A d-allulose-3-epimerase mutant, and a preparation method and application thereof

Abstract

The application belongs to the technical field of genetic engineering and enzyme engineering, and discloses a D-psicose-3-epimerase mutant, which is obtained by performing point mutation on one site in the 79th, 115th, 141st, 193rd or 202nd amino acid of a D-psicose-3-epimerase parent; specifically, a mutation primer for point mutation is designed, a vector carrying a wild-type D-psicose-3-epimerase gene is used as a template to perform point mutation and construct a plasmid vector containing the mutant, and then the plasmid vector is transformed into a host cell to obtain the D-psicose-3-epimerase mutant; and the D-psicose-3-epimerase mutant is applied to the production of psicose. The thermal stability and catalytic activity of the D-psicose-3-epimerase mutant are significantly improved, and the D-psicose-3-epimerase mutant has good industrial application value.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering technology, and in particular relates to a D-allulose-3-epimerase mutant, its preparation method and application. Background Technology

[0002] Allulose is a relatively rare natural hexulose, a functional rare sugar with the molecular formula C6H. 12 O6 has a relative molecular mass of 180.16. Allulose is a white, odorless, and non-hygroscopic powder with a melting point of 96°C. It is highly soluble in water; at 25°C, the solubility of allulose in 100g of water is 291g. Allulose has 70% the sweetness of sucrose, but its caloric value is very low, less than 1.6 kJ / g, approximately 10% of the caloric value of sucrose.

[0003] Allulose has a sweetness and texture similar to or close to sucrose, but its calories are far lower. Therefore, experts both domestically and internationally predict that allulose will become a new generation of healthy, functional sweeteners. Besides being a sucrose substitute, allulose possesses unique physiological properties, including inhibiting blood sugar spikes, lowering blood lipids, and suppressing abdominal fat accumulation. Its potential health benefits are gradually being discovered. Allulose was approved as a GRAS (Generally Recognized As Safe) substance by the US FDA in 2011, and in 2019, the FDA removed it from the list of total sugars and added sugars, allowing it to be used as a zero-sugar sweetener. After more than a decade of product research and market validation abroad, the US food industry considers allulose to be the most promising sucrose substitute.

[0004] D-psicose 3-epimerase (DAEase or DPE) is the most active epimerase in the ketose 3-epimerase family for synthesizing D-allulose from D-fructose. Therefore, DAEase is currently the key enzyme used in the industrial production of D-allulose.

[0005] Directed evolution of proteins refers to the use of various experimental techniques to simulate natural evolutionary mechanisms (random mutation, recombination, and natural selection) in vitro. This involves inducing gene mutations through mutation and recombination with one or more parent proteins, and then selecting valuable non-natural protein molecules by screening for specific functions or properties of specific enzymes. Directed evolution of proteins has significantly accelerated the pace of modifying the original functions of proteins and developing new functions, and its applications are widespread in industrially relevant enzyme catalysis.

[0006] Because unmodified DAEase enzymes derived from wild strains have limitations in terms of thermal stability and catalytic activity, their application cost is relatively high, limiting their industrial application. Chinese patent CN113373135A obtained a mutant with enhanced fermentation enzyme activity through directed evolution, with optimal conditions of: temperature 55℃, pH 8.0, and 0.1 mMCo. 2+ The allulose conversion rate reached 29%. Chinese patent CN103849612A, using gene mutation technology, obtained a mutant with a 55℃ half-life 1.88 times that of the wild type. However, resources of highly catalytically active DAEase enzymes remain scarce, hindering the widespread adoption of D-allulose. Modifying wild-type DAEase enzymes using protein-directed evolution technology to obtain highly active DAEase enzyme mutants suitable for industrial applications will be key to the biological production of D-allulose. Summary of the Invention

[0007] The first technical problem to be solved by the present invention is to provide a D-allulose-3-epimerase mutant, which is obtained by point mutation at one of the amino acid positions 79, 115, 141, 193 or 202 of the D-allulose-3-epimerase parent.

[0008] The second technical problem to be solved by this invention is to provide a method for preparing a D-allulose-3-epimerase mutant. The method involves designing site-directed mutagenesis primers, using a vector carrying the D-allulose-3-epimerase gene as a template to perform site-directed mutagenesis and constructing a plasmid vector containing the mutant. The vector is then transformed into a host cell to express the D-allulose-3-epimerase mutant.

[0009] The third technical problem to be solved by this invention is: to provide an application of a D-allulose-3-epimerase mutant in allulose production.

[0010] To solve the above-mentioned technical problems, the specific technical solution of the present invention is as follows:

[0011] A D-allulose-3-epimerase mutant is derived from the D-allulose-3-epimerase of the strain Prolixibacteraceaebacterium, with the amino acid sequence shown in the sequence listing SEQ. The sequence of NO.1 (NCBI accession number MCK9413541.1) was obtained by making a point mutation at amino acid positions 79, 115, 141, 193, or 202. Among them, mutant D79E changed amino acid position 79 from aspartic acid (Asp) to glutamic acid (Glu), mutant G115V changed amino acid position 115 from glutamic acid (Gla) to valine (Val), mutant T141N changed amino acid position 141 from threonine (Thr) to asparagine (Asn), mutant E193D changed amino acid position 193 from glutamic acid (Glu) to aspartic acid (Asp), and mutant R202K changed amino acid position 202 from arginine (Arg) to lysine (Lys).

[0012] A method for preparing a D-allulose-3-epimerase mutant includes the following steps:

[0013] a. Based on the parental amino acid sequence of D-allulose-3-epimerase (NCBI accession number MCK9413541.1), determine amino acids 79, 115, 141, 193, or 202 as mutation sites, and design site-directed mutagenesis primers:

[0014] The site-directed mutagenesis primers for introducing the D79E mutation are:

[0015] Forward primer: 5'-ATGATATCGCCTCCAAT GAA GAAAGAATCAG-3' (underlined bases are mutant bases)

[0016] Reverse primer: 5'-TTTTGCCTGATTCTTTC TTC ATTGGAGGCGA-3' (underlined bases are mutant bases);

[0017] The site-directed mutagenesis primers for introducing the G115V mutation are:

[0018] Forward primer: 5'-TCTACAGTTCCTGGCCC GTG ACCTTGCCAGC-3' (underlined bases are mutant bases)

[0019] Reverse primer: 5'-TCCCCGGCTGGCAAGGT CAC GGGCCAGGAAC-3' (underlined bases are mutant bases);

[0020] The site-directed mutagenesis primers for introducing the T141N mutation are:

[0021] Forward primer: 5'-TGAAAGAGGTGATGAAA AAT GTTGAAGATTG-3' (underlined bases are mutant bases)

[0022] Reverse primer: 5'-ACATCGCAATCTTCAAC ATT TTTCATCACCT-3' (underlined bases are mutant bases);

[0023] The site-directed mutagenesis primers for introducing the E193D mutation are:

[0024] Forward primer: 5'-TTCACATGAACATCGAA GAT GATAATATTTA-3' (underlined bases are mutant bases)

[0025] Reverse primer: 5'-GCATTGTAAATATTATC ATC TTCGATGTTCA-3' (underlined bases are mutant bases);

[0026] The site-directed mutagenesis primers for introducing the R202K mutation are:

[0027] Forward primer: 5'-TTTACAATGCCATTGTC AAG GCCGGGGATAA-3' (underlined bases are mutant bases)

[0028] Reverse primer: 5'-CCAAGTTTATCCCCGGC CTT GACAATGGCAT-3' (underlined bases are mutant bases);

[0029] b. Using the parental amino acid sequence of D-allulose-3-epimerase as a template, PCR amplification was performed using the primers from step a, and the PCR products were digested with DpnI and then transferred into plasmid vectors.

[0030] c. The plasmid vectors obtained in step b were transformed into host cells to obtain D-allulose-3-epimerase mutants, namely mutant D79E, mutant G115V, mutant T141N, mutant E193D and mutant R202K.

[0031] Preferably, the plasmid vector in step b is any one of the PUC series, PET series, or PGEX series.

[0032] Furthermore, the plasmid vector in step b is a PET series plasmid vector.

[0033] Preferably, the host cell in step c is a bacterial cell or a fungal cell.

[0034] Furthermore, in step c, the host cell is a Gram-positive bacterial cell or a Gram-negative bacterial cell.

[0035] Furthermore, in step c, the host cell is BL21(DE3).

[0036] Application of a D-allulose-3-epimerase mutant: Application of the D-allulose-3-epimerase mutant in allulose production.

[0037] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0038] The thermostability and catalytic activity of the D-allulose-3-epimerase mutant obtained in this invention are significantly improved. Compared with the parent enzyme, the specific enzyme activity of mutant E193D increased from 312.5 U / mg to 532.6 U / mg, the optimal reaction temperature increased from 55℃ to 62.5℃, the half-life was extended from 87.46 min to 182.37 min, and the allulose conversion rate increased from 28.8% to 33.6%. Furthermore, the mutant of this invention can be converted under weakly acidic pH conditions, which can reduce the Maillard reaction of sugars at high temperatures and reduce the formation of byproducts, thus possessing higher industrial application value. Attached Figure Description

[0039] Figure 1 This is a graph showing the relationship between the relative enzyme activity and time of the wild-type and mutant D-allulose-3-epimerase in Example 5 of the present invention. Detailed Implementation

[0040] The present invention will be further illustrated below with reference to the embodiments.

[0041] In specific embodiments of the present invention, the obtained mutants are defined as follows:

[0042] The naming principle for mutants is to use "original amino acid residue + residue position + substituted amino acid residue" in the amino acid sequence to represent the mutant obtained by the substitution (mutation). For example, "M15G" represents a mutant obtained by replacing the 15th amino acid residue in the parent amino acid sequence with glycine (Gly).

[0043] The high-performance liquid chromatography (HPLC) conditions for allulose detection were as follows: differential refractive index detector, NH2 column (APS-2HYPERSIL, Thermo Scientific), mobile phase (water:acetonitrile = 1:4), flow rate: 0.8 mL / min, column temperature: 40℃.

[0044] Example 1: Expression of wild-type D-allulose-3-epimerase

[0045] The pET24a(+) plasmid and the plasmid DPE / pMD18T containing the wild-type D-allulose-3-epimerase gene (NCBI accession number MCK9413541.1, which was constructed earlier or can be purchased directly) were digested with NdeI and EcoRI, respectively. After the digestion products were excised and recovered from the gel, they were ligated with T4 ligase. The ligation products were transformed into E. coli JM109 competent cells and cultured at 37°C for 8 hours. Transformants were picked and cultured in LB medium containing 30 mg / L kanamycin solution with shaking. The plasmid was extracted and verified by enzyme digestion to obtain the expression plasmid DPE / pET24a(+).

[0046] The plasmid DPE / pET24a(+) was transformed into E. coli BL21(DE3) host bacteria, plated on LB agar plates containing kanamycin (30 mg / L), and incubated at 37°C for 8 h, named DPE / pET24a(+) / E. coli BL21(DE3). Single colonies were picked and incubated overnight at 37°C in liquid LB medium containing 30 mg / L kanamycin, and the glycerol tubes were stored.

[0047] DPE / pET24a / BL21(DE3) was inoculated from preserved glycerol tubes into LB liquid medium (containing 100 mg / L kanamycin) and grown for 8 h. Seed culture was then inoculated into TB liquid fermentation medium (containing 100 mg / L kanamycin) at a 5% inoculation rate. After culturing *E. coli* at 37°C for 2 h, 0.01 mmol / L IPTG (isopropyl thio-β-D-galactopyranoside) was added for induction, and fermentation continued at 25°C in a shaker for 24 h. A certain volume of fermentation broth was centrifuged at 4°C and 12000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were collected. The bacterial cell pellet was resuspended in 20 mmol / L HEPES buffer and mixed thoroughly. The cell walls of the bacterial cells were disrupted using an ultrasonic cell disruptor (operating conditions: ψ6 probe, 10 min working time, 2 s working, 3 s rest, 20% power). Crude enzyme solution was obtained after disruption.

[0048] Example 2: Preparation and expression of D-allulose-3-epimerase mutant

[0049] (1) Expression of mutants

[0050] Based on the gene sequence of D-allulose-3-epimerase, primers for introducing D79E, G115V, T141N, E193D, and R202K mutations were designed and synthesized to perform site-directed mutagenesis on the DPE gene. Sequencing was then performed to confirm whether the coding gene of the D-allulose-3-epimerase mutant was correct. The vector carrying the mutant gene was introduced into E. coli for expression to obtain a single-mutant D-allulose-3-epimerase.

[0051] PCR amplification of the site-directed mutant encoding gene: Rapid PCR technology was used with the expression vector DPE / pET-24a(+) carrying the wild-type D-allulose-3-epimerase gene as a template.

[0052] The site-directed mutagenesis primers for introducing the D79E mutation are:

[0053] Forward primer: 5'-ATGATATCGCCTCCAAT GAA GAAAGAATCAG-3' (underlined bases are mutant bases) reverse primer: 5'-TTTTGCCTGATTCTTTC TTC The site-directed mutagenesis primer for introducing the G115V mutation into ATTGGAGGCGA-3' (underlined bases) is:

[0054] Forward primer: 5'-TCTACAGTTCCTGGCCC GTG ACCTTGCCAGC-3' (underlined bases are mutant bases) reverse primer: 5'-TCCCCGGCTGGCAAGGT CAC The site-directed mutagenesis primer for introducing the T141N mutation into GGGCCAGGAAC-3' (underlined bases are mutant bases) is:

[0055] Forward primer: 5'-TGAAAGAGGTGATGAAA AAT GTTGAAGATTG-3' (underlined bases are mutant bases) reverse primer: 5'-ACATCGCAATCTTCAAC ATT The site-directed mutagenesis primer for introducing the E193D mutation into TTTCATCACCT-3' (underlined bases) is:

[0056] Forward primer: 5'-TTCACATGAACATCGAA GAT GATAATATTTA-3' (underlined bases are mutant bases) reverse primer: 5'-GCATTGTAAATATTATC ATC The site-directed mutagenesis primer for introducing the R202K mutation into TTCGATGTTCA-3' (underlined bases) is:

[0057] Forward primer: 5'-TTTACAATGCCATTGTC AAG GCCGGGGATAA-3' (underlined bases) reverse primer: 5'-CCAAGTTTATCCCCGGC CTT GACAATGGCAT-3' (underlined bases are mutant bases)

[0058] The PCR reaction system consisted of: 10 μL of 5×PS buffer, 4 μL of dNTPs Mix (2.5 mM), 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), 1 μL of template DNA, 0.5 μL of PrimerStar HS (5 U / μL), and double-distilled water to a final volume of 50 μL.

[0059] PCR amplification conditions were as follows: pre-denaturation at 94℃ for 4 min; followed by 30 cycles (98℃ for 10 s, 60℃ for 5 s, 72℃ for 8.5 min); extension at 72℃ for 10 min; and incubation at 4℃. PCR products were detected by 1% agarose gel electrophoresis.

[0060] Strain construction: PCR products were digested with DpnI and transformed into competent Escherichia coli JM109 cells. After being cultured overnight in LB solid medium (containing 30 μg / mL kanamycin), clones were selected and cultured in LB liquid medium (containing 30 μg / mL kanamycin), and plasmids were extracted. All mutant plasmids were correctly sequenced. The mutant plasmids were transformed into competent Escherichia coli BL21(DE3) cells to finally obtain recombinant strains that could express mutants D79E, G115V, T141N, E193D, and R202K.

[0061] (2) Expression of mutants

[0062] The recombinant strains that could express mutants D79E, G115V, T141N, E193D, and R202K obtained in (1) were inoculated into LB liquid medium (containing 100 mg / L kanamycin) and grown for 8 h. The seed culture was then inoculated into TB liquid fermentation medium (containing 100 mg / L kanamycin) at a 5% inoculation rate. After E. coli was cultured at 37 °C for 2 h, 0.01 mmol / L IPTG (isopropyl thio-β-D-galactopyranoside) was added for induction, and fermentation was continued at 25 °C in a shaker for 24 h. A certain volume of fermentation broth was centrifuged at 4 °C and 12000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were collected. The bacterial cell pellet was resuspended in 20 mmol / L HEPES buffer and mixed. The cell walls of the bacterial cells were broken using an ultrasonic cell disruptor (operating conditions of the ultrasonic cell disruptor: ψ6 working probe, working time 10 min, working for 2 s and stopping for 3 s, working power of 20%), and crude enzyme solution was obtained after disruption.

[0063] Example 3: Isolation and purification of D-allulose-3-epimerase

[0064] The DPEase obtained in Example 2 was processed through Ni 2+ Purification was performed using a chromatography column (the plasmid containing wild-type D-allulose-3-epimerase was also expressed using the method in Example 2, and then isolated and purified using this method). The specific steps are as follows:

[0065] After washing and resuspending the bacterial cells in 50 mM PBS buffer (pH 7.4), the autoclave was sterilized three times with 20% alcohol, followed by three washes with deionized water. The cells were then homogenized using an autoclave at a pressure between 1000 and 1100 bar. The cell lysis buffer was centrifuged at 4°C and 5000 rpm for 15 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane.

[0066] Pre-wash Ni with at least 10 column volumes of buffer 1 2+ The chromatography column was prepared, and the filtered crude enzyme solution was loaded onto the column three times. Impurities were eluted with 40 mL of 10 mM imidazole solution (0.68 g imidazole in 1000 mL buffer 1), 20 mL of 50 mM imidazole solution (1.36 g imidazole in 1000 mL buffer 1), and 10 mL of 100 mM imidazole solution (6.81 g imidazole in 1000 mL buffer 1). The target protein was then eluted with 4 mL of 200 mM imidazole solution (13.61 g imidazole in 1000 mL buffer 1). Finally, residual protein was thoroughly eluted with buffer 2 (500 mM imidazole solution, 34.04 g imidazole in 1000 mL buffer 1). The purified protein sample was dialyzed twice at 4°C with 20 mM HEPES buffer, pH 7.0, for 6-8 hours each time.

[0067] Example 4: Determination of specific enzyme activities of wild-type and mutant D-allulose-3-epimerase

[0068] Determination of DPEase enzyme activity: A 10% (w / v) fructose solution prepared with 20 mM MES Buffer pH 6.0 was used as the substrate, and 0.01 mM Mn was added. 2+ Take 10 mL and add it to a test tube. Preheat at 55℃ for 10 min. Add the purified enzyme with a final concentration of 0.2 μmol / L. React at 55℃ for 10 min. Quickly remove the test tube and heat it in a boiling water bath for 15 min to inactivate the enzyme and terminate the reaction. Detect the content of each component in the reaction solution by HPLC.

[0069] Enzyme activity unit definition: The amount of enzyme required to generate 1 μmol of trehalose in 1 min under reaction conditions of pH 6.0 and 55℃ is defined as 1 enzyme activity unit.

[0070] The wild-type and mutant D-allulose-3-epimerase were determined using the methods described above. The results are shown in Table 1.

[0071] Table 1. Specific enzyme activities of wild-type and mutant DPEase enzymes

[0072] strain name Enzyme activity U / mg fold relative to wild type wild type 312.5 1 D79E 293.7 0.94 G115V 357.2 1.14 T141N 427.9 1.37 E193D 532.6 1.70 R202K 447.8 1.43

[0073] As can be seen from Table 1, most of the mutants in this invention have significantly increased specific enzyme activity compared with wild-type enzymes, with mutant E193D showing a 1.7-fold increase in specific enzyme activity.

[0074] Example 5: Stability comparison between wild-type and mutant D-allulose-3-epimerase

[0075] (1) Effect of pH on DPEase activity

[0076] Using a 10% (w / v) fructose solution prepared with 20 mM MES Buffer (pH 5.0–7.0) or Tris-HCl Buffer (pH 7.5–9.0) as the substrate, 0.01 mM Mn was added. 2+ Take 10 mL of the solution and add it to a test tube. Preheat the tube for 10 min at different pH values ​​(5.0-9.0). Then add purified wild-type and mutant D-allulose-3-epimerase at a final concentration of 0.2 μmol / L. Mix thoroughly and react for 10 min. Immediately remove the tube and incubate in a boiling water bath for 15 min to inactivate the enzyme and terminate the reaction. Determine the optimal reaction pH and set the group with the highest enzyme activity as 100% relative enzyme activity. The optimal pH values ​​for wild-type and mutant DPEase are shown in Table 2.

[0077] Table 2. Optimal pH values ​​for wild-type and mutant DPEase enzymes

[0078]

[0079]

[0080] As shown in Table 2, the optimal reaction pH for both the wild type and the mutant is 6.0, with no significant difference.

[0081] (2) Effect of reaction temperature on DPEase enzyme activity

[0082] Using a 10% (w / v) fructose solution prepared with 20 mM MES Buffer pH 6.0 as the substrate, 0.01 mM MMN was added.2+ Take 10 mL of the solution and add it to a test tube. Preheat the tube at different temperatures (40-75℃) for 10 min each. Then add purified wild-type and mutant D-allulose-3-epimerase at a final concentration of 0.2 μmol / L, respectively. Mix thoroughly and react for 10 min. Quickly remove the tube and incubate in a boiling water bath for 15 min to inactivate the enzyme and terminate the reaction. Detect the D-allulose content in the reaction solution by HPLC. The optimal reaction temperature data for wild-type and mutant DPEase are shown in Table 3.

[0083] Table 3 Optimal reaction temperatures for wild-type and mutant DPEase enzymes

[0084] strain name Optimal reaction temperature (°C) wild type 55.0 D79E 57.5 G115V 55 T141N 60.0 E193D 62.5 R202K 60.0

[0085] As can be seen from Table 3, compared with the wild-type enzyme, the optimal reaction temperature of most mutants in this invention is increased to a certain extent, with mutant E193D increasing from 55℃ to 62.5℃.

[0086] (3) Study on the thermostability of wild-type and mutant DPEase enzymes

[0087] Wild-type and mutant DPEase enzyme solutions (D79E, G115V, T141N, E193D, and R202K) were incubated at 60°C. Samples were taken periodically, and enzyme activity was immediately measured at 55°C using the method described above. The enzyme activity measured in the untreated enzyme solution was recorded as 100% relative enzyme activity. A curve showing the relationship between residual enzyme activity and time was plotted (see...). Figure 1 The half-life of DPEase wild-type and its mutants at this temperature was obtained from the curve, and the specific data are shown in Table 4.

[0088] Table 4 Half-life of wild-type and mutant DPEase enzymes

[0089] Mutant name Half-life (min) Wild enzymes 87.46 D79E 102.34 G115V 118.46 T141N 151.76 E193D 182.37 R202K 146.85

[0090] As shown in Table 4, the half-life of the wild-type enzyme at 60℃ is 87.46 min, while the half-life of the mutant enzymes is prolonged to some extent. Among them, E193D has the longest half-life, reaching 182.37 min, indicating that some mutants obtained in this invention have high potential for industrial application.

[0091] Example 6: Application of wild-type and mutant D-allulose-3-epimerase in allulose production

[0092] Using a 50% (w / v) fructose solution prepared with 20 mM MES Buffer pH 6.0 as the substrate, 0.01 mM MMN was added. 2+Take 200 mL of the solution and add it to a stoppered Erlenmeyer flask. Add 400 U of DPEase enzyme produced by the purified wild-type and mutant strains D79E, G115V, T141N, E193D, and R202K, respectively. Place the flask in a water bath and shake at 60℃ for 10 h. After the reaction, take a sample and boil for 10 min to inactivate the enzyme. HPLC was used to determine the allulose content. The data obtained are shown in Table 5.

[0093] Table 5. Allulose conversion rate of wild-type enzymes and mutant enzymes.

[0094] strain name Allulose conversion rate (%) wild type 28.8 D79E 30.1 G115V 30.9 T141N 31.8 E193D 33.6 R202K 31.4

[0095] As shown in Table 5, compared with the wild-type enzyme, the allulose conversion rate of the mutant D-allulose-3-epimerase was improved to varying degrees, with the mutant E193D showing the highest allulose conversion rate at 33.6%.

[0096] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A D-allulose-3-epimerase mutant, characterized in that, is obtained by point mutation of the sequence of SEQ NO. 1 with NCBI accession number MCK9413541.1 at the 202th amino acid; wherein the mutant R202K is that the 202th amino acid is changed from arginine to lysine.

2. The method for preparing the D-allulose-3-epimerase mutant as described in claim 1, characterized in that: The method comprises the following steps: a. determining the 202th amino acid as a mutation site based on the sequence of SEQ NO. 1 D-allulose-3-epimerase parent amino acid sequence, and designing a mutation primer for site-directed mutagenesis: Forward primer: 5'-TTTACAATGCCATTGTC AAG GCCGGGGATAA-3' Reverse primer: 5'-CCAAGTTTATCCCCGGC CTT GACAATGGCAT-3' b. taking the D-allulose-3-epimerase parent amino acid sequence as a template, performing PCR amplification with the primer of step a respectively, and transforming the PCR product after DpnI digestion into a pET24a(+) plasmid vector; c. transforming the plasmid vector obtained in step b into BL21(DE3) host cells respectively to obtain D-allulose-3-epimerase mutant R202K.

3. The application of the D-allulose-3-epimerase mutant as described in claim 1, characterized in that: The application of the D-allulose-3-epimerase mutant in allulose production.

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