D-psicose-3-epimerase mutant and application thereof

Abstract

The invention relates to a D-psicose-3-epimerase mutant, a coding gene of the D-psicose-3-epimerase mutant, a recombinant plasmid containing the coding gene, a genetically engineered bacterium containing the recombinant plasmid and application of the D-psicose-3-epimerase mutant, and compared with an existing D-psicose-3-epimerase mutant, the D-psicose-3-epimerase mutant has the advantage that the catalytic activity and the conversion rate are remarkably improved.

Description

Technical Field

[0001] This application belongs to the technical field of genetic engineering, and particularly relates to a D-allulose-3-epimerase mutant, its encoding gene, a recombinant plasmid containing the encoding gene, a genetically engineered bacterium containing the recombinant plasmid, and their applications. Background Art

[0002] Rare sugars are a class of monosaccharides and their derivatives that exist in nature but have extremely low contents. There are many types of rare sugars, among which representative ones include D-allulose, D-tagatose, D-glucose, etc. Most rare sugars can be used as low-calorie sweeteners and also have unique physiological functions, showing broad application prospects in the fields of medicine, diet, health care, etc. D-allulose, also known as D-ribo-2-hexulose, belongs to a kind of rare sugar and its sweetness is 70% of that of sucrose. A large number of studies have confirmed the important significance of D-allulose to human health. For example, it can control obesity and diabetes, lower blood sugar, and reduce blood lipids.

[0003] There are two types of preparation methods for D-allulose: chemical synthesis method and enzyme-catalyzed synthesis method. D-allulose can be chemically synthesized from D-fructose under the catalysis of molybdate ions, or ethanol and triethylamine can be heated together to synthesize it, but chemical synthesis has many deficiencies. The process is often complex with by-products and has certain dangers. The enzyme-catalyzed synthesis method is the most popular synthesis method at present; D-allulose can be synthesized in one step from D-fructose, a cheap monosaccharide, through D-allulose-3-epimerase. The enzyme-catalyzed synthesis method also has many advantages, such as excellent substrate selectivity, mild reaction conditions, low cost, and the product can be purified more conveniently from easily available substrates.

[0004] However, at present, the activity of natural D-allulose-3-epimerase is relatively low. At the same time, the isomerization reversible reaction of D-fructose to D-allulose catalyzed by biocatalysts has an insurmountable thermodynamic bottleneck, resulting in a relatively low conversion rate of fructose and certain limitations in industrial applications. Summary of the Invention

[0005] The purpose of this application is to provide a D-allulose-3-epimerase mutant, its encoding gene, a recombinant plasmid containing the encoding gene, a genetically engineered bacterium containing the recombinant plasmid, and their applications, aiming to solve the technical problems of low catalytic activity and conversion rate of the existing D-allulose-3-epimerase mutant.

[0006] To achieve the above purpose, the technical solution of this application is:

[0007] The first aspect of the present application provides a D-psicose-3-epimerase mutant. This D-psicose-3-epimerase mutant uses the amino acid sequence of the original D-psicose-3-epimerase shown in SEQ ID NO.1 as a vector, and performs the following combined mutations on the amino acid sequence:

[0008] i: mutate histidine at position 38 to leucine;

[0009] ii: mutate alanine at position 64 to tyrosine;

[0010] iii: mutate isoleucine at position 66 to alanine;

[0011] iv: mutate aspartic acid at position 256 to glutamic acid.

[0012] In an optional implementation manner of the first aspect, the D-psicose-3-epimerase mutant has the amino acid sequence shown in SEQ ID NO.2.

[0013] In an optional implementation manner of the first aspect, the nucleotide sequence of the original enzyme of the D-psicose-3-epimerase is shown in SEQ ID NO.3.

[0014] The second aspect of the present application provides a coding gene for the D-psicose-3-epimerase mutant described in the first aspect. This coding gene is shown in SEQ ID NO.4.

[0015] The third aspect of the present application provides a recombinant plasmid containing the coding gene for the D-psicose-3-epimerase mutant described in the second aspect.

[0016] In an optional implementation manner of the third aspect, the expression vector of the recombinant plasmid is pETDuet-1.

[0017] The fourth aspect of the present application provides a genetically engineered bacterium containing the coding gene for the D-psicose-3-epimerase mutant described in the second aspect.

[0018] In an optional implementation manner of the fourth aspect, the expression host of the genetically engineered bacterium is E.coli BL21(DE3).

[0019] The fifth aspect of the present application provides the use of the genetically engineered bacterium described in the fourth aspect in catalyzing the synthesis of D-psicose from fructose.

[0020] Compared with the prior art, the advantages or beneficial effects of the present application at least include:

[0021] The D-psicose-3-epimerase mutant provided in the first aspect of the present application is obtained by performing the above-mentioned combinatorial mutations on the amino acid sequence of the original D-psicose-3-epimerase shown in SEQ ID NO.1, resulting in directional structural and functional changes in the amino acids and their related nucleotide sequences, significantly improving the catalytic activity and substrate conversion rate of the D-psicose-3-epimerase mutant, which is conducive to industrial production. Among them, the test results of the examples show that the specific enzyme activity of the D-psicose-3-epimerase mutant reaches 218 U / ml, and when the substrate concentration is 400 g / L, its substrate conversion rate reaches 37.5%. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for the description of the embodiments will be briefly introduced below.

[0023] Figure 1 It is the high performance liquid chromatography (HPLC) diagram of the substrate fructose and D-psicose provided in the embodiments of the present application;

[0024] Figure 2 It is the standard curve diagram of D-psicose provided in the embodiments of the present application.

[0025] Figure 3 It is the protein expression diagram of the original enzyme and mutant enzyme of D-psicose-3-epimerase (M: protein marker, 1 is the original enzyme without induction, 2 is the original enzyme with induction, 3 is

[0026] the mutant His38LeuAla64Tyr Ile66AlaAsp256Glu without induction, 4 is

[0027] the mutant His38LeuAla64Tyr Ile66AlaAsp256Glu with induction) DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present application without creative efforts shall fall within the scope of protection of the present application.

[0029] In the following description of the embodiments, the term "and / or" is used to describe the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent: the case of A alone, the case of B alone, and the case of both A and B existing simultaneously. Where A and B may be singular or plural. The character " / " generally represents an "or" relationship between the associated objects before and after.

[0030] In the following description of this embodiment, the term "at least one" means one or more, and "a plurality" means two or more. "At least one of the following" or its similar expressions refer to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can both represent: a, b, c, a - b (i.e., a and b), a - c, b - c, or a - b - c, where a, b, and c can be single or multiple respectively.

[0031] Those skilled in the art should understand that in the following description of the embodiments of this application, the sequence numbers do not imply the order of execution. Some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, without constituting any limitation to the implementation process of the embodiments of this application.

[0032] The terms used in the embodiments of this application are only for the purpose of describing specific embodiments, and are not intended to limit this application. The singular forms "a" and "the" used in the embodiments of this application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0033] In a first aspect, an embodiment of this application provides a D - allulose - 3 - epimerase mutant. This D - allulose - 3 - epimerase mutant uses the amino acid sequence of the original D - allulose - 3 - epimerase shown in SEQ ID NO.1 as a vector, and performs the following combined mutations on the amino acid sequence:

[0034] i: Mutate the histidine at position 38 to leucine;

[0035] ii: Mutate the alanine at position 64 to tyrosine;

[0036] iii: Mutate the isoleucine at position 66 to alanine;

[0037] iv: Mutate the aspartic acid at position 256 to glutamic acid.

[0038] It should be noted that the amino acid names in the sequence shown in SEQ ID NO.1 match the single - letter abbreviations of the amino acids in Table 1 below.

[0039] Table 1 - Names and Abbreviation Expressions of Amino Acids

[0040]

[0041] Therefore, in the embodiments of the present application, based on the amino acid sequence of the original D-psicose-3-epimerase shown in SEQ ID NO.1, after performing the above-mentioned combined mutations on specific sites in the amino acid sequence, the amino acid sequence of the D-psicose-3-epimerase quadruple mutant obtained is shown in SEQ ID NO.2. Among them, the amino acid sequence of the mutant in the embodiments of the present application is represented by replacing the corresponding amino acid sites in the amino acid sequence of the original D-psicose-3-epimerase with mutant amino acids, and the numbering of the mutation sites corresponds to the sites of the amino acid sequence of the original D-psicose-3-epimerase.

[0042] Meanwhile, the embodiments of the present application provide a nucleotide sequence based on the amino acid sequence of the original D-psicose-3-epimerase described above. The nucleotide sequence of the original D-psicose-3-epimerase is shown in SEQ ID NO.3. Based on the specific nucleotide sequences of each amino acid, after performing the above-mentioned combined mutations, the nucleotide sequence of the D-psicose-3-epimerase mutant obtained is shown in SEQ ID NO.4. Among them, the nucleotide sequence of the mutant in the embodiments of the present application is represented by replacing the corresponding nucleotide sites in the nucleotide sequence of the original D-psicose-3-epimerase with the nucleotides of the mutant amino acids, and the numbering of the mutation sites corresponds to the sites of the nucleotide sequence of the original D-psicose-3-epimerase.

[0043] Those skilled in the art should understand that the above-mentioned methods for modifying combined mutations are all implemented according to the relevant genetic engineering techniques known in the art. The embodiments of the present application do not make special limitations on specific gene mutation techniques, as long as those skilled in the art can obtain the target mutant through the above-mentioned combined mutations.

[0044] Among them, in the embodiments of the present application, by performing the above-mentioned combined mutations on the amino acid sequence of the original D-psicose-3-epimerase shown in SEQ ID NO.1, directional structural and functional changes are produced in the amino acids and their related nucleotide sequences, thereby greatly improving the catalytic activity and substrate conversion rate of the D-psicose-3-epimerase mutant, which is helpful for industrial production.

[0045] In the second aspect, the embodiments of the present application also provide the coding gene of the D-psicose-3-epimerase quadruple mutant described in the first aspect. This coding gene is shown in SEQ ID NO.4.

[0046] Among them, in the embodiment of the present application, the D-allulose-3-epimerase mutant described in the first aspect is encoded to form a coding gene carrying the D-allulose-3-epimerase mutant. Specifically, the coding gene carrying the mutant enzyme is synthesized by a total synthesis method based on genetic engineering technology. Among them, the coding gene of the D-allulose-3-epimerase quadruple mutant is shown as SEQ ID NO.4; at the same time, for convenience of description, the embodiment of the present application represents the coding gene of the D-allulose-3-epimerase mutant as DPE10.

[0047] In the third aspect, the embodiment of the present application also provides a recombinant plasmid containing the coding gene of the D-allulose-3-epimerase mutant described in the second aspect.

[0048] Among them, the embodiment of the present application provides a preparation method of the above-mentioned recombinant plasmid, preferably including:

[0049] After double digestion of the coding gene of the D-allulose-3-epimerase mutant and the expression vector pETDuet of Escherichia coli with restriction endonucleases BamHI and KpnI for 3-6 hours respectively, the digested products are recovered and ligated with T4 DNA ligase at a temperature of 16 °C for 16 hours to obtain a recombinant plasmid containing the coding gene of the D-allulose-3-epimerase mutant; at the same time, for convenience of description, the embodiment of the present application represents the recombinant plasmid as pETDuet-DPE10.

[0050] It should be noted that the specific sources of T4 DNA ligase, restriction endonucleases BamHI and KpnI in the embodiment of the present application are not particularly limited. For example, they can be obtained by purchasing from Fermetens company, or synthesized according to the genetic engineering methods known in the relevant field.

[0051] In the fourth aspect, the embodiment of the present application also provides a genetically engineered bacterium containing the coding gene of the D-allulose-3-epimerase mutant described in the second aspect.

[0052] Among them, the embodiment of the present application preferably uses Escherichia coli as the host to express the recombinant plasmid to synthesize the genetically engineered bacterium, specifically including:

[0053] S41: Transform the recombinant plasmid pETDuet-DPE10 into the E. coli BL21(DE3) recipient bacterium, and spread it on an LB agar plate containing ampicillin resistance (mass concentration of 100 μg / mL). After that, culture it at 37 °C for 12 h to grow single colonies on the plate; randomly pick single colonies and perform monoclonal culturing, then inoculate them into an LB liquid medium and culture for 12 h, and extract the plasmid for sequencing. According to the sequencing results, screen out the genetically engineered bacterium, denoted as the positive clone E. coli BL21(DE3) / pETDuet-DPE10.

[0054] S42: Inoculate the genetically engineered bacterium into 4 mL of LB medium containing kanamycin with a mass concentration of 100 μg / mL, and initially culture it at a temperature of 37 °C and a rotation speed of 200 r / min for 12 h; inoculate the initially cultured genetically engineered bacterium into 30 mL of new LB medium containing kanamycin with a mass concentration of 100 μg / mL at an inoculation amount of 1 vt.%, and culture it at a temperature of 37 °C and a rotation speed of 200 r / min until the optical density (OD 600 ) is 0.6 - 0.8; then add isopropyl-β-D-thiogalactoside (IPTG) inducer to a final concentration of 0.1 mM, and induce and culture it at a temperature of 28 °C and a rotation speed of 200 r / min for 16 h; finally, centrifuge and collect the bacterial cells under the conditions of 5000×g for 5 min, resuspend and wash them with Tris-SO4 buffer solution with pH = 7.0, and centrifuge at 13000×g for 1 min to collect the wet bacterial cells, and store them at -20 °C for standby. Resuspend 0.5 of the wet bacterial cells in 10 ml of PBS (pH 7.5) buffer solution, resuspend the bacterial cells after resuspension with an ultrasonic disruptor under ice bath conditions, centrifuge the disrupted solution, and the supernatant is the crude enzyme solution.

[0055] It should be noted that the composition of the LB agar plate includes: tryptone 10.0 g / L, yeast extract 5.0 g / L, NaCl 10.0 g / L; the composition of the LB solid medium includes: tryptone 10.0 g / L, yeast extract 5.0 g / L, NaCl 10.0 g / L, agar 15.0 g / L. Among them, both the LB agar plate and the LB solid medium are autoclaved at 121 °C for 20 min.

[0056] In the embodiment of the present application, the encoding gene encodes the D-allulose-3-epimerase mutant described above, and the recombinant plasmid carrying the encoding gene is expressed. Research shows that the activity of synthesizing D-allulose from fructose catalyzed by this crude enzyme solution is 5 times higher than that of the original enzyme, and the substrate conversion rate reaches more than 37.5%.

[0057] In a specific embodiment, the expression host of the genetically engineered bacterium is preferably E. coli BL21(DE3).

[0058] In a fifth aspect, the embodiments of the present application further provide an application of the crude enzyme solution described in the fourth aspect in catalyzing the synthesis of D-allulose from fructose.

[0059] Example 1: Preparation of D-allulose by catalytic resolution of the substrate fructose using crude enzyme solutions of different D-allulose-3-epimerase mutants

[0060] The specific method for catalytic resolution of the substrate fructose by the crude enzyme solution in the embodiments of the present application includes:

[0061] S51: Total reaction system volume (1 mL): Add 500 μL of a fructose solution with a concentration of 800 mg / mL, 10 μL of 10 mM CoCl2, and 490 μL of the crude enzyme solution into each 2 mL centrifuge tube. Insert the centrifuge tubes into a floating plate and place them in a water bath at 55 °C for reaction. Samples are taken every 20 min during the reaction. After the reaction is completed, place each centrifuge tube in boiling water for 10 min to stop the reaction. After completion, centrifuge each sample at 12,000 rpm and 25 °C for 10 min. Dilute 10-fold with ultrapure water, take the supernatant of each sample with a syringe, pass through a 0.22 μm membrane, and inject it into a liquid phase bottle for liquid phase detection of the D-allulose content.

[0062] High performance liquid analysis of fructose and D-allulose specifically includes:

[0063] The chromatographic column is waters sugar-Pak I; the specification is 10 μm 6.5 mm × 300 mm; the column temperature is 80 °C; the detector is RID; the detection temperature is 55 °C; the sample injection volume is 10 μL; the mobile phase is water; the flow rate is 0.4 mL / min.

[0064] According to Figure 1 It can be known that the retention time of the standard sample D-fructose is about 14.8 min, and the retention time of D-allulose is about 20.5 min. Calculate the concentration of D-allulose from the peak area according to the standard curve. Then calculate the enzyme activity. The unit of enzyme activity is defined as: the amount of enzyme required to catalyze D-fructose to produce 1 μmol of D-allulose per 10 min is defined as one U. The specific enzyme activity is the enzyme activity divided by the volume of the crude enzyme.

[0065] Enzyme activity calculation method: Assume the standard curve equation is y = Ax + B, the concentration of D-allulose is x, and the peak area is y. The molar mass M of D-allulose is 180.16 g / mol, and the time h is in units of 20 min.

[0066] Concentration of D-allulose: Unit: mg / mL

[0067] Unit: U / mL

[0068] Table 1 Comparison of catalytic performance between different mutants and the original enzyme

[0069]

[0070] As can be seen from the results in Table 1, the specific enzyme activity of the above-mentioned D-psicose-3-epimerase combinatorial quadruple mutant is 218 U / ml, the yield of D-psicose catalyzed by it is 150.1 g / L, and the conversion rate of fructose is 37.5%. The specific enzyme activity of the crude enzyme solution containing the original D-psicose-3-epimerase is 43 U / ml, and its conversion rate of fructose under the same conditions is 18.2%. Therefore, the specific enzyme activity of the crude enzyme solution containing the D-psicose-3-epimerase combinatorial quadruple mutant is 5 times that of the crude enzyme solution containing the original D-psicose-3-epimerase, and the conversion rate of fructose is 2 times that of the original enzyme.

[0071] In addition, single-point mutation of the original D-psicose-3-epimerase can improve the specific enzyme activity and conversion rate to a certain extent, but the mutants with single mutations at each site are far less effective in catalytic synthesis and resolution than the combinatorial mutant.

[0072] The various embodiments in this specification are described in a progressive manner. The same or similar parts between the various embodiments can be referred to each other, and each embodiment focuses on the differences from other embodiments.

[0073] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit the present application; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the present application.

[0074] The sequences used in the above embodiments of the present application are shown in the following sequences. It should be understood that the following sequences are only exemplary sequences of the embodiments of the present application, rather than any limitation to the solutions of the present application. The nucleic acid sequences in the following sequences can be represented as DNA sequences or RNA sequences. When it is represented as an RNA sequence, "T" therein represents uridine.

[0075] Amino acid sequence of the original enzyme

[0076] SEQ ID NO.1

[0077]

[0078] Four - mutant enzyme amino acid sequence:

[0079] SEQ ID NO.2

[0080]

[0081] Original enzyme nucleotide sequence

[0082] SEQ ID NO.3

[0083]

[0084]

[0085] Four - mutant enzyme nucleotide sequence SEQ ID NO.4

[0086]

[0087] Mutant His38Leu amino acid sequence SEQ ID NO.5

[0088]

[0089] Mutant Ala64Tyr amino acid sequence SEQ ID NO.6

[0090]

[0091]

[0092] Mutant Ile66Ala amino acid sequence SEQ ID NO.7

[0093]

[0094] Mutant Asp256Glu amino acid sequence SEQ ID NO.8

[0095]

[0096] Mutant His38Leu nucleotide sequence SEQ ID NO.9

[0097]

[0098] Mutant Ala64Tyr nucleotide sequence SEQ ID NO.10

[0099]

[0100]

[0101] Mutant Ile66Ala nucleotide sequence SEQ ID NO.11

[0102]

[0103] Mutant Asp256Glu nucleotide sequence SEQ ID NO.12

[0104]

Claims

1. A mutant of D-psicose-3-epimerase, characterized in that it comprises one or more of the following mutations: i: Mutating the histidine at position 38 to leucine; ii: Mutating the alanine at position 64 to tyrosine; iii: Mutating the isoleucine at position 66 to alanine; iv: Mutating the aspartic acid at position 256 to glutamic acid.

2. The mutant according to claim 1, wherein the D-psicose-3-epimerase comprises the following mutations: i: Mutating the histidine at position 38 to leucine; ii: Mutating the alanine at position 64 to tyrosine; iii: Mutating the isoleucine at position 66 to alanine; iv: Mutating the aspartic acid at position 256 to glutamic acid.

3. The mutant of D-psicose-3-epimerase according to any one of claims 1-2, wherein the sequence of the D-psicose-3-epimerase is as shown in SEQ ID NO.

1.

4. The mutant of D-psicose-3-epimerase according to any one of claims 1-2, wherein the sequence of the mutant of D-psicose-3-epimerase includes or is SEQ ID NO.2; or a sequence having 99%, 98%, 97%, 96%, 95% or 90% identity with SEQ ID NO.

2.

5. A nucleic acid encoding the mutant of D-psicose-3-epimerase according to any one of claims 1-4, preferably, the nucleic acid is DNA or RNA.

6. The nucleic acid according to claim 5, wherein the coding gene includes or is as shown in SEQ ID NO.4; or a sequence having 99%, 98%, 97%, 96%, 95% or 90% identity with SEQ ID NO.

4.

7. Use of the mutant of D-psicose-3-epimerase according to any one of claims 1-4 or the nucleic acid according to any one of claims 5-6 in the preparation of D-psicose.

8. A recombinant plasmid or host bacterium expressing the mutant of D-psicose-3-epimerase according to any one of claims 1-4.

9. The recombinant plasmid or host bacterium according to claim 8, wherein the expression vector of the recombinant plasmid is pETDuet-1 or the host bacterium is E. coli BL21(DE3).

10. Use of the recombinant plasmid or host bacterium according to any one of claims 8-9 in the preparation of D-psicose.

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