illustrate preferred implementations of this application, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Therefore, the scope of protection of this patent application should be determined by the appended claims.

Claims

1. A mutan aldo-2-epimerase mutant, characterized in that, The amino acid sequence of the aldohexose-2-epimerase mutant is shown in any one of SEQ ID NO: 1 to SEQ ID NO:

5.

2. A polynucleotide, comprising, The polynucleotide encodes the aldohexose-2-epimerase mutant of claim 1.

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

4. A host cell, characterized in that, The host cell comprises the recombinant vector of claim 3.

5. The host cell of claim 4, wherein The host cell is a bacterial cell, and the bacterial cell is Escherichia coli or Bacillus subtilis.

6. A whole cell catalyst characterized in that, The preparation steps of the whole cell catalyst comprise: The transformed host cell is cultured, so that the aldohexose-2-epimerase mutant accumulates in the bacterial cells of the transformed host cell, and the bacterial cells are collected to obtain the whole cell catalyst, wherein the recombinant vector contains the nucleotide sequence encoding the aldohexose-2-epimerase mutant of claim 1.

7. An immobilized cell, characterized in that, The carrier and the host cell immobilized on the carrier are included, and the host cell synthesizes the aldohexose-2-epimerase mutant of claim 1.

8. Use of the aldohexose-2-epimerase mutant of claim 1 or the polynucleotide of claim 2 or the recombinant vector of claim 3 or the host cell of claim 4 or 5 in any one of the following: A1) producing D-mannose; A2) preparing a product for producing D-mannose; A3) catalyzing the conversion of D-glucose into D-mannose.

9. A method of synthesizing D-mannose, characterized by, The steps comprise: The aldohexose-2-epimerase mutant of claim 1 or the transformed host cell containing the aldohexose-2-epimerase mutant of claim 1 is contacted with D-glucose, so that the D-glucose is converted into D-mannose.

10. The method of synthesizing D-mannose according to claim 9, wherein, The reaction temperature is 45-55℃, and the reaction pH value is 7-10.

Images