A high-throughput screening method for D-psicose 3-epimerase mutant library and mutants obtained

Abstract

The present invention discloses a high-throughput screening method for a D-psicose 3-epimerase mutant library and the mutants obtained, and belongs to the field of enzyme engineering technology. The high-throughput screening method provided by the present invention is based on the fact that in an acidic Si(IV)-Mo(VI) color developing solution, D-fructose can reduce yellow molybdate Mo(VI) to generate blue molybdate Mo(V / VI), and its color depth is proportional to the content of D-fructose within a certain range, and at the same concentration level, D-psicose does not interfere with the determination of D-fructose. At the same time, the present invention also provides a D-psicose 3-epimerase four-point mutant, compared with the wild type of the enzyme, its residual enzyme activity after incubation at 60°C for 1h is increased by 69.8%, the optimum temperature is increased by 5°C, and the half-life value at 60°C is increased from 0.64h to 7.30h, so its industrial application potential is significantly improved.

Description

Technical Field

[0001] The invention relates to a high-throughput screening method for a D-psicose 3-epimerase mutant library and the obtained mutants, belonging to the technical field of enzyme engineering. Background Art

[0002] D-allulose is the C-3 diastereomer of D-fructose and is the hottest rare sugar currently being studied. The sweetness of D-allulose is about 70% of sucrose, but its energy value is only 10% of sucrose, making it an ideal sucrose substitute. In addition, D-allulose has a variety of excellent physiological functions, including preventing obesity, lowering blood sugar, lowering blood lipids, anti-caries, anti-oxidation, and treating diabetes and atherosclerotic diseases. In addition, adding D-allulose to food can also improve the gel properties and improve the flavor of food through the Maillard reaction. At present, D-allulose has been certified as a generally recognized safe food (GRAS) by the U.S. Food and Drug Administration (FDA), and in 2019, the FDA allowed D-allulose to be excluded from the total sugar and added sugar content in food labels.

[0003] D-psicose can be synthesized by chemical methods, but there are problems such as harsh reaction conditions, difficult product separation, and chemical pollution. Compared with chemical synthesis, the biotransformation method has the advantages of mild reaction conditions, environmental friendliness, and high specificity. D-psicose 3-epimerase (DAEase) can catalyze the epimerization reaction of D-fructose at the C-3 position to directly generate D-psicose. It is currently the most efficient and common method for producing D-psicose.

[0004] Since most reported DAEases have poor thermal stability and are difficult to meet the needs of practical industrial applications, molecular modification studies to improve the thermal stability of DAEase have been carried out at home and abroad, mainly based on protein fusion technology and rational design methods, including temperature factor (B-factor) analysis, disulfide bond design, subunit interface interaction analysis and computer-assisted folding free energy (ΔΔG Fold ) calculations, etc.

[0005] Directed evolution technology is an effective method for enhancing enzyme function, but its application potential in DAEase molecular modification has not been fully explored. Its main bottleneck is the lack of fast and efficient screening methods. Existing DAEase high-throughput screening methods have problems such as complex operation and high screening costs. Summary of the invention

[0006] The present invention utilizes the difference in reducing ability between D-fructose and D-psicose in an acidic Si(IV)–Mo(VI) colorimetric solution to establish a high-throughput screening method based on colorimetric quantification, effectively coupling the genotype and phenotype of DAEase mutants. The present invention also provides a D-psicose 3-epimerase four-point mutant obtained by multiple rounds of directional screening using the screening method and D-psicose 3-epimerase from the microorganism Clostridium cellulolyticum as a parent, wherein both the thermal stability and enzyme activity of the four-point mutant are enhanced.

[0007] The purpose of the present invention is to provide a high-throughput screening method for a D-psicose 3-epimerase mutant library, as well as a D-psicose 3-epimerase quadruple-point mutant with enhanced thermal stability and enzyme activity, an encoding gene, a recombinant vector and a genetically engineered bacterium.

[0008] The technical solution adopted by the present invention is:

[0009] The first object of the present invention is to provide a high-throughput screening method for a D-psicose 3-epimerase mutant library based on colorimetric quantification, the specific steps of which are as follows:

[0010] 1) Using the DAEase encoding gene as a mutation template, based on the error-prone PCR method, a low-fidelity DNA polymerase (rTaq, Mn 2+ (0.1 mM) and Mg 2+ (6 mM) to generate and adjust the mutation rate, obtain a random mutation library of DAEase, and then inoculate the single colony to be screened into a 96-well deep-well plate to obtain a fermentation broth after inducing expression of recombinant DAEase;

[0011] 2) centrifuging the fermentation liquid obtained in step 1) to obtain bacterial precipitate, then adding lysis solution (containing 1 mg / mL lysozyme and 1 μL / mL DNase I) to disrupt cells, centrifuging the disrupted cell liquid to obtain the supernatant as a crude enzyme liquid, and heat-treating it at 60° C. for 10 min before performing an enzyme reaction; the enzyme reaction is to add the crude enzyme liquid to a reaction system containing D-fructose to catalyze D-fructose to generate D-psicose;

[0012] 3) After the enzyme reaction for 10 minutes, add 4 times the volume of Si(IV)-Mo(VI) colorimetric solution, heat at 70°C for 30 minutes to develop color, cool to room temperature, and measure the absorbance at a wavelength of 750nm. The average enzyme activity of the wild-type or exogenous DAEase is set as 100% relative enzyme activity, and mutants with a relative enzyme activity greater than 120% are identified as positive mutants.

[0013] The second object of the present invention is to provide a D-psicose 3-epimerase mutant, which is obtained by mutating one or more of the 38th, 42nd, 70th, and 119th amino acids of the parent enzyme D-psicose 3-epimerase having an amino acid sequence as shown in SEQ ID NO.2.

[0014] In one embodiment of the present invention, the D-psicose 3-epimerase is derived from Clostridium cellulolyticum.

[0015] In one embodiment of the present invention, the nucleotide sequence encoding the parent enzyme D-psicose 3-epimerase is shown as SEQ ID NO.1.

[0016] In one embodiment of the present invention, the mutant is based on the amino acid sequence shown in SEQ ID NO.2, with the serine at position 38 mutated to phenylalanine, the phenylalanine at position 42 mutated to asparagine, the alanine at position 70 mutated to proline and the threonine at position 119 mutated to proline, and is named S38F / F42N / A70P / T119P.

[0017] In one embodiment of the present invention, the amino acid sequence of the D-psicose 3-epimerase mutant S38F / F42N / A70P / T119P is shown in SEQ ID NO.4.

[0018] In one embodiment of the present invention, the nucleotide sequence encoding the D-psicose 3-epimerase mutant S38F / F42N / A70P / T119P is shown in SEQ ID NO.3.

[0019] The third object of the present invention is to provide a gene encoding the D-psicose 3-epimerase mutant S38F / F42N / A70P / T119P.

[0020] The fourth object of the present invention is to provide a recombinant vector carrying the mutant gene.

[0021] In one embodiment of the present invention, the recombinant vector uses pET28a as an expression vector.

[0022] The fifth object of the present invention is to provide a genetically engineered bacterium expressing the mutant S38F / F42N / A70P / T119P.

[0023] In one embodiment of the present invention, the genetically engineered bacteria uses Escherichia coli as a host.

[0024] In one embodiment of the present invention, the genetically engineered bacteria uses pET28a plasmid as an expression vector.

[0025] The sixth object of the present invention is to provide a recombinant cell expressing the above-mentioned D-psicose 3-epimerase mutant, or carrying the above-mentioned gene, or carrying the above-mentioned recombinant vector.

[0026] In one embodiment of the present invention, the recombinant cell uses bacteria or fungi as host cells.

[0027] In one embodiment of the present invention, the genetically engineered bacteria uses Escherichia coli as a host.

[0028] In one embodiment of the present invention, the genetically engineered bacteria uses pET28a plasmid as an expression vector.

[0029] The seventh object of the present invention is to provide a method for improving the thermal stability of D-psicose 3-epimerase, wherein the serine at position 38 of the D-psicose 3-epimerase having an amino acid sequence as shown in SEQ ID NO.2 is mutated to phenylalanine, the phenylalanine at position 42 is mutated to asparagine, the alanine at position 70 is mutated to proline, and the threonine at position 119 is mutated to proline.

[0030] An eighth object of the present invention is to provide a method for preparing psicose, comprising: using the above-mentioned D-psicose 3-epimerase mutant or the above-mentioned recombinant cell and D-fructose as a substrate to react and prepare psicose.

[0031] The ninth objective of the present invention is to provide the application of the high-throughput screening method in the molecular modification of D-psicose 3-epimerase or the genetically engineered bacteria in the industrial production of D-psicose.

[0032] The tenth object of the present invention is to provide use of the D-psicose 3-epimerase mutant or the recombinant cell in the preparation of psicose or a product containing psicose.

[0033] The present invention also provides a method for preparing the D-psicose 3-epimerase mutant S38F / F42N / A70P / T119P, which is a high-throughput screening method based on colorimetric quantification. The D-psicose 3-epimerase mutant S38F / F42N / A70P / T119P is obtained by screening according to the above-mentioned high-throughput screening method of a D-psicose 3-epimerase mutant library based on colorimetric quantification.

[0034] Beneficial Effects

[0035] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0036] The present invention provides an efficient and rapid high-throughput screening method for a D-psicose 3-epimerase mutant library. At the same time, the present invention also provides a D-psicose 3-epimerase quadruple mutant S38F / F42N / A70P / T119P obtained by screening using the screening method. Compared with the wild type of the enzyme, the residual enzyme activity of the mutant after incubation at 60°C for 1 hour is increased by 69.8%, the optimum temperature is increased by 5°C, and the half-inactivation temperature (T 50 5 ) value increased from 54.76℃ to 60.96℃, and the half-life (t 1 / 2 ) value increased from 0.64h to 7.30h, and its specific enzyme activity was 1.23 times that of the wild-type enzyme, which has broad application prospects in the industrial production of allulose. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 : Standard curve of the reaction between ketose and color developing solution.

[0038] Figure 2 : Comparison of the optimal temperatures of the four-point mutant enzyme and the wild-type enzyme.

[0039] Figure 3 : Comparison of the half-inactivation temperatures of the four-point mutant enzyme and the wild-type enzyme.

[0040] Figure 4 : Comparison of half-lives of the four-point mutant enzyme and the wild-type enzyme. DETAILED DESCRIPTION

[0041] The culture medium involved in the following examples is as follows:

[0042] LB medium (1L): 10g NaCl, 10g tryptone and 5g yeast extract, sterilized at 121℃ for 20min. Add kanamycin sulfate (Kan) to a final concentration of 50μg / mL before use. If it is prepared as a solid medium, add 1.5% (w / v) agar powder before sterilization.

[0043] ZYM-5052 medium (1 L): Add 40 mL ZYP-B (20X), 40 mL ZYP-C (20X) and 8 mL MgSO4 (100X) to 712 mL ZYM-A (8 g tryptone, 4 g yeast extract).

[0044] 20X ZYP-B (400 mL): 54 g KH2PO4, 143 g Na2HPO4 and 26.4 g (NH4)2SO4;

[0045] 20X ZYP-C (400 mL): 40 g glycerol, 4.4 g glucose and 16 g lactose;

[0046] 100X MgSO4: (400mL): 2.46g MgSO4.

[0047] ZYM-A, ZYP-B (20X), ZYP-C (20X) and MgSO4 (100X) were sterilized separately and mixed before use.

[0048] The preparation method of the solution involved in the following embodiments:

[0049] Si(IV)-Mo(VI) colorimetric solution: The composition of Si(IV)-Mo(VI) colorimetric solution is: 600mM Na2MoO4 and 50mMNa2SiO3, and the pH is adjusted to 4.5-5.5. The colorimetric solution should be clear and transparent before use. If it is turbid, it should be re-prepared.

[0050] The detection methods involved in the following embodiments are as follows:

[0051] Detection method of D-psicose 3-epimerase activity:

[0052] Enzyme reaction system: 50 g / L D-fructose (50 mM Tris HCl buffer, pH 7.5, containing 1 mM Co 2+ ) as substrate, add appropriate concentration of DAEase wild-type enzyme or mutant enzyme. The total volume of enzyme reaction is 1 mL.

[0053] Enzyme activity definition (U): The amount of enzyme required to catalyze the synthesis of 1 μmol of D-psicose per minute.

[0054] Specific enzyme activity detection method: react at 60°C for 5 minutes and boil for 10 minutes to terminate the enzyme reaction. The content of the product D-psicose was determined by HPLC to calculate the specific enzyme activity of the DAEase wild-type enzyme or mutant enzyme. The results are shown in Table 1.

[0055] Residual enzyme activity detection method: Place the wild-type DAEase enzyme or mutant enzyme in a 60°C water bath for 60 minutes, then add it to the above enzyme assay reaction system to determine the enzyme activity. The activity of each recombinant enzyme that has not been heat treated is defined as 100%, and the residual enzyme activity after heat treatment is calculated.

[0056] Example 1: Preparation of D-psicose 3-epimerase mutant

[0057] The specific steps are as follows:

[0058] 1. Establishment of a high-throughput screening method for D-psicose 3-epimerase mutant library

[0059] In the acidic Si(IV)–Mo(VI) colorimetric solution, D-fructose can reduce yellow molybdate Mo(VI) to generate blue molybdate Mo(V / VI), the color depth of which is proportional to the content of D-fructose within a certain range. At the same concentration level, D-psicose will not interfere with the determination of D-fructose.

[0060] (1) Prepare a series of D-fructose and D-psicose standard solutions with different concentrations (concentrations range from 0 to 20 mM, specifically 0, 2, 4, 6, 8, 10, 12, 16, and 20 mM). Take 40 μL of D-fructose or D-psicose standard solution, add 160 μL of freshly prepared Si(IV)–Mo(VI) colorimetric solution, mix, heat at 70°C for 30 min, and place at room temperature for 10 min after heating. Then, use the reaction solution of pure water and colorimetric solution as a blank control, and measure its absorbance (OD) at a wavelength of 750 nm using an enzyme marker. 750 ).

[0061] (2) The concentration of D-fructose or D-psicose (mM) was used as the horizontal axis and the OD 750 Draw a standard curve with y as the ordinate. The standard curve of the reaction between D-fructose and D-psicose and the color developing solution is shown in Figure 1 After linear fitting, the calculation formula of D-fructose was obtained as y=0.0374x-0.0059, R 2 It is 0.9987, indicating that the colorimetric method can be used as a detection method for high-throughput screening.

[0062] 2. Construction of random mutation library of D-psicose 3-epimerase

[0063] The specific steps are as follows:

[0064] (1) Construction of pET28a-Ptac-Cred

[0065] Using plasmid pAGM22082_cRed (Addgene #117225) as template and T7_tac_F / T7_tac_R as primers, pET28a-Ptac-CRed was constructed. The details are as follows:

[0066] ① Primer design: The primers used are as follows:

[0067] T7_tac_F: 5'-ATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAA-3';

[0068] T7_tac_R: 5'-CGAGCCGATGATTAATTGTCAACCTATAGTGAGTCGTATTAATTTCGCG-3';

[0069] ②PCR amplification:

[0070] The PCR reaction system had a total volume of 20 μL and was specifically composed of: 10 μL PrimeSTAR Max Premix (2×), 10 ng template plasmid pAGM22082_cRed, and 0.2 μM each of the forward and reverse primers.

[0071] The PCR reaction program was as follows: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 15 s; annealing at 56°C for 15 s; extension at 72°C for 7 min; 30 cycles; final extension at 72°C for 10 min; and storage at 4°C.

[0072] ③PCR product nucleic acid electrophoresis verification and template digestion:

[0073] The correctness of the PCR product band size was verified by agarose gel electrophoresis. After verification, 1 μL of Q.cut Dpn I and 2 μL of Q.cut Buffer (10×) were added to the PCR product system, and the enzyme digestion reaction was performed at 37°C for 2 hours to remove the template plasmid in the system. The digestion product was then transferred into E.coli DH5α competent medium, and the plasmid pET28a-Ptac-CRed was obtained after plasmid extraction.

[0074] (2) Construction of pET28a-Ptac-CCDPE plasmid

[0075] The gene fragment encoding DAEase from Clostridium cellulolyticum was synthesized, and a 6-histidine tag was added to the C-terminus of the target gene fragment during the synthesis process. The recombinant gene fragment was then introduced into the E. coli expression vector pET28a-Ptac-CRed using the Bsa I restriction endonuclease to obtain the recombinant plasmid pET28a-Ptac-CCDPE. The synthesis of the recombinant gene fragment was commissioned to Sangon Biotech (Shanghai) Co., Ltd.

[0076] (3) Using the pET28a-Ptac-CCDPE plasmid containing the gene encoding DAEase (SEQ ID NO. 2) from Clostridium cellulolyticum as a template, a low-fidelity DNA polymerase (rTaq) was used based on the error-prone PCR method, and different concentrations of Mn 2+ and Mg 2+ Generates and regulates mutation rate.

[0077] The primers used are as follows, and the underlined part is the Bsa I restriction site:

[0078] Forward primer F: 5'-TT GGTCTC AAATGAAACATGGTATATACTACGCATATTGGG-3';

[0079] Reverse primer R: 5'-TT GGTCTC TAAGCTTAGTGGTGGTGGTGGTGGTGGGAGTGTTTAT-3';

[0080] The error-prone PCR reaction system had a total volume of 50 μL and was specifically composed of 2.5 U rTaq DNA polymerase, 50 ng plasmid template, 6 mM MgCl2, 0.1 mM MnCl2, 0.2 mM dATP, 0.2 mM dGTP, 1 mM dCTP, 1 mM dTTP, and 0.2 μM each of the forward and reverse primers.

[0081] The error-prone PCR reaction program was as follows: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 30 s; annealing at 56°C for 30 s; extension at 72°C for 60 s; 30 cycles; final extension at 72°C for 10 min; and storage at 4°C.

[0082] After 1% agarose electrophoresis, the error-prone PCR products were recovered using a gel recovery kit, and then the recovered error-prone PCR products were connected to the pET28a-Ptac-CRed expression vector through the GoldenGate cloning method and transformed into Escherichia coli TOP10 competent cells to obtain a random mutation library.

[0083] 3. High-throughput screening of random mutation library of D-psicose 3-epimerase

[0084] (1) Use a sterile toothpick or a high-throughput picker to pick mutant library clones and three wild-type or DAEase clones (as controls), inoculate into 96-well deep-well plates containing 200 μL LB medium (containing 50 μg / mL Kan) per well, seal the plate, and culture overnight at 37°C, 200 rpm;

[0085] (2) Transfer 20 μL of overnight culture solution to a 96-well deep-well plate containing 1 mL of ZYM-5052 medium (containing 100 μg / mL Kan) and culture at 20°C, 200 rpm for 24 h to express the recombinant enzyme;

[0086] (3) Place the 96-well deep-well plate in a refrigerated well plate centrifuge and centrifuge at 4°C, 4000 rpm for 10 min. Discard the supernatant, resuspend the bacterial pellet in 150 μL lysis buffer (containing 1 mg / mL lysozyme and 1 μL / mL DNase I), and place in a constant temperature shaker (30°C, 200 rpm) and incubate for 1 h to lyse the cells;

[0087] (4) Centrifuge at 4000 rpm for 30 min at 4°C, take the supernatant as the crude enzyme solution, heat treat at 60°C for 10 min, and then perform enzyme reaction. After 10 min of enzyme reaction, add 4 times the volume of Si(IV)–Mo(VI) colorimetric solution, heat at 70°C for 30 min to develop color, cool to room temperature, and measure the absorbance at 750 nm.

[0088] Since the higher the DAEase activity, the lower the residual D-fructose content in the reaction, the D-fructose content in the enzyme reaction solution was obtained according to the standard curve of the reaction between D-fructose and the colorimetric solution, and then the recombinant enzyme activity was determined. The average enzyme activity of the wild-type or starting DAEase was set as 100% relative enzyme activity, and mutants with a relative enzyme activity greater than 120% were identified as positive mutants, and further rescreened by high-performance liquid chromatography analysis. The sequencing results showed that the mutants with enhanced thermal stability and enzyme activity obtained by the final rescreening with Clostridium cellulolyticum DAEase as the starting template contained four point mutations: S38F, F42N, A70P, and T119P.

[0089] That is, the recombinant bacteria containing mutants were obtained: Escherichia coli TOP10 / pET28a-Ptac-S38F / F42N / A70P / T119P, Escherichia coli TOP10 / pET28a-Ptac-S38F, Escherichia coli TOP10 / pET28a-Ptac-F42N, Escherichia coli TOP10 / pET28a-Ptac-A70P, Escherichia coli TOP10 / pET28a-Ptac-T119P and the recombinant bacteria containing the wild type Escherichia coli TOP10 / pET28a-Ptac-CCDPE.

[0090] Example 2: Expression and purification of mutant enzymes and wild-type enzymes

[0091] The specific steps are as follows:

[0092] (1) The wild-type recombinant bacteria and mutant recombinant bacteria obtained in Example 1 were inoculated into 4 mL of LB medium containing Kan resistance (final concentration of 50 μg / mL), respectively, and cultured in a constant temperature shaking incubator at 37° C. and 200 rpm for 12-14 h to prepare seed solutions.

[0093] (2) Then, at a 2% (v / v) inoculation volume, the seed solution was transferred to 4 mL to 200 mL of LB medium containing Kan resistance (final concentration of 50 μg / mL), and cultured in a constant temperature shaking incubator at 37°C and 200 rpm for 2-3 h. During the culture period, the OD 600 Value, when OD 600When the value reached 0.6-0.8, IPTG with a final concentration of 1 mM was added as an inducer, and the cells were cultured at low temperature at 28°C and 200 rpm in a constant temperature shaking incubator for 6-8 h to induce overexpression of the recombinase.

[0094] The induced bacterial solution was centrifuged at 4°C, 8000 rpm for 10 min, the supernatant was discarded, and the bacterial precipitate was collected.

[0095] (3) Add 20 mL of Lysis buffer (100 mM NaCl, 50 mM Tris, pH 7.5) to the obtained bacterial precipitate, gently pipette and fully resuspend, and place in an ice box for fixation. Place the ice box in an ultrasonic cell disruptor for ultrasonic disruption, and set the program as follows: power 200 W, time 15 min (on 2 s, off 3 s).

[0096] The obtained cell disrupted liquid was centrifuged at 4°C and 8000 rpm for 10 min. After the centrifugation, it was filtered with a 0.45 μm water filter membrane to obtain the crude enzyme solution.

[0097] (4) Purification:

[0098] Connect the constant flow pump, Ni 2+ The pipelines between the affinity chromatography column, UV detector and other devices were set to a flow rate of 1 mL / min with a constant flow pump and tested for leaks with deionized water. After the pure enzyme system was ready, two column volumes of Binding Buffer (50 mM Tris, 500 mM NaCl, pH 7.5) were used to balance the Ni 2+ Affinity chromatography column, and then pump the crude enzyme solution into the chromatography column at a flow rate of 0.5 mL / min.

[0099] After the crude enzyme solution has completely entered the chromatography column, continue to use Binding Buffer to wash away unadsorbed proteins and other impurities. After the detector readings are stable, pump in Washing Buffer (50mM imidazole, 50mM Tris, 500mM NaCl, pH7.5) to wash away the weakly binding proteins. After the detector readings are stable again, pump in Elution Buffer (500mM imidazole, 50mM Tris, 500mM NaCl, pH 7.5) to elute the adsorbed recombinant enzyme, and collect the effluent according to the signal value of the UV detector to obtain the recombinant enzyme.

[0100] The obtained recombinant enzyme solution was transferred to a dialysis bag with a molecular weight cutoff of 10 kDa, clamped with a dialysis clip and placed in Dialysis buffer I, placed in a chromatography cabinet at 4°C, dialyzed for 18 hours, and fresh Dialysis buffer I (10mM EDTA·2Na, 50mM Tris, pH 7.5) was replaced every 6 hours to remove imidazole and other metal ions in the recombinant enzyme solution. The dialysis bag was then transferred to Dialysis buffer II (50mM Tris, pH 7.5), and dialyzed for 18 hours, and fresh Dialysis buffer II was replaced every 6 hours. After the dialysis was completed, the pure enzyme solution was obtained, collected in a 10mL EP tube, and stored in a refrigerator at 4°C for later use.

[0101] The following were prepared respectively: S38F / F42N / A70P / T119P pure enzyme solution, S38F pure enzyme solution, F42N pure enzyme solution, A70P pure enzyme solution, T119P pure enzyme solution and wild-type CCDPE pure enzyme solution.

[0102] Example 3: Comparison of specific enzyme activity and residual enzyme activity between mutant enzyme and wild-type enzyme.

[0103] The specific steps are as follows:

[0104] (1) The specific enzyme activities of S38F / F42N / A70P / T119P pure enzyme solution, S38F pure enzyme solution, F42N pure enzyme solution, A70P pure enzyme solution, T119P pure enzyme solution and wild-type CCDPE pure enzyme solution were tested respectively. The results are shown in Table 1.

[0105] (2) The residual enzyme activities of S38F / F42N / A70P / T119P pure enzyme solution, S38F pure enzyme solution, F42N pure enzyme solution, A70P pure enzyme solution, T119P pure enzyme solution and wild-type CCDPE pure enzyme solution at 60°C were detected respectively;

[0106] Detection method: The wild-type DAEase enzyme or mutant enzyme was placed in a 60°C water bath for 60 minutes, and then added to the above-mentioned enzyme assay reaction system to determine the enzyme activity. The activity of each recombinant enzyme that was not heat-treated was defined as 100%, and the residual enzyme activity after heat treatment was calculated. The results are shown in Table 1.

[0107] Table 1: Comparison of specific and residual enzyme activities of mutant enzymes and wild-type enzymes

[0108]

[0109]

[0110] The results showed that the specific enzyme activity and residual enzyme activity of the four single-point mutants and quadruple-point mutants were all improved compared with the wild-type enzyme. Among them, the residual enzyme activity of the quadruple-point mutant S38F / F42N / A70P / T119P was 69.8% higher than that of the wild-type enzyme, indicating that its thermal stability was significantly improved.

[0111] Example 4: Comparison of thermal stability between the four-point mutant enzyme and the wild-type enzyme.

[0112] The optimum temperature, half inactivation temperature (T 50 5 ) and half-life (t 1 / 2 ) and other indicators to compare the thermal stability of wild-type enzymes and mutant enzymes. The specific steps are as follows:

[0113] 1. Comparison of optimum temperature

[0114] The wild-type CCDPE pure enzyme and the mutant enzyme S38F / F42N / A70P / T119P pure enzyme in Example 2 were subjected to enzyme reaction in a temperature range of 50-80°C (a temperature point was set every 5°C).

[0115] Except for changing the reaction temperature, other reaction and detection conditions are the same as the comparative enzyme activity detection method in Example 3. The enzyme activity at the optimal reaction temperature is set as 100% relative enzyme activity to calculate the relative enzyme activity at other temperatures. The optimal reaction temperature is the temperature corresponding to the highest enzyme activity.

[0116] The results are as follows Figure 2 As shown, the optimum temperature of the quadruple mutant S38F / F42N / A70P / T119P (MT4) increased to 70°C, which was 5°C higher than that of the wild-type enzyme.

[0117] 2. Comparison of half-inactivation temperatures

[0118] The wild-type CCDPE pure enzyme and the mutant enzyme S38F / F42N / A70P / T119P pure enzyme in Example 2 were incubated at a temperature range of 45-70° C. for 5 min and then the enzyme activity was measured.

[0119] The initial enzyme activity without heat treatment was set as 100% relative enzyme activity to calculate the residual enzyme activity of the wild-type enzyme and mutant enzyme after 5 min of heat treatment at various temperatures.

[0120] The Boltzmann function was used for nonlinear fitting to obtain the temperature corresponding to when the residual enzyme activity decreased to half of the initial enzyme activity, which was the half-inactivation temperature (T 50 5 )value.

[0121] The results are as follows Figure 3As shown, the half-inactivation temperature (T 50 5 ) value increased from 54.76℃ to 60.96℃, which was 6.2℃ higher than that of the wild-type enzyme.

[0122] 3. Half-life comparison

[0123] The wild-type CCDPE pure enzyme and the mutant enzyme S38F / F42N / A70P / T119P pure enzyme in Example 2 were placed in a 60° C. water bath for constant temperature incubation, and the enzyme activities were measured at regular intervals.

[0124] The initial enzyme activity without heat treatment was set as 100% relative enzyme activity to calculate the residual enzyme activity of the wild-type enzyme and mutant enzyme after different heat treatment times.

[0125] The heat treatment time (h) was used as the horizontal axis and the logarithmic value of the residual enzyme activity of the wild-type enzyme and the mutant enzyme was used as the vertical axis to plot. After linear fitting, the inactivation rate constant (k d ) value. Then calculate the half-life (t 1 / 2 )value:

[0126] t 1 / 2 =Ln2 / k d .

[0127] The results are as follows Figure 4 As shown, the half-life (t 1 / 2 ) value increased from 0.64h to 7.30h, which is 11.4 times that of the wild-type enzyme.

[0128] In summary, the thermal stability of the four-point mutant S38F / F42N / A70P / T119P (MT4) was significantly enhanced, and it has stronger industrial application prospects compared with the wild-type enzyme.

[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 D-psicose 3-epimerase mutant is obtained by mutating the 38th serine of the D-psicose 3-epimerase with an amino acid sequence as shown in SEQ ID NO.2 to phenylalanine, the 42nd phenylalanine to asparagine, the 70th alanine to proline, and the 119th threonine to proline.

2. A gene encoding the D-psicose 3-epimerase mutant according to claim 1.

3. A recombinant vector carrying the gene according to claim 2.

4. The recombinant vector according to claim 3, characterized in that The recombinant vector uses pET28a plasmid as an expression vector.

5. A recombinant cell expressing the D-psicose 3-epimerase mutant of claim 1, or carrying the gene of claim 2, or carrying the recombinant vector of claim 3 or 4.

6. The recombinant cell according to claim 5, characterized in that The recombinant cell uses bacteria or fungi as host cells.

7. A method for improving the thermal stability of D-psicose 3-epimerase, characterized in that: The method comprises the following steps: mutating the serine at position 38 of the D-psicose 3-epimerase whose amino acid sequence is shown in SEQ ID NO. 2 to phenylalanine, mutating the phenylalanine at position 42 to asparagine, mutating the alanine at position 70 to proline, and mutating the threonine at position 119 to proline.

8. A method for preparing D-psicose, characterized in that: The method comprises the following steps: using the mutant according to claim 1 or the recombinant cell according to claim 5 or 6, and using D-fructose as a substrate to react and prepare D-psicose.

9. Use of the mutant according to claim 1 or the recombinant cell according to claim 5 or 6 in preparing D-psicose or a product containing D-psicose.

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