D-psicose 3-epimerase mutant with improved enzyme activity and thermal stability

Abstract

The invention discloses a D-psicose 3-epimerase mutant with improved enzyme activity, and belongs to the technical field of gene engineering and enzyme engineering. According to the invention, a site-directed mutagenesis technology is utilized to carry out molecular modification on a wild type DPE coding gene to obtain a series of mutant enzymes, the enzyme activity of the mutant enzymes is improved by 1.08-1.64 times compared with that of the wild enzymes, the enzyme activity of the wild type DPE is optimized and improved, the enzyme activity of the combined mutant is improved by 1.28-1.64 times, and the enzyme activity of the combined mutant is improved by 1.28-1.64 times compared with that of the wild enzymes. The optimal temperature of the combined mutant DPEA25V / C212N is increased by 5 DEG C, the residual enzyme activity of the combined mutant DPEA25V / C212N after heat preservation at 60 DEG C for 6 hours is still 43.55%, the half-life period of the combined mutant DPEA25V / C212N is 5.76 times that of a wild type, and the D-fructose conversion rate of the combined mutant DPEA25V / C212N after catalysis for 6 hours reaches 30.26% and is higher than that of the wild type DPEA25V / C212N by 28.81%, so that better use conditions are created for the enzyme in practical application.

Description

Technical Field

[0001] The invention relates to a D-psicose 3-epimerase mutant with improved enzyme activity, belonging to the technical field of genetic engineering and enzyme engineering. Background Art

[0002] D-psicose 3-epimerase (EC: 5.1.3.30), referred to as DPE, can catalyze the mutual conversion of hexose C3 isomers, such as D-fructose and D-psicose. DPE is widely present in Bacillus and Clostridium. DPE from different sources has obvious differences in molecular weight range, optimal pH, optimal temperature and other enzymatic properties.

[0003] At present, DPE is mainly used in the production of allulose, which has the advantages of high conversion rate, simple process control, mild reaction conditions, and abundant raw materials. Compared with chemical synthesis, the enzymatic synthesis of D-psicose has the advantages of environmental protection and sustainability. However, due to some defects of DPE itself, such as low specific enzyme activity, poor thermal stability, and low conversion rate, its application range is limited. Nowadays, a series of D-psicose 3-isomerases with excellent properties have been obtained through computer mining technology and protein engineering technology. For example, Feng et al. improved the thermal stability of D-psicose 3-isomerase from Clostridium cellulolyticum through high-throughput screening. The half-life of its triple-point mutant H56R / Q277R / S293R at 60°C was increased from 9.23min to 44.82min, and the specific enzyme activity was 338.5U / mg, which was increased by 1.58 times. Zhao et al. modified DPE from Clostridium bolteae by introducing a new disulfide bond-constructed mutant C175-A209C. 1 / 2 The time at 55°C increased from 0.37h to 4.5h, which improved their industrial application potential, but still had certain limitations. In order to further improve the enzyme activity and thermal stability, based on the expression platform obtained in Bacillus subtilis, the semi-rational design of the computer was used to modify the DPE molecules, in order to obtain DPE with enzymatic properties more suitable for industrial application. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a D-psicose 3-epimerase mutant with improved enzyme activity. The D-psicose 3-epimerase gene from Clostridium scindens 35704 (the nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2) is molecularly modified, and site-directed mutagenesis technology is used to obtain a D-psicose 3-epimerase mutant with significantly improved enzyme activity, thereby solving the limitation of DPE's own enzymatic performance defects on application.

[0005] The first technical solution provided by the present invention is a D-psicose 3-epimerase mutant, which is obtained by mutating one or more of the amino acids at positions 22, 25, 46, 165, 212 and 267 of a D-psicose 3-epimerase parent having an amino acid sequence as shown in SEQ ID NO.2.

[0006] In one embodiment, the mutant is obtained by mutating the 22nd valine, the 25th alanine, the 46th glutamine, the 165th glycine, the 212th cysteine ​​and / or the 267th glycine of the D-psicose 3-epimerase parent having an amino acid sequence as shown in SEQ ID NO.2.

[0007] In one embodiment, the mutant is obtained by mutating the valine V at position 22 of the D-psicose 3-epimerase parent having an amino acid sequence as shown in SEQ ID NO.2 to isoleucine I to obtain the mutant V22I.

[0008] In one embodiment, the mutant is obtained by mutating alanine A at position 25 of the D-psicose 3-epimerase parent having an amino acid sequence as shown in SEQ ID NO.2 to valine V to obtain mutant A25V.

[0009] In one embodiment, the mutant is obtained by mutating the 46th glutamine Q of the D-psicose 3-epimerase parent with the amino acid sequence as shown in SEQ ID NO.2 to isoleucine I to obtain the mutant Q46I.

[0010] In one embodiment, the mutant is obtained by mutating the glycine G at position 165 of the D-psicose 3-epimerase parent having an amino acid sequence as shown in SEQ ID NO.2 to alanine A to obtain the mutant G165A.

[0011] In one embodiment, the mutant is obtained by mutating the cysteine ​​C at position 212 of the D-psicose 3-epimerase parent having an amino acid sequence as shown in SEQ ID NO.2 to asparagine N to obtain the mutant C212N.

[0012] In one embodiment, the mutant is obtained by mutating the glycine G at position 267 of the D-psicose 3-epimerase parent having an amino acid sequence as shown in SEQ ID NO.2 to aspartic acid D to obtain the mutant G267D.

[0013] In one embodiment, the mutant is obtained by mutating the 25th alanine A of the D-psicose 3-epimerase parent with the amino acid sequence as shown in SEQ ID NO.2 to valine V and the 212th cysteine ​​C to asparagine N to obtain the mutant A25V / C212N.

[0014] In one embodiment, the mutant is obtained by mutating the valine V at position 22 of the D-psicose 3-epimerase parent having an amino acid sequence as shown in SEQ ID NO.2 to isoleucine I and the alanine A at position 25 to valine V to obtain the mutant V22I / A25V.

[0015] The second technical solution provided by the present invention is a gene encoding the mutant described in the first technical solution.

[0016] The third technical solution provided by the present invention is a recombinant plasmid carrying the gene described in the second technical solution.

[0017] In one embodiment, the expression vector of the recombinant plasmid includes but is not limited to pP43 series plasmids.

[0018] In one embodiment, the pP43 series plasmid is pP43NMK-Phag-RBS4.

[0019] The fourth technical solution provided by the present invention is a recombinant microbial cell expressing the mutant described in the first technical solution, or containing the gene described in the second technical solution, or transformed with the recombinant plasmid described in the third technical solution.

[0020] In one embodiment, the recombinant microbial cell includes, but is not limited to, bacteria or fungi.

[0021] In one embodiment, the microorganism is Bacillus subtilis.

[0022] In one embodiment, the recombinant microbial cell uses Bacillus subtilis WB800 as a host and pP43NMK-Phag-RBS4 as a vector.

[0023] The fifth technical solution provided by the present invention is a method for improving the enzymatic activity and / or thermal stability of D-psicose 3-epimerase, wherein the method comprises performing any of the following mutations on the D-psicose 3-epimerase parent with the amino acid sequence as shown in SEQ ID NO.2:

[0024] (1) mutating valine V at position 22 to isoleucine I;

[0025] (2) mutating alanine A at position 25 to valine V;

[0026] (3) mutating glutamine Q at position 46 to isoleucine I;

[0027] (4) mutating glycine G at position 165 to alanine A;

[0028] (5) mutating cysteine ​​C at position 212 to asparagine N;

[0029] (6) Mutation of glycine G at position 267 to aspartic acid D;

[0030] (7) mutating alanine A at position 25 to valine V and cysteine ​​C at position 212 to asparagine N;

[0031] (8) The valine V at position 22 was mutated to isoleucine I and the alanine A at position 25 was mutated to valine V.

[0032] The sixth technical solution provided by the present invention is a method for preparing D-psicose, wherein D-fructose is used as a substrate, the D-psicose 3-epimerase mutant described in the first technical solution is used as a catalyst to form a catalytic system to catalyze the production of D-psicose.

[0033] In one embodiment, the catalytic system further comprises 1 mM Mn 2+ .

[0034] In one embodiment, the catalytic reaction conditions are 65° C. and 200 r / min for 6 hours.

[0035] The seventh technical solution provided by the present invention is the use of the D-psicose 3-epimerase mutant described in the first technical solution, the gene described in the second technical solution, the recombinant plasmid described in the third technical solution, the recombinant microorganism described in the fourth technical solution, the method described in the fifth technical solution, or the method described in the sixth technical solution in the production of D-psicose.

[0036] The technical effects of the present invention are as follows:

[0037] The present invention uses D-psicose 3-epimerase from Clostridium scindens 35704 as a parent enzyme for molecular modification, mutates the amino acids at positions 22, 25, 46, 165, 212, 267, 22 / 25, and 25 / 212, and obtains a series of mutants with improved enzyme activities. The enzyme activities of a series of D-psicose 3-epimerase mutants provided by the present invention are significantly improved compared with wild enzymes, the mutated enzymes still maintain a moderate pH, and the enzyme activities can reach 618.1 U / mg, 606.1 U / mg, 596.1 U / mg, 653.1 U / mg, 611.5 U / mg, 725.6 U / mg, 908.9 U / mg, and 710.2 U / mg, which are 1.08 to 1.64 times higher than the wild-type enzyme activities.

[0038] The present invention measured the thermal stability of the mutants at positions 25 and 212. Compared with the wild enzyme, the optimum temperature of the combined mutants increased by 5°C, the residual enzyme activity after being incubated at 60°C for 6 hours was still 43.55%, and the half-life was 5.76 times that of the wild type.

[0039] The present invention conducted an enzyme conversion experiment on the combined mutants at positions 25 and 212, and the results showed that the combined mutants could effectively improve the conversion rate of the substrate, and the D-fructose conversion rate reached 30.26% after catalysis for 6 hours, which was higher than 28.81% of the wild type.

[0040] The present invention optimizes and improves the enzyme activity of wild-type D-psicose 3-epimerase, creates better use conditions for the enzyme in practical applications, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the construction map of the recombinant plasmid pP43NMK-Phag-RBS4-DPE (pP43NMK-hag-R4-dpe).

[0042] Figure 2 is the relative enzyme activity of the wild enzyme DPE and the mutant enzyme.

[0043] Figure 3 Comparison of the thermal stability of the wild enzyme DPE and the single-point mutant enzyme at 50°C.

[0044] Figure 4 Comparison of the thermal stability of wild enzyme DPE and mutant enzyme at 55℃.

[0045] Figure 5 Comparison of the thermal stability of wild enzyme DPE and mutant enzyme at 60℃.

[0046] Figure 6is the half-life of the wild enzyme DPE and the mutant enzyme.

[0047] Figure 7 Substrate conversion rates of wild-type enzyme DPE and mutant enzyme at 65 °C. DETAILED DESCRIPTION

[0048] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0049] Test method:

[0050] 1. High performance liquid chromatography (HPLC) detection and analysis:

[0051] The formation of D-psicose was detected by high performance liquid chromatography with a differential detector (RID). The chromatographic column was Sugar-Pak I and the operating conditions were as follows: column temperature 85°C; the mobile phase was water with 50 mg / L EDTA-2Na added; the mobile phase flow rate was 0.4 mL / min, and the sample injection volume was 10 μL.

[0052] 2. D-psicose 3-epimerase activity detection method

[0053] Enzyme activity assay conditions: 1 ml reaction system includes D-fructose (50 g / L), Tris-HCl buffer (50 mM, pH 7.5) and 0.25 μM enzyme. React at 60°C for 5 min, boil for 10 min to inactivate the enzyme. Centrifuge the sample at 12000 r / min for 5 min, filter the supernatant through a 0.22 μm filter membrane, prepare the sample, and analyze it using high performance liquid chromatography (HPLC).

[0054] Enzyme activity definition: Under the above reaction conditions: the amount of enzyme required to produce 1 μmol of D-psicose per minute.

[0055] The enzymatic activity calculation formula of D-psicose 3-epimerase is as follows:

[0056]

[0057] Materials used in the examples:

[0058] (I) Culture medium

[0059] LB liquid culture medium: yeast powder 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L, high pressure sterilization at 121°C for 30 min.

[0060] LB solid medium: yeast powder 5.0 g / L, tryptone 10.0 g / L, NaCl 10.0 g / L, agar powder 15 g / L, high pressure sterilization at 121°C for 30 min.

[0061] Fermentation medium: glucose 15 g / L, peptone 10 g / L, yeast extract powder 20 g / L, NaCl 8 g / L, Na2HPO4·12H2O1 g / L, MgSO4·7H2O 1 g / L, adjust pH to 7.0, and sterilize at 115°C for 30 min.

[0062] (II) Buffer

[0063] Lysis buffer: 50mmol / L Tris, 100mmol / L NaCl, pH 7.5;

[0064] Binding Buffer: 50mmol / L Tris, 500mmol / L NaCl, pH 7.5;

[0065] Washing Buffer: 50mmol / L Tris, 500mmol / L NaCl, 50mmol / L imidazole, pH 7.5;

[0066] Elution Buffer: 50mmol / L Tris, 500mmol / L NaCl, 500mmol / L imidazole, pH 7.5;

[0067] Dialysis buffer A: 50 mmol / L Tris, 10 mmol EDTA·2H2O-2Na, pH 7.5;

[0068] Dialysis buffer B: 50 mmol / L Tris, pH 7.5.

[0069] (III) Materials and reagents

[0070] The restriction endonucleases, Dpn I enzyme, PCR reagents, etc. used were purchased from TaKaRa Biotechnology Co., Ltd.; primers were purchased from Anshengda Biotechnology Co., Ltd.; plasmid extraction kits, genome extraction kits, agarose purification kits, Escherichia coli E.coil DH5α, and Bacillus subtilis B.subtilis WB800 strains were all preserved in the laboratory and were all commercial strains; other reagents were analytical grade reagents purchased domestically or abroad.

[0071] Example 1D-Design of mutation sites of D-psicose 3-epimerase

[0072] After multiple sequence alignment, homology alignment, and thermal stability design combined with online servers EsPript (https: / / espript.ibcp.fr / ESPript / cgi-bin / ESPript.cgi)), DeepDDG (http: / / protein.org.cn / ddg.html), mCSM-PPI 2 (https: / / biosig.lab.uq.edu.au / mcsm_ppi2 / ), and FireProt (http: / / loschmidt.chemi.cz / fireprot), mutation sites were selected; the D-psicose 3-epimerase protein structure was simulated using SWISS-MODEL software to obtain the tertiary structure model of D-psicose 3-epimerase. The amino acid sites to be mutated were determined to be valine at position 22, alanine at position 25, glutamine at position 46, glycine at position 165, cysteine ​​at position 212, and glycine at position 267.

[0073] Example 2: Site-directed mutagenesis of D-psicose 3-epimerase and construction of recombinant Bacillus subtilis

[0074] (1) Construction of wild-type plasmid

[0075] The D-psicose 3-epimerase from the microorganism Clostridium scindens 35704 has a nucleotide sequence as shown in SEQ ID NO.1 and an amino acid sequence as shown in SEQ ID NO.2.

[0076] The laboratory has constructed the D-psicose 3-epimerase encoding gene with the nucleotide sequence shown in SEQ ID NO.1 between the Eag I and Pst I restriction sites of the plasmid vector pP43NMK-Phag-RBS4 (disclosed in patent CN114350699A) to obtain a recombinant plasmid named pP43NMK-Phag-RBS4-DPE (i.e., pP43NMK-hag-R4-dpe, as Figure 1 As shown, it has been disclosed in patent CN114350699A).

[0077] (2) Construction of mutant plasmid

[0078] Primers were designed based on the gene encoding DPE from Clostridium scindens 35704 as shown in SEQ ID NO.1, and site-directed mutagenesis was performed using pP43NMK-Phag-RBS4-DPE constructed in step (1) as a template to construct a mutant plasmid:

[0079] V22I-F:GCGGTATatcGAGAAGGCGGCAAAGCTTGGAT;

[0080] V22I-R: CCTTCTCgatATACCGCTTGTAATCTGCTGCC;

[0081] A25V-F:AGAGAAGgtcGCAAAGCTTGGATTCGATATACTG;

[0082] A25V-R:GCTTTGCgacCTTCTCTACATACCGCTTGTAATCTGC;

[0083] Q46I-F:TTCTGCGatcGAGGTAAAGGAACTGAAAAAATGCG;

[0084] Q46I-R:TTACCTCgatCGCAGAATAGTCCCGGCAGTGGC;

[0085] G165A-F:GGAAGAAgccGTGAAGTTTCGTGACGGAAGTAGG;

[0086] G165A-R:ACTTCACggcTTCTTCCGAAGTATTTAAGATATGGC;

[0087] C212N-F:GAGaacAACCGCATGGTACCCGGAAAGGGCCG;

[0088] C212N-R:ACCATGCGGTTgttCTCGCCGGTGTGGAAGTG;

[0089] G267D-F:AAGGGCCGgatGAGGACCGGCTGGATGAGGA;

[0090] G267D-R:TCCCatcCGCGCCCTTGCTGATGTCTCTCCA.

[0091] The lowercase letters represent the codons corresponding to the 22nd valine, 25th alanine, 46th glutamine, 165th glycine, 212th cysteine, and 267th glycine encoded by the mutant gene. In addition, pP43NMK-Phag-RBS4-DPE was used as a template to construct mutants with any two-point combination, among which the combined mutants higher than any single-point mutant in the combined mutants were V22I / A25V and A25V / C212N.

[0092] Table 1 PCR amplification system

[0093]

[0094] After PCR amplification, 0.5 μL DpnⅠ restriction endonuclease (10 U / μL) was added to the reaction solution and incubated at 37°C for 2 h to eliminate the template. The PCR product was transformed into E.coil DH5α cells, coated on LB plates, and a single colony was picked into LB liquid culture medium to extract the plasmid. The correct mutant plasmid pP43NMK-Phag-RBS4-DPE was obtained by sequencing. V22I 、pP43NM K-Phag-RBS4-DPE A25V 、pP43NMK-Phag-RBS4-DPE Q46I 、pP43NMK-Phag-RBS4-DPE G165A 、pP43NMK-Phag-RBS4-DPE C212N 、pP43NMK-Phag-RBS4-DPE G267D 、pP43NMK-Phag-RBS4-DPE V22I / A25V 、pP43NMK-Phag-RBS4-DPE A25V / C212N The successfully constructed mutant plasmid and the wild-type plasmid of step (1) were transformed into B. subtilis WB800 to obtain the mutant strain B. subtilis WB800 / pP43NMK-Phag-RBS4-DPE V22I , B.subtilis WB800 / pP43NMK-Phag-RBS4-DPE A25V , B.subtilis WB800 / pP43NMK-Phag-RBS4-DPE Q46I , B.subtilis WB800 / pP43NMK-Phag-RBS4-DPE G165A , B.subtilis WB800 / pP43NMK-Phag-RBS4-DPE C212N , B.subtilis WB800 / pP43NMK-Phag-RBS4-DPE G267D , B.subtilis WB800 / pP43NMK-Phag-RBS4-DPE V22I / A25V , B.subtilis WB800 / pP43NMK-Phag-RB S4-DPE A25V / C212N and strain B. subtilis WB800 / pP43NMK-Phag-RBS4-DPE expressing the wild-type enzyme.

[0095] Example 3: Expression and purification of wild-type enzymes and mutant enzymes

[0096] The B. subtilis WB800 / pP43NMK-Phag-RBS4-DPE and single colonies of each mutant prepared in Example 2 were picked and placed in a fermentation medium containing 50 μg / mL kanamycin. After culturing at 37° C. and 200 r / min for 12 h, the fermentation broth was collected.

[0097] After the collected fermentation liquid was centrifuged at 8000r / min for 10min, the supernatant was discarded, and the cells were washed three times with Tris-HCl buffer. 15mL Tris-HCl buffer was added to suspend the cells. 20ul 20mg / ml lysozyme buffer was added to each ml of cells. The cells were kept warm in a 37℃ water bath for 30min, and then ultrasonically disrupted for 10min (power 200W, disruption for 1s, rest for 2s). Centrifuged at 4℃ and 8000r / min for 10min, the supernatant was collected, which was the crude enzyme solution, and filtered with a 0.22μm water membrane. Binding Buffer was used to treat Ni 2+ The chelating agarose resin column is pre-balanced; crude enzyme solution is added and balanced with Binding Buffer and Washing Buffer respectively; the enzyme is eluted with Elution Buffer and recovered; the recovered enzyme solution is dialyzed in dialysis buffer and stored in a refrigerator at 4°C.

[0098] Under this condition, the original enzyme activity was defined as 100%, and the mutant species were plotted as a percentage of relative enzyme activity. The results of enzyme activity evaluation were as follows: Figure 2 Compared with the specific enzyme activity of 553.7U / mg of the wild enzyme DPE, the specific enzyme activities of the mutants were: V22I was 618.1U / mg, A25V was 606.1U / mg, Q46I was 596.1U / mg, G165A was 653.1U / mg, C212N was 611.4U / mg, G267D was 725.6U / mg, V22I / A25V was 908.9U / mg, and A25V / C212N was 710.2U / mg.

[0099] Example 4: Thermal stability test of wild enzyme and mutant enzyme

[0100] The specific steps are as follows:

[0101] (1) Thermal stability of WT and single-point mutants at 50°C

[0102] In order to obtain mutants with improved enzyme activity and thermal stability, the enzyme activity of A25V / C212N was first determined. Under standard reaction conditions, the specific enzyme activity of A25V / C212N was 710.2 U / mg, which was 1.28 times that of the wild type. Secondly, the thermal stability of WT and all single-point mutants was evaluated at 50°C. Figure 3 The stability of A25V, C212N and G267D after 45 min of incubation was higher than that of the wild type. Based on this, A25V, C212N and G267D were screened to study their thermal stability. Among them, G267D was heated to 1 mM N 2+ , which was inactivated after incubation at 50°C for 8 h, so G267D was discarded and A25V and C212N were combined to measure the activity of the 2-mercaptoethanol in the presence of 1 mM Mn at 55°C and 60°C. 2+ The thermal stability of the enzyme solution was tested.

[0103] (2) Detection of residual enzyme activity at 55°C

[0104] The pure enzyme solution containing WT and the pure enzyme solution containing A25V / C212N prepared in Example 2 were respectively incubated at 55°C for 18 hours and then the residual enzyme activity was measured. The initial enzyme activity at 0 hours of incubation at 55°C was set as 100%. The results are as follows: Figure 4 And as shown in Table 2.

[0105] Table 2 Residual enzyme activity of wild-type and mutant D-psicose 3-epimerase at 55°C

[0106]

[0107] (3) Residual enzyme activity detection at 60°C

[0108] The pure enzyme solution containing WT and the pure enzyme solution containing A25V / C212N prepared in Example 2 were respectively incubated at 60°C for 6 hours and then the residual enzyme activity was measured. The initial enzyme activity at 0 hours of incubation at 60°C was set as 100%. The results are as follows: Figure 5 The half-lives (t 1 / 2 ), t 1 / 2 =Ln2 / k d , Ln(E t / E0)=-k d ×t (t is time (h), kd is the inactivation rate constant (h -1 ), Et / E0 is the relative enzyme activity (%). Figure 6 And as shown in Table 4.

[0109] Table 3 Residual enzyme activity of wild-type and mutant D-psicose 3-epimerase at 60°C

[0110]

[0111] Table 4 Half-life of wild-type and mutant D-psicose 3-epimerase at 60°C

[0112]

[0113] Embodiment 5:

[0114] Add fructose to the conversion beaker to a final concentration of 50% (w / w), add Tris to a final concentration of 50 mM, adjust the pH to 7.5, add water, stir and heat to 65°C, add 1 mM manganese ions, add 20 mg / L of WT and A25V / C212N mutant pure enzyme solution, react under stirring, take samples regularly and use HPLC detection and area normalization method to calculate the conversion rate, such as Figure 7 And as shown in Table 5.

[0115] Table 5D - Fructose conversion results

[0116]

[0117] Comparative Example 1:

[0118] According to the same strategy as in Examples 1 to 3, the conserved sites W14 (tryptophan at position 14), E149 (glutamic acid at position 149), E243 (glutamic acid at position 243), G65 (glycine at position 65), and Y6 (tyrosine at position 6) were screened and obtained. The mutants W14S (tryptophan at position 14 mutated to serine), E149M (glutamic acid at position 149 mutated to methionine), E243H (glutamic acid at position 243 mutated to histidine), G65A (glycine at position 65 mutated to alanine), and Y6F (tyrosine at position 6 mutated to phenylalanine) were constructed according to the following primers, respectively, to obtain the plasmid pP43NMK-Phag-RBS 4-DPE W14S 、pP43NMK-Phag-RBS4-DPE E149M、 pP43NMK-Phag-RBS4-DPE E243H 、pP43NMK-Pha g-RBS4-DPE G65A 、pP43NMK-Phag-RBS4-DPE Y6F , and constructed the recombinant strain B. subtilis WB800 / pP43N MK-Phag-RBS4-DPE W14S , B.subtilisWB800 / pP43NMK-Phag-RBS4-DPE E149M, B.subtilis WB800 / pP43NMK-Phag-RBS4-DP E243H , B.subtilis WB800 / pP43NMK-Phag-RBS4-DPE G65A , B.subtilis WB800 / pP43NMK-Phag-RBS4-DPE Y6F The recombinant bacteria were cultured in the same manner as in Example 3, and the crude enzyme solution and the specific enzyme activity after purification were detected, respectively. The results showed that the crude enzyme activity and specific enzyme activity of W14S, E149M, E243H, G65A and Y6F were almost undetectable, indicating that the mutants W14S, E149M, E243H, G65A and Y6F had lost their enzyme activity. W14S, E149M, E243H, G65A and Y6F are important residues with catalytic ability of the enzyme, and the enzyme activity was not improved after mutation but lost instead.

[0119] E149M-F:GGGAATGatgGTCCTGAACCGCTTTGAGAGCC;

[0120] E149M-R:TCAGGACcatCATTCCCAGATTGATGCCATACT;

[0121] W14S-F:ACAGGAAagtGCAGCAGATTACAAGCGGTAT G；

[0122] W14S-R:CTGCTGCactTTCCTGTTCCCAGTACGCGTAA;

[0123] E243H-F: GGTTATGcacCCATTTGTACGCATGGGCGGAC;

[0124] E243H-R: CAAATGGgtgCATAACCACGGTTCCGTCATACT;

[0125] G65A-F: TGACCGCGgccTATGGTCCCGCCTTCAATCAT;

[0126] G65A-R:ACCATAggcCGCGGTCAGCTGGATACCGTTAT;

[0127] Y6F-F:TTttcTACGCGTACTGGGAACAGGAATGGGCA;

[0128] Y6F-R:CCAGTACGCGTAgaaAATACCATGCTTCATATGTGCACC

[0129] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A D-psicose 3-epimerase mutant, characterized in that: The mutant is obtained by mutating the 22nd valine, the 25th alanine, the 46th glutamine, the 165th glycine, the 212th cysteine ​​and / or the 267th glycine of the D-psicose 3-epimerase parent, and the amino acid sequence of the parent is shown in SEQ ID NO.

2.

2. The mutant according to claim 1, characterized in that The mutant is a mutant of a parent subjected to any one of the mutations (1) to (8): (1) Valine V at position 22 was mutated to isoleucine I to obtain the mutant V22I; (2) alanine A at position 25 was mutated to valine V to obtain mutant A25V; (3) glutamine Q at position 46 was mutated to isoleucine I to obtain mutant Q46I; (4) glycine G at position 165 was mutated to alanine A to obtain mutant G165A; (5) cysteine ​​C at position 212 was mutated to asparagine N to obtain mutant C212N; (6) Glycine G at position 267 was mutated to aspartic acid D to obtain mutant G267D; (7) Alanine A at position 25 was mutated to valine V and cysteine ​​C at position 212 was mutated to asparagine N to obtain the mutant A25V / C212N; (8) The valine V at position 22 was mutated to isoleucine I and the alanine A at position 25 was mutated to valine V to obtain the mutant V22I / A25V.

3. A gene encoding the mutant according to claim 1 or 2.

4. A recombinant plasmid carrying the gene according to claim 3.

5. The recombinant plasmid according to claim 4, characterized in that The expression vector of the recombinant plasmid is a pP43 series plasmid.

6. A recombinant microbial cell expressing the mutant according to claim 1 or 2, or containing the gene according to claim 3, or transformed with the recombinant plasmid according to claim 4 or 5.

7. The recombinant microbial cell according to claim 6, characterized in that The recombinant microbial cell uses Bacillus subtilis WB800 as a host.

8. A method for improving the enzymatic activity and / or thermal stability of D-psicose 3-epimerase, characterized in that: The method is to perform any of the following mutations on a parent D-psicose 3-epimerase having an amino acid sequence as shown in SEQ ID NO.2: (1) mutating valine V at position 22 to isoleucine I; (2) mutating alanine A at position 25 to valine V; (3) mutating glutamine Q at position 46 to isoleucine I; (4) mutating glycine G at position 165 to alanine A; (5) mutating cysteine ​​C at position 212 to asparagine N; (6) Mutation of glycine G at position 267 to aspartic acid D; (7) mutating alanine A at position 25 to valine V and cysteine ​​C at position 212 to asparagine N; (8) The valine V at position 22 was mutated to isoleucine I and the alanine A at position 25 was mutated to valine V.

9. A method for preparing D-psicose, characterized in that: The method uses D-fructose as a substrate and the D-psicose 3-epimerase mutant according to claim 1 or 2 as a catalyst to form a catalytic system to catalytically generate D-psicose.

10. Use of the D-psicose 3-epimerase mutant of claim 1 or 2, the gene of claim 3, the recombinant plasmid of claim 4 or 5, the recombinant microorganism of claim 6 or 7, the method of claim 8 or the method of claim 9 in the production of D-psicose.

Images