Mutants of D-psicose 3-epimerase gene from Antarctica and their application

Abstract

The present invention relates to a mutant of an Antarctic-derived D-psicose 3-epimerase gene and its application, belonging to the field of biotechnology and enzyme engineering. The protein encoded by the D-psicose 3-epimerase gene can catalyze high-concentration D-fructose to produce D-psicose, with a yield of 145 g / L, and has broad application prospects in the food industry; adding low-concentration Co in the reaction system 2+ , which can significantly improve the activity and thermal stability of the enzyme; mutating the 270th alanine of D-psicose 3-epimerase to arginine can significantly improve the heat resistance of the mutant; adding a cellulose binding domain gene sequence to the C-terminus of the D-psicose 3-epimerase gene, the protein encoded by the gene can quickly bind to cellulose to generate an immobilized enzyme, which can be reused 20 times, greatly saving production costs.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a mutant of an Antarctic-derived D-psicose 3-epimerase gene and application thereof. Background Art

[0002] With the improvement of social productivity, sugars have become easier to obtain, but a high-sugar diet has brought about serious health problems such as obesity, diabetes, and cardiovascular disease. Sweetness is one of the indispensable senses of human beings. Using low-calorie sweeteners to replace sugars is a best-of-both-worlds solution. The sweetness of D-psicose is 70% of that of sucrose, while the calories are only one-tenth of sucrose. Its taste is close to that of sucrose, and it can replace sucrose 1:1. It is widely used in dairy products and baked goods and is an ideal sweetener. The content of D-psicose in nature is extremely low, and it is difficult to extract from natural sources, so its production mainly relies on biosynthesis. D-psicose is a diastereomer of fructose and can be produced by D-psicose 3-diastereomerase catalyzing fructose isomerization. The catalytic reaction is generally carried out in high fructose concentrations (500 g / L to 700 g / L). Limited by the substrate and product equilibrium constants, the conversion rate of the reaction is usually between 20% and 34%. Although many D-psicose 3-epimerase have been reported, the thermal stability and catalytic efficiency of existing enzymes need to be improved. Through rational design of enzymes, the biological properties of enzymes can be further improved and enzymes that are more suitable for industrial production can be obtained. Summary of the invention

[0003] The present invention provides a mutant encoding gene of D-psicose 3-epimerase, which can rapidly generate D-psicose in high-concentration fructose and has broad application prospects in D-psicose biosynthesis. 2+ , improving enzyme activity and thermal stability; the mutant can significantly improve the heat resistance of the wild-type enzyme by mutating the 270th alanine of D-psicose 3-isomerase to arginine.

[0004] The present invention is achieved through the following technical solutions:

[0005] The present invention provides a gene encoding a D-psicose 3-epimerase mutant, and the nucleotide sequence of the gene is shown in SEQ ID No: 1.

[0006] A mutant of D-psicose 3-epimerase, whose amino acid sequence is shown in SEQ ID No: 2, wherein the 270th alanine in the amino acid sequence of wild D-psicose 3-epimerase is mutated to arginine.

[0007] A recombinant expression vector comprises the nucleotide sequence shown in SEQ ID No: 1; the expression vector is pQE-a270r.

[0008] A recombinant bacterium comprises the nucleotide sequence shown in SEQ ID No: 1, and the strain is Escherichia coli JM109.

[0009] The present invention also provides use of the recombinant D-psicose 3-epimerase mutant in the biosynthesis of D-psicose.

[0010] Furthermore, 1 mM Co was added to the reaction system in the application. 2+ .

[0011] The present invention also provides a fusion enzyme encoding a D-psicose 3-epimerase mutant, wherein the nucleotide sequence of the gene of the fusion enzyme is shown in SEQ ID No: 3, and the amino acid sequence thereof is shown in SEQ ID No: 4. The fusion enzyme is characterized in that a cellulose binding domain gene is fused to the C-terminus of the gene encoding the D-psicose 3-epimerase mutant.

[0012] A recombinant expression vector comprises the nucleotide sequence shown in SEQ ID No: 3, and the recombinant expression vector is pET-a270r-cbd.

[0013] A recombinant bacterium comprises a nucleotide sequence shown in SEQ ID No: 3, and the host strain is Escherichia coli BL21 (DE3).

[0014] The present invention also provides a method for preparing an immobilized enzyme using the above-mentioned fusion enzyme, and the steps of the method are as follows: after the crude enzyme solution of the fusion enzyme and microcrystalline cellulose are oscillated and mixed at 25°C and 700 rpm for 5-30 min, the supernatant is removed by centrifugation at 12000 rpm for 1 min, and the remaining solid is washed with 50 mM pH 7.5 Tris-HCl buffer to obtain the immobilized enzyme.

[0015] The beneficial effects of the present invention compared with the prior art are as follows:

[0016] The present invention provides a gene sequence having D-psicose 3-epimerase activity. The recombinant D-psicose 3-epimerase expressed by the gene can be used to synthesize D-psicose 3-epimerase in a low concentration of Co 2+ In the presence of β-D-fructose, it has a higher optimum temperature than known enzymes and can catalyze high concentrations of D-fructose to rapidly produce D-psicose;

[0017] The present invention provides a D-psicose 3-epimerase mutant. The enzyme activity of the recombinant mutant is similar to that of a wild enzyme. After being warm-bathed at 50°C for 1 hour, the activity of the wild enzyme decreases to 40% of the original, while the activity of the recombinant mutant can retain 90%. The recombinant mutant greatly improves the thermal stability of the wild enzyme.

[0018] The present invention also provides a gene of a fusion enzyme mutant of D-psicose 3-epimerase. The fusion enzyme encoded by the gene has a cellulose binding domain and can quickly bind to microcrystalline cellulose to generate a stable immobilized enzyme, thereby achieving repeated use of the enzyme and reducing the production cost of D-psicose biosynthesis.

[0019] The present invention also provides a method for rapidly preparing an immobilized enzyme, wherein the immobilized enzyme is added to a 100 g / L D-fructose solution, reacted at 40°C for 10 min, centrifuged, washed three times with 50 mM Tris-HCl buffer (pH 7.5), and then put into reaction again. After repeating 20 times, the immobilized enzyme still maintains 26% of the initial enzyme activity. The enzyme reusability is improved, and the method has broad application prospects in the biosynthesis of D-psicose. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The electrophoresis diagram of the expression and purification of recombinant D-psicose 3-epimerase; Lane 1, protein marker; Lane 2, E. coli lysate containing pQE-dpema4; Lane 3, E. coli lysate containing pQE-dpema4 after IPTG induction; Lane 4, crude enzyme; Lane 5, pure enzyme;

[0021] Figure 2 is the optimum temperature diagram of recombinant D-psicose 3-epimerase;

[0022] Figure 3 is the optimum temperature of recombinant D-psicose 3-epimerase (containing Co 2+ Reaction system) diagram;

[0023] Figure 4 is a line graph of the thermal stability of the recombinant D-psicose 3-epimerase;

[0024] Figure 5 is a bar graph of metal ion stability of recombinant D-psicose 3-epimerase;

[0025] Figure 6 To investigate the effect of temperature and Co on the biosynthesis of D-psicose by recombinant D-psicose 3-epimerase 2+ Impact change graph;

[0026] Figure 7The electrophoresis diagram of the expression and purification of mutant A270R; Lane 1, protein marker; Lane 2, bacterial lysate of E. coli containing pQE-a270r after IPTG induction; Lane 3, bacterial lysate of E. coli containing pQE-a270r; Lane 4, purified mutant A270R;

[0027] Figure 8 This is the thermal stability change diagram of mutant A270R at 50°C;

[0028] Fig. 9 Schematic diagram of the gene connection structure of the fusion enzyme;

[0029] Fig.10 The electrophoresis diagram of fusion enzyme expression and purification; Lane 1, protein marker; Lane 2, E. coli lysate containing pET-a270r-cbd; Lane 3, E. coli lysate containing pET-a270r-cbd after IPTG induction; Lane 4, crude recombinant fusion enzyme; Lane 5, pure recombinant fusion enzyme after immobilized enzyme was washed with urea;

[0030] Fig.11 This is a graph showing the reusability of the immobilized enzyme. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further explained below by examples, but the protection scope of the present invention is not limited in any form by the examples.

[0032] Example 1 Construction of D-psicose 3-epimerase gene expression vector

[0033] Using metagenomic DNA extracted from Antarctic seawater as a template, primer 1 (5'-CATG CCATGG GAATGAACATCG-3', SEQ ID NO.7) and primer 2 (5'-GA AGATCTGTGCAGCCTGATCGAGT-3', SEQ ID NO.8) was used for PCR (the underlined parts are restriction enzyme sites NcoI and BglII, respectively) to obtain the D-psicose 3-epimerase gene. The PCR product was double-digested and ligated to the expression plasmid pQE-60. The linker was transformed into Escherichia coli DH5α, and the positive clones were screened by the antibiotic ampicillin and sequenced to obtain the recombinant expression vector pQE-dpema4 with the new D-psicose 3-epimerase gene. The D-psicose 3-epimerase gene has a total of 870 bases (SEQ ID NO.5) and encodes a total of 289 amino acids (SEQ ID NO.6). The theoretical relative molecular mass is 31kDa and the theoretical isoelectric point is 4.88. Its amino acid sequence has a 100% similarity with the gene numbered WP_042541372.1 in the NCBI database.

[0034] Example 2 Preparation method of recombinant D-psicose 3-epimerase crude enzyme solution

[0035] The recombinant plasmid described in Example 1 was transformed into Escherichia coli JM109 to obtain a genetically engineered strain of D-psicose 3-epimerase. The strain was inoculated into LB liquid culture medium containing 50 μg / mL ampicillin and cultured at 37°C overnight. 1% of the culture solution was inoculated and cultured until OD 600 The value was 1, IPTG was added to a final concentration of 0.2 mM, and the mixture was induced at 20°C for 16 hours. The cells were collected by centrifugation at 9000 rpm for 15 min at 4°C, and the cells were resuspended in Tris-HCl buffer (50 mM, pH 7.5). The cells were disrupted by ultrasound, and the disrupted liquid was centrifuged at 9000 rpm for 15 min at 4°C, and the supernatant was collected to obtain the crude enzyme solution.

[0036] Example 3 Purification method of recombinant D-psicose 3-epimerase

[0037] After the affinity chromatography column Ni-NTA Agarose was rinsed with equilibration buffer (50 mM Tris-HCl, 500 mM NaCl, pH=7.5) for 5 column volumes, the crude enzyme solution obtained in Example 2 was filtered through a 0.22 μm filter membrane and then entered the column, and then rinsed with equilibration buffer for 5 column volumes. The nonspecific bands were removed by rinsing with equilibration solution containing 50 mM imidazole for 5 column volumes. Gradient elution was performed with an equilibration solution containing 50-250 mM imidazole, and the elution peak was collected. The buffer was replaced with Tris-HCl buffer (50 mM, pH 7.5) by dialysis to obtain pure enzyme. The protein purity was detected by SDS-PAGE, and the results are shown in FIG. Figure 1As shown, the purified recombinant enzyme is a single band, and the molecular weight is consistent with the theoretical molecular weight. The protein concentration was determined by the BCA method, with BSA as the standard protein.

[0038] Example 4 Enzymatic properties of recombinant D-psicose 3-epimerase

[0039] The optimal reaction temperature of recombinant D-psicose 3-epimerase was determined using D-fructose as substrate in the temperature range of 10-90°C. The relationship between reaction temperature and relative enzyme activity is shown in Figure 2 As shown in the figure, the optimal reaction temperature of recombinant D-psicose 3-epimerase is 60℃, and it can maintain more than 50% activity in the range of 20℃ to 65℃, and still has about 40% activity at 70℃. 2+ The optimal reaction temperature of recombinant D-psicose 3-epimerase was increased to 85℃ ( Figure 3 ), it can be seen that Co 2+ It can significantly improve the thermal stability of enzymatic reactions, exceeding that of most known thermostable enzymes. The thermal stability of recombinant D-psicose 3-epimerase was achieved by subjecting the pure enzyme to 40 and 50°C for 4 hours and measuring its residual enzyme activity. Figure 4 As shown in the figure, the recombinant D-psicose 3-epimerase showed good stability at 40°C, with the activity remaining above 60%. o After 2 hours of action of C, the activity dropped below 30%.

[0040] Different metal ions (Mn 2+ Mg 2+ 、Co 2+ , Ca 2+ 、Zn 2+ 、Ni 2+ , Cu 2+ ), and the residual enzyme activity was determined. The experimental results are as follows Figure 5 As shown. 2+ The activity of recombinant D-psicose 3-epimerase increased 4-fold in the presence of Mg 2+ 、Co 2+ 、Ni 2+ It can also significantly improve enzyme activity, Cu 2+ It has a strong inhibitory effect on recombinant D-psicose 3-epimerase.

[0041] Example 5 Application of recombinant D-psicose 3-epimerase in the production of D-psicose

[0042] The reaction system was D-fructose (500 g / L), Tris-HCl buffer (50 mM, pH 7.5) and D-psicose 3-epimerase (60 nM), and the reaction was carried out in a constant temperature shaker at 40°C and 180 rpm for 6 hours. Figure 6 As shown in the figure, after 1 hour of reaction, the conversion rate of recombinant D-psicose 3-isomerase was 18% and the yield was 90 g / L; 2+ After 1 hour of reaction, the yield reached 23% and the output was 115 g / L. After 5 hours of reaction, in the presence of low concentration Co 2+ In the presence of Co, the yield of D-psicose reached 29% and the yield was 145 g / L. After 7 hours of reaction, in the absence of Co 2+ In the system, the yield of D-psicose reached 29%. 2+ The presence of 1,2-dimerase can significantly improve the activity and catalytic efficiency of D-psicose 3-isomerase.

[0043] Example 6 Construction of mutant A270R expression vector

[0044] Site-directed mutagenesis primers were designed, and the recombinant expression vector pQE-dpema4 was used as a template. Site-directed mutagenesis primer 3 (5'-CGAGCTCGGTCGCCACGCCAACG-3', SEQ ID NO.9) and primer 4 (5'-ACGTTGTCGGTCCAC-3', SEQ ID NO.10) were added to perform PCR to generate a linearized plasmid. The linearized plasmid was ligated and transformed into DH5α competent cells. Positive clones were screened by the antibiotic ampicillin and verified by sequencing to obtain the recombinant expression vector pQE-a270r carrying the D-psicose 3-epimerase mutant gene.

[0045] Example 7 Preparation of Crude Enzyme Solution of Mutant A270R

[0046] The recombinant plasmid described in Example 6 was transformed into Escherichia coli JM109 to obtain a mutant A270R genetically engineered strain. The strain was inoculated into LB liquid culture medium containing 50 μg / mL ampicillin and cultured overnight at 37°C. 1% of the culture solution was inoculated and cultured to an OD600 value of 1, IPTG was added to a final concentration of 0.2 mM, and induced at 20°C for 16 hours. The cells were collected by centrifugation at 4°C, 9000 rpm for 15 min, and the cells were resuspended in Tris-HCl buffer (50 mM, pH 7.5). The cells were ultrasonically disrupted, and the disrupted liquid was centrifuged at 4°C, 9000 rpm for 15 min, and the supernatant was collected to obtain a crude enzyme solution.

[0047] Example 8 Purification method of mutant A270R

[0048] After the affinity chromatography column Ni-NTA Agarose was rinsed with equilibration buffer (50 mM Tris-HCl, 500 mM NaCl, pH = 7.5) for 5 column volumes, the crude enzyme solution obtained in Example 2 was filtered through a 0.22 μm filter membrane and then entered the column, and then rinsed with equilibration buffer for 5 column volumes. The nonspecific bands were removed by rinsing with equilibration solution containing 50 mM imidazole for 5 column volumes. Gradient elution was performed with an equilibration solution containing 50-250 mM imidazole, and the elution peak was collected. The buffer was replaced with Tris-HCl buffer (50 mM, pH 7.5) by dialysis to obtain pure enzyme. The purity of the mutant was detected by SDS-PAGE. The experimental results are shown in Figure 2. Figure 7 shown.

[0049] Example 9 Thermal stability of mutant A270R

[0050] The mutant A270R and the wild-type enzyme were placed in a water bath at 50°C for 1 hour. The changes in enzyme activity before and after the water bath were as follows: Figure 8 As shown, the initial enzyme activity of mutant A270R is equivalent to that of the wild enzyme, indicating that the single mutation has no effect on the activity of the enzyme. After incubation at 50°C, mutant A270R can maintain 90% of the initial enzyme activity, which is about twice that of the wild enzyme, indicating that the mutant significantly improves the thermal stability of the wild enzyme.

[0051] Example 10 Construction of fusion enzyme expression vector

[0052] like Fig. 9 As shown, the fusion enzyme is composed of a D-psicose 3-epimerase mutant (A270R) gene, a flexible chain linker (amino acid sequence: RSLAAALEG), and a cellulose binding domain (CBD). The nucleotide sequence is shown in SEQ ID No: 3. It is obtained by total gene synthesis. The artificially synthesized DNA is connected to the linearized plasmid pET-24a through an In-fusion reagent and transformed into the cloning strain DH5α. The positive strains after kanamycin screening are cloned and sequenced to obtain the expression plasmid pET-a270r-cbd with the fusion enzyme gene.

[0053] Example 11 Expression of fusion enzyme

[0054] The recombinant plasmid described in Example 10 was transformed into Escherichia coli BL21 (DE3) to obtain a genetically engineered strain expressing the fusion enzyme. The strain was inoculated into LB liquid culture medium containing 50 μg / mL kanamycin and cultured overnight at 37°C. 1% of the culture medium was inoculated and cultured until OD 600value of 0.4, add IPTG to a final concentration of 0.4 mM, and induce at 20℃ for 20 hours. Centrifuge at 4℃, 9000 rpm for 15 min to collect the bacteria, and resuspend the bacteria in Tris-HCl buffer (50 mM, pH 7.5). Ultrasonic cells were broken, and the broken liquid was centrifuged at 4℃, 9000 rpm for 15 min. The supernatant was collected to obtain the crude enzyme solution of fusion enzyme. The expression of fusion enzyme was detected by SDS-PAGE. The results are shown in Fig.10 As shown, after induction, the fusion enzyme was successfully expressed.

[0055] Example 12 Preparation of immobilized enzyme

[0056] The crude fusion enzyme described in Example 11 was mixed with a certain amount of microcrystalline cellulose under the conditions of 25°C and 700 rpm for 25 minutes, or any time between 5 and 30 minutes, and then centrifuged at 12,000 rpm for 1 minute to remove the supernatant. The remaining solid was washed three times with 50 mM Tris-HCl (pH 7.5) buffer to obtain an immobilized enzyme. High concentration of urea can denature the protein and elute it from the medium. After the immobilized enzyme was eluted with 8 mol / L urea, the electrophoresis diagram ( Fig.10 ) showed a single band at the position corresponding to the fusion enzyme, indicating that the fusion enzyme could be specifically adsorbed onto microcrystalline cellulose and immobilized.

[0057] Example 13 Reusability of immobilized enzyme

[0058] The immobilized enzyme described in Example 12 was added to a 100 g / L D-fructose solution and reacted in a 40°C water bath for 10 min. The amount of D-psicose produced in the supernatant after the reaction was detected by high performance liquid chromatography, which was recorded as one reuse. The immobilized enzyme after the reaction was washed three times with 50 mM Tris-HCl buffer (pH 7.5), and then reacted again, and reused 20 times. The experimental results are shown in FIG. Fig.11 As shown, after repeated use for 5, 10, and 20 times, the immobilized enzyme can maintain 58%, 44%, and 26% of the initial enzyme activity, respectively.

[0059] A270R (SEQ ID NO.1)

[0060] ATGAACATCGGATGCCACGGGCTCGTCTGGACCGGACACTTCGATGCCGACGGCATCCGACTCTCGGTGGAGCAGACGAAGGCGGCAGGCTTCGACCTGATCGAGTTCCCGCTCATGGATCCGTTCACGTTCGATGTCGCCGCCGCGAAGGAGGCGCTCGAAGAGCACGACCTGGCCGTCAGCGCCTCGCTCGGTCTCTCGGGGGCGACGGATGTGACCAGCTCTGATCCGGCAGTCGTCGCCGCGGGGGAGGCGCTGCTGATGAAGGCCGTCGATGTGCTGGCCGAGCTGGGCGGCCGGCACTTCTGCGGAGTGATCTACAGCGCGATGCAGAAGTACATGGACCCGGTGACGACAGAAGGGCTCGAGAGCAGCCGTCGCACCATCGCCCGCGTCGCGGATCACGCGGCCGAGCGAGGGATCTCGGTCTCGCTCGAGGTCGTGAACCGCTATGAGACCAACGTGCTGAACACCGCGCGGCAGGCACTCGCCTACCTCGCGGAGGTCGATCGGCCGAACCTCGGCATCCACCTCGACACGTACCACATGAACATCGAGGAGTCGGATATGTTCGCGCCGGTCCTCGATGCCGCACCCGCCCTCCGCTACGTGCACATCGGCGAGAGCCACCGCGGCTATCTCGGCACCGGAACGGTCGACTTCGACAACTTCTTCAAGGCGCTGGGGCGCATCGGCTATGACGGCCCGATCGTGTTCGAGTCGTTCTCCTCGGCGGTGGTCGCCCCCGACCTCAGCCGGATGCTCGGCATCTGGCGCAACCTGTGGACCGACAACGTCGAGCTCGGT CGC CACGCCAACGCCTACATCCGCGACAAGCTCGTCGCGGTCGACTCGATCAGGCTGCAC。

[0061] A270R(SEQ ID NO.2)

[0062] MNIGCHGLVWTGHFDADGIRLSVEQTKAAGFDLIEFPLMDPFTFDVAAAKEALEEHDLAVSASLGLSGATDVTSSDPAVVAAGEALLMKAVDVLAELGGRHFCGVIYSAMQKYMDPVTTEGLESSRRTIARVADHAAERGISVSLEVVNRYETNVLNTARQALAYLAEVDRPNLGIHLDTYHMNIEESDMFAPVLDAAPALRYVHIGESHRGYLGTGTVDFDNFFKALGRIGYDGPIVFESFSSAVVAPDLSRMLGIWRNLWTDNVELG R HANAYIRDKLVAVDSIRLH.

[0063] A270R-cbd (SEQ ID NO.3)

[0064] ATGAACATCGGATGCCACGGGCTCGTCTGGACCGGACACTTCGATGCCGACGGCATCCGACTCTCGGTGGAGCAGACGAAGGCGGCAGGCTTCGACCTGATCGAGTTCCCGCTCATGGATCCGTTCACGTTCGATGTCGCCGCCGCGAAGGAGGCGCTCGAAGAGCACGACCTGGCCGTCAGCGCCTCGCTCGGTCTCTCGGGGGCGACGGATGTGACCAGCTCTGATCCGGCAGTCGTCGCCGCGGGGGAGGCGCTGCTGATGAAGGCCGTCGATGTGCTGGCCGAGCTGGGCGGCCGGCACTTCTGCGGAGTGATCTACAGCGCGATGCAGAAGTACATGGACCCGGTGACGACAGAAGGGCTCGAGAGCAGCCGTCGCACCATCGCCCGCGTCGCGGATCACGCGGCCGAGCGAGGGATCTCGGTCTCGCTCGAGGTCGTGAACCGCTATGAGACCAACGTGCTGAACACCGCGCGGCAGGCACTCGCCTACCTCGCGGAGGTCGATCGGCCGAACCTCGGCATCCACCTCGACACGTACCACATGAACATCGAGGAGTCGGATATGTTCGCGCCGGTCCTCGATGCCGCACCCGCCCTCCGCTACGTGCACATCGGCGAGAGCCACCGCGGCTATCTCGGCACCGGAACGGTCGACTTCGACAACTTCTTCAAGGCGCTGGGGCGCATCGGCTATGACGGCCCGATCGTGTTCGAGTCGTTCTCCTCGGCGGTGGTCGCCCCCGACCTCAGCCGGATGCTCGGCATCTGGCGCAACCTGTGGACCGACAACGTCGAGCTCGGT CGCCACGCCAACGCCTACATCCGCGACAAGCTCGTCGCGGTCGACTCGATCAGGCTGCACAGATCTCTGGCGGCGGCGCTGGAAGGCCTGGTTCCGCGTGGCAGCGGCCCGCCGACCCCGACCCCGACCAGCGGCCCGGCGGGGTGCCAGGTTCTGTGGGGCGTTAACCAGTGGAACACCGGCTTCACCGCGAACGTTACCGTTAAAAACACCTCCAGCGCGCCGGTTGATGGCTGGACCCTGACCTTCAGCTTCCCGTCTGGCCAGCAGGTTACTCAGGCTTGGAGTTCAACTGTTACCCAGTCTGGTTCTGCAGTTACTGTTCGTAACGCTCCGTGGAACGGTTCTATTCCGGCTGGCGGTACCGCTCAATTCGGTTTTAATGGGAGCCACACCGGTACTAACGCAGCACCGACTGCTTTTAGCCTGAATGGTACACCGTGCACCGTTGGTAGATCTAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGA。

[0065] A270R-cbd(SEQ ID NO.4)

[0066] MNIGCHGLVWTGHFDADGIRLSVEQTKAAGFDLIEFPLMDPFTFDVAAAKEALEEHDLAVSASLGLSGATDVTSSDPAVVAAGEALLMKAVDVLAELGGRHFCGVIYSAMQKYMDPVTTEGLESSRRTIARVADHAAERGISVSLEVVNRYETNVLNTARQALAYLAEVDRPNLGIHLDTYHMNIEESDMFAPVLDAAPALRYVHIGESHRGYLGTGTVDFDNFFKALGRIGYDGPIVFESFSSAVVAPDLSRMLGIWRNLWTDNVELG RHANAYIRDKLVAVDSIRLHRSLAAALEGLVPRGSGPPTPTPTSGPAGCQVLWGVNQWNTGFTANVTVKNTSSAPVDGWTLTFSFPSGQQVTQAWSSTVTQSGSAVTVRNAPWNGSIPAGGTAQFGFNGSHTGTNAAPTAFSLNGTPCTVGRSKLAAALEHHHHHH。

[0067] dpema4(SEQ ID NO.5)

[0068] ATGAACATCGGATGCCACGGGCTCGTCTGGACCGGACACTTCGATGCCGACGGCATCCGACTCTCGGTGGAGCAGACGAAGGCGGCAGGCTTCGACCTGATCGAGTTCCCGCTCATGGATCCGTTCACGTTCGATGTCGCCGCCGCGAAGGAGGCGCTCGAAGAGCACGACCTGGCCGTCAGCGCCTCGCTCGGTCTCTCGGGGGCGACGGATGTGACCAGCTCTGATCCGGCAGTCGTCGCCGCGGGGGAGGCGCTGCTGATGAAGGCCGTCGATGTGCTGGCCGAGCTGGGCGGCCGGCACTTCTGCGGAGTGATCTACAGCGCGATGCAGAAGTACATGGACCCGGTGACGACAGAAGGGCTCGAGAGCAGCCGTCGCACCATCGCCCGCGTCGCGGATCACGCGGCCGAGCGAGGGATCTCGGTCTCGCTCGAGGTCGTGAACCGCTATGAGACCAACGTGCTGAACACCGCGCGGCAGGCACTCGCCTACCTCGCGGAGGTCGATCGGCCGAACCTCGGCATCCACCTCGACACGTACCACATGAACATCGAGGAGTCGGATATGTTCGCGCCGGTCCTCGATGCCGCACCCGCCCTCCGCTACGTGCACATCGGCGAGAGCCACCGCGGCTATCTCGGCACCGGAACGGTCGACTTCGACAACTTCTTCAAGGCGCTGGGGCGCATCGGCTATGACGGCCCGATCGTGTTCGAGTCGTTCTCCTCGGCGGTGGTCGCCCCCGACCTCAGCCGGATGCTCGGCATCTGGCGCAACCTGTGGACCGACAACGTCGAGCTCGGTGCGCACGCCAACGCCTACATCCGCGACAAGCTCGTCGCGGTCGACTCGATCAGGCTGCACTGA。

[0069] dpema4(SEQ IDNO.6)

[0070] MNIGCHGLVWTGHFDADGIRLSVEQTKAAGFDLIEFPLMDPFTFDVAAAKEALEEHDLAVSASLGLSGATDVTSSDPAVVAAGEALLMKAVDVLAELGGRHFCGVIYSAMQKYMDPVTTEGLESSRRTIARVADHAAERGISVSLEVVNRYETNVLNTARQALAYLAEVDRPNLGIHLDTYHMNIEESDMFAPVLDAAPALRYVHIGESHRGYLGTGTVDFDNFFKALGRIGYDGPIVFESFSSAVVAPDLSRMLGIWRNLWTDNVELGAHANAYIRDKLVAVDSIRLH.

Claims

1. A gene encoding a D-psicose 3-epimerase mutant, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID No:

1.

2. A mutant of D-psicose 3-epimerase, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID No:

2.

3. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleotide sequence shown in SEQ ID No:

1.

4. A recombinant bacterium, characterized in that: The recombinant bacteria comprises the nucleotide sequence shown in SEQ ID No: 1, and the host strain is Escherichia coli JM109.

5. Use of the gene encoding the D-psicose 3-epimerase mutant according to claim 1 in the biosynthesis of D-psicose.

6. The use according to claim 5, characterized in that: 1 mM Co was added to the reaction system in the application 2+ .

7. A fusion enzyme encoding a D-psicose 3-epimerase mutant, characterized in that: The nucleotide sequence of the gene of the fusion enzyme is shown in SEQ ID No:3; its amino acid sequence is shown in SEQ ID No:4, which contains the nucleotide sequence shown in SEQ ID No:

1.

8. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleotide sequence shown in SEQ ID No:

3.

9. A recombinant bacterium, characterized in that: It comprises the nucleotide sequence shown in SEQ ID No: 3, and the host strain is Escherichia coli BL21 (DE3).

10. A method for preparing an immobilized enzyme using the fusion enzyme according to claim 7, characterized in that: The method comprises the following steps: mixing the crude enzyme solution of the fusion enzyme with microcrystalline cellulose under oscillation at 25°C and 700 rpm for 5-30 min, centrifuging at 12000 rpm for 1 min to remove the supernatant, and washing the remaining solid with 50 mM pH 7.5 Tris-HCl buffer to obtain an immobilized enzyme.

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