D-psicose 3-epimerase mutant, method for producing same, and application

Abstract

Provided are D-psicose 3-epimerase mutants, methods for producing the same, and applications thereof. [Solution] A D-psicose 3-epimerase mutant consisting of a specific amino acid sequence is provided. The specific enzyme activity of the D-psicose 3-epimerase mutant at 80°C and pH 6.0 increased from 10.21 U / mg of the control (before mutation) to 15.29 U / mg, and the equilibrium conversion rate when 700 g / L of fructose was used as a substrate increased from 29.98% of the control (before mutation) to 36.26%. The D-psicose 3-epimerase mutant provided by the present invention has high catalytic activity and has very broad application prospects in the highly efficient production and manufacturing of D-psicose.

Description

[Technical Field]

[0001] The present invention relates to the fields of genetic engineering and enzyme engineering, and in particular to a D-psicose 3-epimerase mutant, a production method thereof, and applications thereof. [Background technology]

[0002] D-psicose is a low-calorie, rare ketohexose with 70% of the sweetness of sucrose but only 10% of the calories, making it an ideal low-energy sweetener substitute. Furthermore, D-psicose possesses a variety of beneficial physiological activities, including improving glucose tolerance, reducing fat accumulation, scavenging reactive oxygen species, and protecting neuronal function. These beneficial functions make D-psicose a natural functional ingredient with broad application prospects in food processing and medical health.

[0003] The amount of D-psicose found in nature is extremely low, making direct extraction expensive. While it is possible to obtain D-psicose through chemical synthesis, it has many drawbacks, including the high cost of the substrate, the presence of by-products, and environmental pollution. Compared to chemical synthesis, enzymatic biosynthesis of D-psicose boasts high synthetic efficiency, strong substrate specificity, mild reaction conditions, and no environmental pollution. Enzymatic biosynthesis of D-psicose primarily uses D-psicose 3-epimerase to catalyze the conversion of D-fructose to D-psicose. Further improvement of the catalytic activity of D-psicose 3-epimerase is currently a research focus for large-scale bioenzymatic synthesis of D-psicose. Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention aims to provide a D-psicose 3-epimerase mutant, a method for producing the same, and applications thereof, in order to solve the above-mentioned problems of the prior art. The D-psicose 3-epimerase mutant of the present invention has high catalytic activity and has very broad application prospects in the highly efficient production and manufacturing of D-psicose. [Means for solving the problem]

[0005] To achieve the above object, the present invention provides the following scheme.

[0006] The present invention provides a D-psicose 3-epimerase mutant, the amino acid sequence of which is shown in SEQ ID NO. 1.

[0007] The present invention also provides genes encoding the above-mentioned D-psicose 3-epimerase mutants.

[0008] Furthermore, the nucleotide sequence of said gene is shown in SEQ ID NO.2.

[0009] The present invention also provides a recombinant expression vector containing the above-described coding gene.

[0010] The present invention also provides a recombinant microbial strain comprising the above-described recombinant expression vector.

[0011] The present invention also provides the use of the above-mentioned encoding gene, recombinant expression vector, or recombinant microbial strain in producing a D-psicose 3-epimerase mutant.

[0012] The present invention also provides a method for producing the above-mentioned D-psicose 3-epimerase mutant, which includes the steps of inducing and expressing the above-mentioned recombinant microbial strain and then extracting and purifying the D-psicose 3-epimerase mutant.

[0013] The present invention also provides the use of the above-mentioned D-psicose 3-epimerase mutant in the production of D-psicose.

[0014] The present invention also provides a method for producing D-psicose, which includes a step of catalyzing D-fructose with the above-mentioned D-psicose 3-epimerase mutant to produce D-psicose.

[0015] The present invention also provides a method for improving the catalytic activity of D-psicose 3-epimerase, the amino acid sequence of which is set forth in SEQ ID NO. 3, The method includes the steps of mutating the amino acid at position 66 of the D-psicose 3-epimerase to glycine and the amino acid at position 108 to alanine. [Effects of the Invention]

[0016] The present invention discloses the following technical effects.

[0017] In the present invention, single-site fixed-point saturation mutations were performed on amino acid residues involved in binding of the substrate D-fructose in D-psicose 3-epimerase TaDAEase derived from a thermophilic archaeon of the Thermofilaceae family, and the enzymatic activity of each mutant was measured to obtain unit point mutants C66G and I108A with improved enzymatic activity.These unit point mutations were then combined to obtain the D-psicose 3-epimerase mutant C66G / I108A with further improved enzymatic activity.

[0018] The specific enzyme activity of the D-psicose 3-epimerase mutant C66G / I108A provided by the present invention at 80°C and pH 6.0 increased from 10.21 U / mg of the control (before mutation) to 15.29 U / mg, and the equilibrium conversion rate when 700 g / L of fructose was used as a substrate increased from 29.98% of the control (before mutation) to 36.26%. The D-psicose 3-epimerase mutant C66G / I108A provided by the present invention has very broad application prospects in the highly efficient production and manufacturing of D-psicose. [Brief explanation of the drawings]

[0019] In order to more clearly describe the embodiments of the present invention or the technical scheme in the prior art, the following briefly describes the drawings that need to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without paying creative labor. [Figure 1] FIG. 1 shows an SDS-PAGE detection image of D-psicose 3-epimerase TaDAEase and mutant C66G / I108A. [Figure 2] FIG. 1 shows HPLC detection of a standard and a D-psicose 3-epimerase TaDAEase enzyme reaction product. [Figure 3] FIG. 1 shows HPLC detection results of the enzyme reaction products of a standard and the D-psicose 3-epimerase mutant C66G / I108A. [Figure 4] FIG. 1 shows the equilibrium conversion rates at which D-psicose 3-epimerase TaDAEase and mutant C66G / I108A catalyze the synthesis of D-fructose. DETAILED DESCRIPTION OF THE INVENTION

[0020] Various exemplary embodiments of the present invention are described in detail below, which should not be construed as limiting the present invention, but should be understood as providing a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terms used herein are for the purpose of describing particular embodiments and are not intended to limit the invention. Furthermore, with respect to numerical ranges herein, it is understood that every intermediate value between the upper and lower limits of that range is also specifically disclosed. Any stated value or intermediate value within a range, as well as smaller ranges between other stated values ​​or intermediate values ​​within that range, are also encompassed by the invention. The upper and lower limits of these smaller ranges may independently be included or excluded within the range.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Only preferred methods and materials are described in the present invention; however, any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials related to the publication. In the event of a conflict between an incorporated publication and the contents of this specification, the contents of this specification shall control.

[0023] Numerous modifications and variations can be made to the specific embodiments of the present specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments will also be apparent to those skilled in the art from the present specification. The present specification and examples are merely illustrative.

[0024] As used herein, the words "including," "including," "having," "containing," and the like are all open-ended terms, i.e., meaning "without being limited to."

[0025] The experimental materials used in the following examples are as follows:

[0026] 1. Strains and Vectors

[0027] Escherichia coli JM109, Escherichia coli BL21-CodonPlus(DE3)-RIL, and the E. coli expression vector pET28a were purchased from Shanghai Biotechnology Co., Ltd.

[0028] 2. Enzymes and other biochemical reagents

[0029] KOD DNA polymerase and KOD-Plus-neo DNA polymerase were from Toyobo, DNA restriction enzymes and T4 DNA ligase were from Fermentase, DNA gel recovery kit and plasmid extraction kit EZNA were from Omega Bio-tek, and QuickMutation TM The gene mutation reagent kit was from Shanghai Biyuntian Biotechnology Co., Ltd., and Chelating Sepharose TM Fast Flow was purchased from GE Healthcare, USA, and the Bradford protein concentration measurement reagent kit was purchased from Shanghai Synchro Bio-Engineering Co., Ltd. Gene synthesis was completed by Shanghai Boyi Bio-Technology Co., Ltd. Polymerase chain reaction primer synthesis and sequencing were completed by Shanghai Synchro Bio-Engineering Co., Ltd. All other chemical reagents were domestically produced or imported analytical pure products.

[0030] 3. Culture medium

[0031] LB medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0. LB medium containing 50 μg / mL kanamycin was used as the screening medium.

[0032] The molecular cloning techniques and protein detection techniques used in the present invention are all conventional techniques in the art. Techniques not described in detail in the following examples are performed according to the relevant parts of the following laboratory manual: Green MR, Sambrook J. Molecular cloning: a laboratory manual [M]. New York: Cold Spring Harbor Laboratory Press, 2012.

[0033] 4. Amino acid and nucleotide sequences

[0034] The amino acid sequence of the mutant C66G / I108A is shown in SEQ ID NO. 1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO. 2. The amino acid sequence of D-psicose 3-epimerase TaDAEase derived from a thermophilic archaeon of the Thermofilaceae family is shown in SEQ ID NO. 3, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO. 4. The gene sequence of ribitol dehydrogenase KpRD derived from Klebsiella pneumoniae MGH 48 is shown in SEQ ID NO. 5. SEQ ID NO.1: MNKIGIYYAYWEHNWAADLLSYPQRVARLGFEILEIKLSVVLAMTERQRRKLKHEAQAHGIGLTFGEALDSQIDISSPRPATRKRGIEYLKRGLDTVHKMGGYLLGGALYGAWNLPAVEGMHKAERLMWSVESMRHVLKTAEDVG VICAIEPVNRFEHFMLNTCAEALEYIKMVESPNLGILLDTFHMNIEEDDIYKAIVSAGKNLVHMHVGEPNRKLPGQGRFPWQELLRALRFINYEGAIVMEPFVQVGGEIGLDIKVWRDLARGQDLDEAAQQSLRFLRALLKLSEF. SEQ ID NO.2: . SEQ ID NO. 3: MNKIGIYYAYWEHNWAADLLSYPQRVARLGFEILEIKLSVVLAMTERQRRKLKHEAQAHGIGLTFCEALDSQIDISSPRPATRKRGIEYLKRGLDTVHKMGGYLLGGILYGAWNLPAVEGMHKAERLMWSVESMRHVLKTAEDVGVICAIEPVNRFEHFMLNTCAEALEYIKMVESPNLGILLDTFHMNIEEDDIYKAIVSAGKNLVHMHVGEPNRKLPGQGRFPWQELLRALRFINYEG AIVMEPFVQVGGEIGLDIKVWRDLARGQDLDEAAQQSLRFLRALLKLSEF. SEQ ID NO.4: . SEQ ID NO. 5: ATGAAGCACTCTGTCTCCTCTATGAATACTTCCCTCAGCGGTAAAGTCGCCGCGATCACCGGCGCGGCGTCCGGTATCGGCCTCGAGTGCGCGAGGACCCTGCTGGGAGCTGGCGCAAAAGTGGTACTGATCGACCGCGAAGGCGAAAAGCTCAACAAACTGGTCGCCGAACTTGGCGAAAACGCCT TCGCCCTGCAGGTCGACCTGATGCAGGCGGACCAGGTCGATAACCTACTGCAGGGCATTTTGCAGCTTACCGGGCGTCTCGATATCTTCCACGCCAACGCCGGCGCCTATATCGGCGGGCCGGTGGCCGAGGGCGACCCGGACGTCTGGGACCGCGTGCTGCACCTCAATATCAACGCCGCCTTCCGC TGCGTGCGCAGCGTCCTGCCGCATCTGATCGCCAAAAATCCGGGATATTATCTTCACCAGCTCGATCGCGGGCGTGGTGCCGGTGATCTGGGAGCCTGTCTATACCGCGTCGAAATTCGCGGTGCAGGCATTTGTGCACACCACTCGTCGCCAGGTGGCGCAGTATGGCGTGCGCGTCGGCGCCG TACTGCCGGGCCCGGTGGTTACCGCTCTGCTGGACGACTGGCCAAAAGCCAAAATGGACGAAGCGCTGGCCAACGGCAGCCTGATGCAGCCGATTGAAGTGGCGGAGTCGGTGCTGTTTATGGTGACGCGTTCGAAAAACGTCACCGTACGCGACATTGTGATCCTGCCGAACAGCGTGGATCTCTGA

[0035] Example 1 Construction and screening of D-psicose 3-epimerase unit point mutants

[0036] 1) Gene synthesis

[0037] Based on the gene sequence of D-psicose 3-epimerase TaDAEase derived from thermophilic archaea of ​​the Thermofilaceae family, Shanghai Boyi Biotechnology Co., Ltd. was commissioned to synthesize the entire gene for D-psicose 3-epimerase TaDAEase, as shown in SEQ ID NO. 4, and the TaDAEase gene was obtained.

[0038] Based on the gene sequence of ribitol dehydrogenase KpRD derived from Klebsiella pneumoniae MGH 48, Shanghai Boyi Biotechnology Co., Ltd. was commissioned to synthesize the entire KpRD gene, as shown in SEQ ID NO. 5, to obtain the kprd gene.

[0039] 2) Construction of expression vectors

[0040] Based on the gene sequence of the D-psicose 3-epimerase TaDAEase, PCR primers F1 and R1 (Table 1) were designed. PCR amplification was performed using the synthetic gene TaDAEase as a template and F1 and R1 as primers. The PCR amplification conditions were as follows: 98°C for 5 minutes; 98°C for 20 seconds, 58°C for 40 seconds, 74°C for 1 minute, 30 cycles; 74°C for 10 minutes. The amplified product was double-digested with BamH I and Xho I and ligated into the vector pET28a to construct the recombinant plasmid pET28a-tadaease.

[0041] Based on the gene sequence of ribitol dehydrogenase KpRD, PCR primers F2 and R2 (Table 1) were designed. PCR amplification was performed using the synthetic gene kprd as a template and F2 and R2 as primers. The PCR amplification conditions were as follows: 98°C for 5 minutes; 30 cycles of 98°C for 20 seconds, 56°C for 40 seconds, and 74°C for 40 seconds; and 74°C for 10 minutes. The amplified product was double-digested with NcoI and NotI and ligated into the vector pET28a to construct the recombinant plasmid pET28a-kprd.

[0042] [Table 1]

[0043] 3) Construction of D-psicose 3-epimerase TaDAEase mutants Mutation site selection: Using the tertiary structure of D-psicose 3-epimerase derived from Agrobacterium tumefaciens (PDB ID: 2HK1) as a template, the protein molecular structure of the D-psicose 3-epimerase TaDAEase was three-dimensionally modeled using Swiss-Model (http: / / swissmodel.expasy.org) to obtain the protein molecular structure of TaDAEase. TaDAEase was docked with the substrate D-fructose using AutoDock 4.2 software to determine the amino acid residues involved in binding to the substrate D-fructose in TaDAEase. From these, non-conserved amino acid residues Y7, C66, and I108 were selected and subjected to fixed-site saturation mutagenesis to construct saturation mutants.

[0044] Construction of fixed-point saturation mutants: QuickMutation TM According to the instructions of the gene fixed-point mutation reagent kit, primers were designed by combining the base sequence of the TaDAEase gene and the amino acid site of the quasi-mutation, as shown in Table 2. The construction procedure for the saturated mutant was as follows: (1) Taking the construction of mutant Y7G as an example, the recombinant plasmid pET28a-tadaease was used as a template, and primers Y7G-F and Y7G-R were used to perform QuickMutation. TM PCR amplification was performed according to the instructions for the gene target mutation reagent kit. The amplified product was treated with Dpn I enzyme and then transformed into E. coli JM109. Transformants were screened on kanamycin-resistant plates to extract the recombinant plasmid. The recombinant plasmid was sent to Shanghai Bioengineering Co., Ltd. for sequencing and comparison with the corresponding gene sequence to confirm successful construction of the recombinant plasmid pET28a-tadaeaseY7G. The construction of all target mutations listed in Table 2 was performed in accordance with the construction method for mutant Y7G.

[0045] [Table 2] TIFF0007805512000004.tif246168TIFF0007805512000005.tif244168TIFF0007805512000006.tif245168TIFF0007805512 000007.tif244168TIFF0007805512000008.tif246168TIFF0007805512000009.tif245168TIFF0007805512000010.tif44168

[0046] 4) Screening of D-psicose 3-epimerase TaDAEase mutants

[0047] Mutant expression: Each mutant expression plasmid was transformed into E. coli BL21-CodonPlus(DE3)-RIL. Mutant monoclonals were picked from freshly transformed plates and inoculated into 48-well microplates containing 300 μL of LB medium (50 μg / mL kanamycin) per well. The microplates were then cultured at 37°C and 200 rpm for 12 hours with shaking to obtain inoculum. The inoculum was then inoculated into a new 48-well microplate containing 300 μL of LB medium (50 μg / mL kanamycin) at a 1% inoculum volume. After 3 hours of culture with shaking at 37°C and 200 rpm, IPTG was added to each well to a final concentration of 0.25 mmol / L, and the culture was continued at 16°C and 200 rpm for 18 hours with shaking. After incubation, the microplate was centrifuged at 4000 rpm for 15 minutes to recover the bacterial cells. 300 μL of 50 mmol / L MES buffer (pH 6.0) was added to each well to resuspend the bacterial cells. 20 μL of 20 mg / mL lysozyme was then added to each well. The microplate was then incubated at 37°C for 2 hours at 200 rpm to lyse the cells and release the recombinant enzyme. After incubation, the microplate was centrifuged at 4000 rpm for 15 minutes, and the supernatant was used as the crude enzyme solution.

[0048] Expression of ribitol dehydrogenase KpRD: The expression vector pET28a-kprd for ribitol dehydrogenase KpRD was transformed into E. coli BL21-CodonPlus(DE3)-RIL and plated on LB solid medium containing 50 μg / mL kanamycin. A single colony was picked from the transformed plate and inoculated into 10 mL of LB liquid medium containing 50 μg / mL kanamycin. The resulting culture was incubated at 37°C for 12 hours with shaking at 200 r / min. The bacterial suspension after activation culture was inoculated into 100 mL of LB liquid medium containing 50 μg / mL kanamycin at a 1% inoculum size and incubated at 37°C for 12 hours with shaking at 200 r / min until the OD of the bacterial suspension reached 100 mL. 600nm The culture was incubated with shaking until the pH reached 0.6, IPTG was added to a final concentration of 0.5 mmol / L, and then the culture was continued with shaking at 30°C and 200 rpm for 6 hours. After expression was complete, the bacterial solution was centrifuged at 4°C and 4000 rpm for 10 minutes, and the bacterial cell pellet was collected and washed with 50 mmol / L MES buffer (pH 6.0). The collected bacterial cells were resuspended in 50 mmol / L MES buffer (pH 6.0) and disrupted by sonication on ice. The ultrasonic cell disrupter parameters were as follows: ultrasonic output 25%, sonication time 3 seconds, and interval 6 seconds. The bacterial cell suspension was sonicated until a homogenous solution was obtained. The disrupted cell solution was centrifuged at 8000 rpm for 10 minutes at 4°C, and the supernatant was collected. Impurities were removed using a 0.45 μm aqueous filter membrane to obtain a crude enzyme solution. 2+ The target protein in the crude enzyme solution was purified using an affinity chromatography column and eluted with 200 mM imidazole elution buffer to obtain purified KpRD enzyme solution. The purified KpRD enzyme solution was dialyzed against 50 mmol / L MES buffer (pH 6.0) as the dialysate, and the dialyzed KpRD enzyme solution was diluted to 0.5 mg / mL with 50 mmol / L MES buffer (pH 6.0) and quantified.

[0049] Mutant screening: A 96-well microplate was prepared, and 20 μL of mutant crude enzyme solution and 100 μL of D-fructose at a mass concentration of 24 g / L were added to each well, and CoCl2 solution was added to a final concentration of 1 mmol / L. The microplate was reacted at 80°C for 30 minutes. Next, 120 μL of KpRD enzyme solution at a mass concentration of 0.5 mg / mL was immediately added to each well, and NADH was added to a final concentration of 5 mmol / L. The microplate was placed at 30°C, and the time-dependent change in absorbance at 340 nm of the reaction system at 0 and 30 minutes was monitored. The change in absorbance at 340 nm at 0 and 30 minutes of the reaction system over time was positively correlated with the amount of D-psicose produced in the reaction system, and △A 340nm The larger the value, the higher the amount of D-psicose produced and the higher the enzyme activity of the mutant.

[0050] △A at 0 and 30 minutes for the reaction system corresponding to the D-psicose 3-epimerase mutant 340nm The results of the measurement of the values ​​are shown in Table 3. Table 3 shows that the enzyme activities of the fixed-point mutants associated with tyrosine at position 7 (Y7) of D-psicose 3-epimerase TaDAEase were all lower than that of TaDAEase, the enzyme activity of fixed-point mutant C66G associated with cysteine ​​at position 66 (C66) of TaDAEase was higher than that of TaDAEase, and the enzyme activities of fixed-point mutants I108A, I108P, and I108F associated with isoleucine at position 108 (I108) of TaDAEase were higher than that of TaDAEase, with mutant I108A in particular having the highest enzyme activity.

[0051] [Table 3]

[0052] Example 2 Construction of mutant C66G / I108A and enzyme activity measurement

[0053] 1) Construction of the mutant C66G / I108A

[0054] The recombinant plasmid pET28a-tadaeaseC66G was used as a template, and primers I108A-F and I108A-R were used to perform QuickMutation. TM PCR amplification was performed according to the instructions in the gene target mutation reagent kit. The amplified product was treated with Dpn I enzyme and transformed into E. coli JM109. Transformants were screened on kanamycin-resistant plates to extract the recombinant plasmid. The recombinant plasmid was sent to Shanghai Bioengineering Co., Ltd. for sequencing and comparison with the corresponding gene sequence to confirm the successful construction of the recombinant plasmid pET28a-tadaeaseC66G / I108A.

[0055] 2) Expression and purification of D-psicose 3-epimerase TaDAEase and its mutant C66G / I108A

[0056] The recombinant plasmids pET28a-tadaease and pET28a-tadaeaseC66G / I108A were transformed into E. coli BL21-CodonPlus(DE3)-RIL, respectively, and plated on LB solid medium containing 50 μg / mL kanamycin. A single colony was picked from the transformed plate and inoculated into 10 mL of LB liquid medium containing 50 μg / mL kanamycin, followed by shaking culture at 37°C and 200 r / min for 12 hours. After activation culture, the bacterial solution was inoculated into 100 mL of LB liquid medium containing 50 μg / mL kanamycin at a 1% inoculum volume and incubated at 37°C and 200 r / min until the OD of the bacterial solution reached 100 μg / mL. 600nm The culture was continued with shaking until the pH reached 0.4, IPTG was added to a final concentration of 0.25 mM, and the culture was continued with shaking at 16°C and 200 r / min for 12 hours.

[0057] After expression was complete, the bacterial solution was centrifuged at 4°C and 4000 r / min for 10 minutes, and the bacterial cell precipitate was collected and washed with 50 mmol / L MES buffer (pH 6.0). The bacterial cells collected by centrifugation were resuspended in 50 mmol / L MES buffer (pH 6.0), and the cells were disrupted using ultrasound on ice. The parameters of the ultrasonic cell disrupter were as follows: ultrasonic output 25%, ultrasonic disruption time 3 seconds, and interval 6 seconds. The bacterial cells were sonicated until the bacterial cell suspension became a homogenous solution. The disrupted cell solution was centrifuged at 4°C and 8000 r / min for 10 minutes, and the supernatant was collected. Impurities were removed using a 0.45 μm aqueous filter membrane to obtain a crude enzyme solution. 2+ The target protein in the crude enzyme solution was purified using an affinity chromatography column and eluted with 250 mM imidazole elution buffer to obtain a purified enzyme solution. The purified enzyme solution was dialyzed using 50 mmol / L MES buffer (pH 6.0) as the dialysate. The purity of the enzyme was detected by SDS-PAGE. The SDS-PAGE results for D-psicose 3-epimerase TaDAEase and the mutant C66G / I108A are shown in Figure 1.

[0058] 3) Enzyme activity measurement of D-psicose 3-epimerase TaDAEase and mutant C66G / I108A

[0059] Ten microliters of the purified enzyme solution was mixed with 990 μL of 50 mmol / L MES buffer (pH 6.0) containing 50 g / L D-fructose and 1 mmol / L CoCl2, and the mixture was incubated at 80°C for 30 minutes. The mixture was then rapidly immersed in a boiling water bath for 5 minutes to terminate the enzyme reaction. The reaction mixture was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was collected and filtered through a 0.22 μm aqueous filter membrane to remove impurities. A high-efficiency liquid sample was prepared, and the concentration of the product, D-psicose, was measured using high-performance liquid chromatography.

[0060] The chromatography column used for high-performance liquid chromatography was a Carbohydrate ES column-w (5 μm, 4.6 × 250 mm), and the detector was a 1260 Infinity evaporative light scattering detector. The conditions for high-performance liquid chromatography were as follows: 75% acetonitrile and 25% deionized water were used as the mobile phase, the flow rate was set to 1.0 mL / min, the column temperature was set to 40 °C, the carrier gas pressure was set to 30 psi, the drift tube temperature was set to 55 °C, the injection volume was 10 μL, and the run time for each sample was 22 minutes.

[0061] Definition of enzyme activity unit (U): The amount of enzyme required to convert D-fructose to produce 1 μM of D-psicose in 1 minute was defined as 1 enzyme activity unit (U).

[0062] The results of measuring the enzyme activity of D-psicose 3-epimerase TaDAEase and the mutant C66G / I108A are shown in Table 4. The enzyme activity of D-psicose 3-epimerase TaDAEase was 10.21 U / mg, and the enzyme activity of the mutant C66G / I108A was 15.29 U / mg. Compared to TaDAEase, the enzyme activity of the mutant C66G / I108A was 0.50-fold improved.

[0063] [Table 4]

[0064] Example 3 Application of D-psicose 3-epimerases TaDAEase and C66G / I108A in the production of D-psicose

[0065] A 1 mL reaction system was prepared containing the enzyme solution (the purified D-psicose 3-epimerase TaDAEase and mutant C66G / I108A enzyme solution prepared in Example 2), the substrate D-fructose, and CoCl2. The enzyme solution was added at 10 U / mL, the final D-fructose concentration was 100 g / L, the final CoCl2 concentration was 1 mmol / L, and the buffer system was 50 mmol / L MES buffer (pH 6.0). The reaction solution was reacted at 80°C for 5 hours and then immediately immersed in a boiling water bath for 5 minutes to terminate the enzyme reaction. Next, the reaction solution was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was collected and filtered through a 0.22 μm aqueous filter membrane to remove impurities. A sample for high-efficiency liquid-phase detection was prepared, and the concentration of the product D-psicose was measured using high-performance liquid chromatography. The measurement method was the same as in Example 2.

[0066] HPLC traces of the standard and the D-psicose 3-epimerase TaDAEase enzyme reaction product are shown in Figure 2, and HPLC traces of the standard and the mutant C66G / I108A enzyme reaction product are shown in Figure 3. The D-psicose concentration in the D-psicose 3-epimerase TaDAEase enzyme reaction product was 30.09 mg / mL, and the D-psicose concentration in the mutant C66G / I108A enzyme reaction product was 39.63 mg / mL. The yield of D-alloketose catalyzed by mutant C66G / I108A was higher.

[0067] Example 4 Equilibrium Conversion Rates of D-Psicose 3-Epimerase TaDAEase and Mutant C66G / I108A at High Concentrations of D-Fructose A 50 mL reaction system was prepared containing enzyme solutions (enzyme solutions of purified D-psicose 3-epimerase TaDAEase and mutant C66G / I108A prepared in Example 2), the substrate D-fructose, and CoCl2. The amount of enzyme solution added to the reaction system was 10 U / mL, the final D-fructose concentration was 700 g / L, the final CoCl2 concentration was 1 mmol / L, and the buffer system was 50 mmol / L MES buffer (pH 6.0). The reaction solution was reacted at 80°C, and samples were taken at regular intervals. The concentration of the product D-psicose was measured by high-performance liquid chromatography (measurement method was the same as in Example 2), and the equilibrium conversion rate of D-psicose was calculated.

[0068] The equilibrium conversion rate was calculated by dividing the concentration of D-psicose in the samples at different time points by the concentration of the substrate D-fructose added before the start of the reaction: equilibrium conversion rate (%) = (D-psicose concentration) / (substrate D-fructose concentration) × 100%

[0069] The results of measuring the equilibrium conversion rates of 700 g / L of D-fructose for D-psicose 3-epimerase TaDAEase and mutant C66G / I108A are shown in Figure 4. The equilibrium conversion rate of 700 g / L of D-fructose for D-psicose 3-epimerase TaDAEase was 29.98%. The equilibrium conversion rate of 700 g / L of D-fructose for mutant C66G / I108A was 36.26%, an improvement of 20.94% compared to TaDAEase.

[0070] The above-described examples are merely illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any changes or modifications made by those skilled in the art to the technical solutions of the present invention without departing from the design spirit of the present invention should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A D-psicose 3-epimerase mutant characterized in that its amino acid sequence is set forth in SEQ ID NO.

1.

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

3. 3. The coding gene according to claim 2, characterized in that the nucleotide sequence of said coding gene is shown in SEQ ID NO.

2.

4. A recombinant expression vector comprising the coding gene of claim 2.

5. A recombinant microbial strain comprising the recombinant expression vector of claim 4.

6. A method for producing the D-psicose 3-epimerase mutant according to claim 1, using the encoding gene according to claim 2 or 3, the recombinant expression vector according to claim 4, or the recombinant microbial strain according to claim 5.

7. A method for producing the D-psicose 3-epimerase mutant according to claim 1, comprising the steps of inducing, expressing, and culturing the recombinant microbial strain according to claim 5, and then extracting and purifying the D-psicose 3-epimerase mutant.

8. A method for using the D-psicose 3-epimerase mutant according to claim 1 in the production of D-psicose.

9. A method for producing D-psicose, comprising a step of catalyzing D-fructose with the D-psicose 3-epimerase mutant according to claim 1 to produce D-psicose.

10. A method for improving the catalytic activity of D-psicose 3-epimerase, wherein the amino acid sequence of the D-psicose 3-epimerase is set forth in SEQ ID NO. 3; The method for improving the catalytic activity of D-psicose 3-epimerase includes the steps of mutating the amino acid at position 66 of the D-psicose 3-epimerase to glycine and mutating the amino acid at position 108 to alanine.

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