A D-psicose-3-epimerase mutant and its application

Abstract

The present invention discloses a D-psicose-3-epimerase mutant and its application. The mutant DAEase-I66V is derived from Agrobacterium tumefaciens as shown in SEQ ID NO: 1. Agrobacterium tumefaciens The D-psicose-3-epimerase was derived by mutating isoleucine I to valine V at amino acid position 66 in its amino acid sequence. Using 400 g / L of L-fructose as a substrate, the mutant produced 88.2 g / L of D-psicose, an approximately 11% increase in activity compared to the original strain. Analysis of the mutant enzyme's activity, stability, and molecular dynamics parameters (RMSD, RMSF, and SASA) revealed improved stability and activity compared to the original strain's DAEase.

Description

Technical Field

[0001] The present invention belongs to the field of enzyme-catalyzed production of D-psicose, and particularly relates to a mutant D-psicose-3-epimerase and applications thereof. Background Art

[0002] D-allulose, the C-3 diastereomer of D-fructose, is a rare ketohexose with very low calories. Unlike traditional sweeteners, D-allulose is mostly excreted in urine or feces after ingestion, eliminating digestive burden. Its sweetness is equivalent to 70% of sucrose, with only 0.3% of its calories. It undergoes a Maillard reaction with proteins in food, and its taste and volumetric properties are similar to those of sucrose, making it an ideal substitute for sucrose in food. Allulose's structure and properties are extremely stable, maintaining its original state even in acidic or alkaline conditions. Its chemical properties are stable at room temperature and pressure. D-allulose has the functional properties of being low in calories, having a low metabolic rate, neuroprotective effects, and lowering blood sugar and lipids. The most significant effect is its blood sugar and lipid-lowering effect. D-allulose enhances energy metabolism and promotes systemic circulation, oxidizing accumulated fat after meals, thereby lowering blood sugar and lipid levels. Secondly, it prevents obesity by significantly inducing the release of glucagon-like peptide-1 receptors, activating vagal nerve afferent signals, and reducing food intake, thereby maintaining a healthy weight. Additionally, D-allulose has antioxidant and neuroprotective properties by scavenging free radicals. As a plant-based material, D-allulose can be used as an additive in waterproof and light-transmitting films for liquid crystal displays and optical devices. In August 2011, with the U.S. Food and Drug Administration (FDA) recognizing D-allulose as safe, it is now legally permitted as a component of food or a food additive in the United States, Australia, and other countries.

[0003] Currently, the main production methods of D-psicose are chemical synthesis and enzyme-catalyzed synthesis.

[0004] (1) Chemical synthesis. Traditional chemical synthesis methods for D-psicose include ring-closing conversion synthesis, alcohol-aldehyde condensation synthesis, catalytic hydrogenation, and Ferrier rearrangement. Fang Zhijie et al. used the reaction of sugar acid lactone with diiodomethane to obtain 1-deoxyiodoketose, which was then hydrolyzed under alkaline conditions to obtain a ketose intermediate. After selective protection and deprotection of the hydroxyl group, D-psicose was successfully synthesized. Although chemical synthesis methods can prepare D-psicose, these methods have problems such as poor economic efficiency, heavy environmental pollution, and insufficient stereoselectivity, making it impossible to achieve large-scale production of D-psicose.

[0005] (2) Enzyme-catalyzed synthesis. Enzymatic reaction to produce D-psicose is currently mostly done by one-step production of D-psicose using D-psicose-3-epimerase. This method is simple and does not produce any other by-products. Compared with chemical synthesis, the enzyme-catalyzed method has high regioselectivity and stereoselectivity, as well as mild reaction conditions, low cost, high economic benefits, and good environmental compatibility. The production of D-psicose from fructose using bioenzymes can greatly reduce the cost of industrial production and is in line with the current mainstream concept of green environmental protection. However, the reversible isomerization reaction from D-fructose to D-psicose has an insurmountable thermodynamic bottleneck, which limits the natural conversion rate to about 30%.

[0006] D-psicose-3-epimerase (EC 5.3.1.3, D-allulose-3-epimerase, DAEase) is a ketohexose isomerase that catalyzes the epimerization of ketohexose at the C-3 position, thereby converting economical sugar substrates into more expensive sugars. Currently, enzymatic production of D-psicose mostly involves a one-step catalysis from D-fructose via D-psicose-3-epimerase. This process is hindered by bottlenecks such as expensive substrates and low enzyme activity and stability. In 1993, Izumori et al. discovered this enzyme in Pseudomonas sp. ST-24. In addition to catalyzing the isomerization between D-fructose and D-psicose, it can also catalyze the conversion of other sugars. In 2005, Kim et al. isolated this enzyme from Agrobacterium tumefaciens. Since then, researchers have isolated DAEases from various sources, including Ruminococcus sp., Arthrobacter globifoemis, and Clostridium bolteae. With the release of more microbial genome data, there are now over 500 annotated DAEase sequences in the NCBI database, but the properties of most sequences have not been thoroughly studied. Most DAEases are weakly alkalophilic enzymes. Metal ions can stabilize the conformation of DAEase, with Mn being the most suitable ion. 2+ or Co 2+ Most enzymes have good thermal stability below 50°C.

[0007] Regarding the catalytic mechanism of DAEase, the crystal structure of A. tumefaciens DAEase (PDB: 2hk0) has been determined. These crystal structures reveal a symmetrical tetrameric structure. Topologically, each subunit follows a TIM-barrel fold composed of eight repeating β-sheets / α-helices, with the β6-α6 loop involved in catalysis. During catalysis, D-fructose enters the catalytic site in a linear form through a narrow channel and undergoes isomerization in an open-chain state. The catalytic mechanism of A. tumefaciens DAEase is known as a hydride transfer reaction. This is due to the interaction of the C-3 position of D-fructose with Glu 150 and Glu 244, and the interaction of amino acid residues in the active site with C-1, C-2, and C-3 of D-fructose, leading to deprotonation of C-3 and the formation of a cis-enedioic acid intermediate. The anionic oxygen (O-2) of this intermediate interacts with the metal ion and Arg 215 to maintain its stability. Glu 150 then directs the rearrangement of C-3 to form D-psicose. In the reverse reaction, the catalytic process begins with the removal of the proton at C-3 by Glu 150, following essentially the same stereochemical mechanism as the forward reaction. Summary of the Invention

[0008] The primary objective of the present invention is to overcome the shortcomings and deficiencies of the prior art. By targeting the low activity of D-psicose-3-epimerase, an amino acid site 66Ile was identified for mutation. Subsequently, through site-directed saturation mutagenesis of 20 common amino acids, a high-quality mutant strain, DAEase-I66V, was identified. Using 400 g / L D-fructose as a substrate, the strain achieved a D-psicose yield of 88.2 g / L, an approximately 11% increase in activity compared to the original strain, and a D-fructose conversion rate of 33%.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A D-psicose-3-epimerase mutant type DAEase-I66V is obtained by mutating the 66th amino acid in the amino acid sequence of the D-psicose-3-epimerase derived from Agrobacterium tumefaciens as shown in SEQ ID NO: 1 from isoleucine I to valine V.

[0011] The amino acid sequence of the D-psicose-3-epimerase mutant DAEase-I66V is shown in SEQ ID NO: 2.

[0012] A polynucleotide encoding the D-psicose-3-epimerase mutant DAEase-I66V, the sequence of which is shown in SEQ ID NO: 4.

[0013] A recombinant vector comprising a gene encoding the D-psicose-3-epimerase mutant DAEase-I66V.

[0014] A recombinant microorganism comprises the above-mentioned recombinant vector and is transformed to produce the D-psicose-3-epimerase mutant DAEase-I66V.

[0015] The recombinant microorganism comprises Escherichia coli or Bacillus subtilis.

[0016] A method for producing D-psicose-3-epimerase using the recombinant microorganism, comprising:

[0017] (a), cultivating the microorganism; and

[0018] (b) Recovering the D-psicose-3-epimerase mutant DAEase-I66V.

[0019] Use of the D-psicose-3-epimerase mutant DAEase-I66V or the recombinant microorganism in producing D-psicose.

[0020] D-fructose is provided, and the D-psicose-3-epimerase mutant DAEase-I66V or the recombinant microorganism is used as a catalyst to induce an enzyme catalytic reaction, and finally the product of the enzyme reaction is purified to obtain D-psicose.

[0021] The enzyme catalytic reaction system comprises: 50 mmol / L Tris-HCl buffer, 5-10 U / mL D-psicose-3-epimerase mutant DAEase-I66V, 400 g / L fructose, and a pH of 7.5-8.0.

[0022] The enzyme-catalyzed reaction temperature is 35-37° C., and the reaction time is 1-2 hours.

[0023] The present invention utilizes bioinformatics computer software to assist simulation, utilizes Autodock Vina to carry out docking simulation between substrate and enzyme molecule, and by analyzing the active pocket and protein crystal structure, thereby screening out an amino acid site 66Ile that can be used for mutation. Further, through the site-directed saturation mutagenesis of 20 common amino acids, a high-quality mutant strain DAEase-I66V was screened out. With 400g / L D-fructose as substrate, the D-psicose yield reached 88.2g / L, which was about 11% higher than the activity of the original strain. Subsequently, the various enzymatic properties of the mutant enzyme, enzyme activity, stability, and molecular dynamics parameters such as RMSD, RMSF, and SASA were explored. The results showed that its stability and activity were improved compared with the original strain DAEase.

[0024] The original D-psicose-3-epimerase is derived from Agrobacterium tumefaciens, and the gene encoding the D-psicose-3-epimerase is shown in SEQ ID NO: 1.

[0025] The site-directed mutagenesis is a technique for accurately improving the catalytic performance of an enzyme by introducing mutations at specific sites in a DNA chain. This technique is primarily based on site-directed mutagenesis mediated by a PCR (polymerase chain reaction), and site-directed saturation mutagenesis is typically used to modify sites within the binding region of an enzyme and a substrate, to improve defects such as the enzyme's substrate range, insufficient activity, and poor stability. Site-directed saturation mutagenesis primarily includes twenty common amino acids, and requires the design of mutagenic primers. Based on the preference for strain codons and the degeneracy of codons, the most suitable codons for the host strain are selected for mutagenic primer design, which can narrow the scope of the screening library and increase the efficiency of screening.

[0026] The enzymatic activity of each type of mutant enzyme in the present invention is measured by its ability to catalyze the synthesis of D-psicose. The enzyme catalytic reaction temperature is 30-35° C. and the reaction time is 1-2 hours.

[0027] The enzyme-catalyzed reaction system comprises: 50-80 mmol / L Tris-HCl buffer, 5-10 U / mL D-psicose-3-epimerase (or mutant enzyme), 400 g / L fructose, and a pH of 7.5-8.0.

[0028] The recombinant Escherichia coli is constructed by inserting D-psicose-3-epimerase and its mutant DAEase-I66V into the genome of the initial strain BL21. The promoter of the expression cassette is the T7 promoter of Escherichia coli; and the terminator is the T7 terminator of Escherichia coli.

[0029] The method for biocatalytic synthesis of D-psicose comprises culturing a recombinant Escherichia coli strain in LB medium to obtain a seed solution; transferring the seed solution to an enriched fermentation medium, and adding an inducer 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG, C9H 18 O5S), and induced the recombinant strain to produce D-psicose-3-epimerase.

[0030] The D-psicose-3-isomerase mutant DAEase-I66V was catalyzed at a temperature of 37°C, a catalytic time of 2 h, an IPTG concentration of 0.1 M, an induction temperature of 18°C, and an induction time of 12 h.

[0031] A recombinant Escherichia coli strain is constructed by inserting a D-psicose 3-epimerase mutant, DAEase-I66V, into the genome of an initial strain BL21. The D-psicose 3-epimerase mutant, DAEase-I66V, is a mutation based on D-psicose 3-epimerase from Agrobacterium tumefaciens, and its encoding gene is shown in SEQ ID NO: 4.

[0032] The recombinant Escherichia coli is used in the production of D-psicose.

[0033] Fructose is used as a substrate, and crude D-psicose-3-epimerase produced by recombinant Escherichia coli is added to carry out an enzymatic reaction to obtain D-psicose; wherein the enzyme is a mutant D-psicose-3-epimerase (D-allulose-3-epimerase, EC 5.3.1.3, DAEase).

[0034] More preferably, the conversion rate can be further increased to above 60% by adding borate. The amount of borate added is 0.01 to 1 mol / L, more preferably 0.125 mol / L.

[0035] The protein sequence of DAEase was input into AlpHaFold2 (https: / / colab.research.google.com / AlpHaFold2) for homology modeling, and a three-dimensional model of the tetrameric protein of DAEase from Agrobacterium tumefaciens coded as 2hk0 was exported; the molecular ligand file of the substrate D-fructose was found in the PDB website (PubChem (nih.gov)) and the three-dimensional model was exported.

[0036] With the help of the software Autodock Vina, the active pocket of DAEase was identified, and a substrate-protein molecular docking experiment was performed. Based on the docking results, six key amino acid sites for substrate-protein binding were identified: 66Ile, 107Ala, 150Glu, 186His, 215Arg, and 244Glu. Next, these six amino acid sites were mutated to alanine, and alanine scanning was performed. According to the catalytic mechanism of DAEase, the three key conserved amino acids required for this hydride transfer reaction are the catalytic key amino acids in the enzyme active center: 150Glu and 244Glu; the metal ion binding sites: 150Glu, 183Asp, 209His, and 244Glu; and the substrate binding sites: 6Tyr, 107Ala, 156Glu, 186His, 215Arg, and 248Phe.

[0037] After screening, of the six amino acid sites docked, only 66Ile was a non-conserved site that could be used for site-directed saturation mutagenesis. After designing mutation primers, based on PCR-mediated site-directed mutagenesis and combined with the codon preference of Escherichia coli, this experiment carried out saturation mutation of 20 common amino acids on isoleucine at position 66, and finally cloned the constructed plasmid in one step. The mutant plasmid after construction and verification was introduced into E. coli BL21 (DE3) competent cells and transformed for fermentation verification. The polyacrylamide gel electrophoresis of the final mutant strain showed that most mutant strain proteins were expressed and of the correct size. In the enzyme activity test experiment of the mutant strains, except for the valine mutant strain, the other mutant strains all had varying degrees of reduced enzyme activity. Therefore, a valine mutant strain was successfully screened as a forward mutant enzyme for the site-directed saturation mutation of Ile 66, and the enzyme activity was increased by about 11% compared to the original DAEase. The detection method was high-performance liquid chromatography.

[0038] The high performance liquid chromatography (HPLC) was a Thermo Fisher Scientific DGP-3600SDN, S / N: 8094557. The detector was a differential refractive index detector, the chromatographic column was an organic acid column (Aminex HPX-87H lonExclusion Column), the column temperature was 50°C, the mobile phase was 0.25 g / L dilute sulfuric acid, the flow rate was 0.5 ml / min, and the injection volume was 20 μL.

[0039] Beneficial effects:

[0040] The present invention uses AlpHaFold2 and other related simulation docking software to predict and analyze the protein structure of the enzyme, uses Autodock Vina to carry out docking simulation between the substrate and the enzyme molecule, and analyzes the active pocket and the protein crystal structure to screen out an amino acid site 66Ile that can be used for mutation. Later, through site-directed saturation mutagenesis, a high-quality mutant strain DAEase-I66V was screened out. With 400g / L D-fructose as a substrate, the D-psicose yield reached 88.2g / L, which was about 11% higher than the activity of the original strain. And explore its enzymatic properties and related molecular dynamics data, including but not limited to enzyme activity, stability, RMSD, RMSF, SASA and other related parameters. Its stability and enzyme activity are both higher than those of the original strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 :Single base (or continuous multiple base) mutation flow chart;

[0042] Figure 2 : Substrate-protein molecular docking and alanine scanning results;

[0043] Figure 3 :SDS protein gel electrophoresis of site-directed saturation mutagenesis;

[0044] Figure 4 : Comparison of enzyme activities between the original strain and the mutant strain of saturated mutant DAEase;

[0045] Figure 5 : Dynamic simulation results of original DAEase and mutant DAEase-I66V at 310 K. A, B, C, and D are the RMSFs of the four chains of the DAEase tetramer, respectively;

[0046] Figure 6 : Dynamic simulation results of original DAEase and mutant DAEase-I66V at 310 K. A: RMSD of the two; B: SASA of the two;

[0047] Figure 7 : The trend line of the optimal temperature enzyme activity decrease of mutant DAEase-I66V and the trend line of the temperature stability enzyme activity decrease curve of mutant DAEase-I66V;

[0048] Figure 8 : Optimal temperature curve (A) and temperature stability curve (B) of mutant DAEase-I66V;

[0049] Figure 9 : Optimal pH curve and pH stability curve of mutant DAEase-I66V;

[0050] Figure 10: Hill regression curves of Km calculation for original DAEase-2hk0 (A) and mutant DAEase-I66V (B). DETAILED DESCRIPTION

[0051] The present invention will be further described below with reference to specific embodiments.

[0052] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0053] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0054] Example 1 Computer-assisted semi-rational design of enzyme modification site-saturation mutagenesis

[0055] The protein sequence of DAEase was input into AlpHaFold2 (https: / / colab.research.google.com / AlpHaFold2) for homology modeling, and a three-dimensional model of the tetrameric protein of DAEase from Agrobacterium tumefaciens coded as 2hk0 was exported. The molecular ligand file of the substrate D-fructose was found in the PDB website (PubChem (nih.gov)) and the three-dimensional model was exported.

[0056] With the help of the software Autodock Vina, the active pocket of DAEase was identified, and a molecular docking experiment with the substrate-protein was performed. Based on the docking results, six key amino acid sites for substrate-protein binding were identified: 66Ile, 107Ala, 150Glu, 186His, 215Arg, and 244Glu. Next, these six amino acid sites were mutated to alanine, and alanine scanning was performed. According to the catalytic mechanism of DAEase, the three key conserved amino acids required for this hydride transfer reaction are the catalytic key amino acids in the enzyme active center: 150Glu and 244Glu; the metal ion binding site: 150Glu, 183Asp, 209His, 244Glu; and the substrate binding site: 6Tyr, 107Ala, 156Glu, 186His, 215Arg, and 248Phe. The homologous sequence 2hk0 of DAEase was then found on the UniProt official website for verification, and consistent results were obtained.

[0057] After screening, only 66Ile was a non-conserved site among the 6 amino acid sites docked and could be used for site-directed saturation mutagenesis. After designing the mutagenesis primers (primers are shown in Table 1), based on PCR-mediated site-directed mutagenesis and combined with the codon preference of E. coli, this experiment carried out saturation mutagenesis of 20 common amino acids at isoleucine at position 66. Finally, the plasmid constructed by one-step cloning (construction method is shown in Figure 1 The constructed and verified mutant plasmid was introduced into competent E. coli BL21 (DE3) cells and transformed for fermentation verification (using 400 g / L fructose as the substrate, Tris-HCl (pH 8.0) buffer, and reaction at 37°C for 120 min). Polyacrylamide gel electrophoresis of the final mutants showed that most mutant proteins were expressed and of the correct size. Enzyme activity tests of the mutants revealed that, with the exception of the valine mutant, all other mutants exhibited varying degrees of reduced enzyme activity (see Table 2). Consequently, a valine mutant was successfully selected as a forward mutant enzyme for site-directed saturation mutagenesis of Ile 66, with an enzyme activity increased by approximately 11% relative to the original DAEase.

[0058] Site-directed mutagenesis is a technique for introducing mutations at specific sites in the DNA chain to precisely improve the catalytic performance of an enzyme. This technique is mainly based on site-directed mutagenesis mediated by the enzyme chain reaction (PCR). Through conventional PCR technology, reverse PCR is performed to amplify the entire chain loop by designing mutations in primers (see Table 1). The specific steps are as follows: Figure 1 shown.

[0059] Primer Design for Single-Base (or Multiple-Base) Site-Directed Mutagenesis: To introduce single-base or multiple base-directed mutations within 50 bp of each other into a plasmid, simply design a pair of primers and amplify the plasmid using inverse PCR. Primers should be designed as follows: 5'-15-21 bp of reverse complementary region + at least 15 bp of non-complementary region - 3'. Amplify the target plasmid using Phanta Max Super-Fidelity DNA Polymerase. After thawing, shake each reaction set thoroughly and return to -20°C immediately after use. The reaction system is shown in Table 3. PCR reaction conditions are shown in Table 4. After the reaction, a small amount of the amplified product should be analyzed by agarose gel electrophoresis. If the target plasmid is amplified correctly, the amplified product contains the original template plasmid. To prevent false positive transformants after transformation, Dpn I digestion must be performed before recombination circularization to remove the methylated template plasmid. Add 1-2 μL of Dpn I, gently pipette to mix, then briefly centrifuge to collect the contents at the bottom of the tube. Incubate at 37°C for 1-2 hours. Then proceed directly to competent transformation. After overnight incubation, if the number of colonies on the recombination reaction plate is significantly higher than the negative control, select several single colonies and inoculate them into LB liquid medium containing appropriate antibiotics and culture overnight. Extract the plasmids for next-generation sequencing.

[0060] As shown in Table 2 and Figure 4 As shown, the fermentation verification of mutant strains of twenty common amino acids showed that, except for valine, the yield of the other strains of amino acid at position 66 showed negative growth. Therefore, valine was selected as the positive mutant strain at position 66 in the present invention.

[0061] Table 1 Primers used in this experiment

[0062]

[0063] Table 2 Comparison of enzyme activities of the original strain and mutant strains of saturated mutant DAEase

[0064]

[0065] Table 3 Mutation reaction system

[0066]

[0067] Table 4 Mutation PCR reaction conditions

[0068]

[0069] Example 2 Molecular dynamics (MD) simulation

[0070] The various properties of mutant enzymes can be characterized by many parameters such as root mean square deviation (RMSD), protein surface solvent contact area (SASA), etc.

[0071] The first is the comparative characterization of RMSD, RMSF and SASA with the original strain. According to the DAEase-WT and DAEase-I66V protein-ligand complexes, the ligand molecules were extracted from the complexes, and Acpype (https: / / www.bio2byte.be / acpype / ) was used to generate the topology file of the ligand for subsequent dynamic simulations. The simulation software used Gromacs 2022.4, the force field used amber14sb, the water molecule model was TIP3P, and the balance ions used sodium ions and chloride ions to construct the initial simulation system. The initial simulation system was subjected to two-step energy minimization. The first step used the steepest descent method (steep) for 10,000 times, and the second step used the conjugate gradient (cg) for 5,000 times. After energy optimization, a 200 ps NPT simulation was performed, and after the NPT simulation, a 100 ns finished product simulation was performed. The temperature control algorithm used was V-rescale, the pressure control algorithm used was Parrinello-rahman, the simulation step size was 2 fs, the temperature was 310 K, and the electrostatic interaction was calculated using the ionic mesh (PME) algorithm. The cutoff radius for calculating the Coulomb interaction and the van der Waals interaction was Hydrogen bonds were constrained using the LINCS algorithm, conformations were saved every 10 ps, ​​and simulation results were completed using the Gromacs embedded program.

[0072] MD simulations can be used to evaluate the stability of protein-ligand complexes. This simulation investigated the stability of protein-ligand complexes by analyzing the root mean square deviation (RMSD) and root mean square fluctuation (RMSF) of the proteins to characterize protein stability. The figure shows the temporal evolution of the RMSD values ​​of the proteins in the complex. The figure shows that the RMSD of the mutant DAEase-I66V is smaller than that of the wild-type 2hk0, and the fluctuation of the RMSD of DAEase-I66V is also relatively small, indicating that the mutant DAEase-I66V protein is more stable than the wild-type 2hk0. Using the SASA simulation website (http: / / mathbio.nimr.mrc.ac.uk / wiki / POPS), the temporal evolution of SASA values ​​of the proteins in the complex was simulated. The SASA values ​​of the mutant DAEase-I66V were found to fluctuate over time, but the SASA value of the mutant DAEase-I66V was smaller than that of the wild-type 2hk0, indicating that the mutant is more stable than the original DAEase.

[0073] Tm value and T 1 / 2 It is an important means to characterize the performance of enzymes. By setting different catalytic time and different catalytic temperatures, the HPLC method is used to quantitatively measure various data in the enzymatic reaction solution, such as Figure 7As shown, the trend line equation of the enzyme activity decrease at the optimum temperature is y = -4.1023x + 245.79. According to the enzyme reaction equation, that is, the Michaelis-Menten equation, Km is equal to the substrate concentration when the initial rate of the enzyme reaction is half of the maximum rate Vmax. Therefore, when y = 50, the result is the Tm value. According to the trend line determination, the theoretical Tm value of DAEase-I66V is 48°C. Figure 7 As shown, the trend line equation of temperature stability is y = -0.2276x + 100.59. When y = 50, the result is T 1 / 2 The value, that is, the half-life of the enzyme activity is 222min. The actual experimental results of the original strain are about 35℃ and 60-90min. Both mutants have certain improvements compared with the original DAEase.

[0074] Example 3 Optimal temperature and temperature stability, optimal pH and pH stability of mutant DAEase-I66V

[0075] The optimal temperature and thermal stability of the mutant strain DAEase-I66V were investigated. The optimal temperature of the mutant was detected by reacting at different reaction temperatures (25°C, 30°C, 37°C, 45°C, 55°C) for 60 minutes, and the catalytic activity of DAEase-I66V on D-fructose was investigated by HPLC. The temperature stability of the mutant was reflected by setting different reaction temperatures (25, 30, 35, 40, 45, 50°C) for 180 minutes and investigating the catalytic activity of DAEase-I66V on D-fructose by HPLC. The enzyme solutions were inactivated by heating at 100°C boiling water for 10 minutes. When the temperature is 37°C, the enzyme activity reaches its maximum. When the temperature is lower than 37°C or higher than 37°C, the enzyme activity is lost, which is manifested as a decrease in the production of D-psicose. Therefore, 37 degrees Celsius is selected as the optimal reaction temperature for the mutant DAEase-I66V. When the temperature is between 25 and 35°C, the enzyme activity can still reach about 80% after 120 minutes. When the temperature reaches 40°C or above, the enzyme activity after 120 minutes is already less than 50%, or even inactivated. Therefore, a temperature of around 35°C can maintain good enzyme activity stability.

[0076] To investigate the optimal pH and pH stability of the mutant DAEase-I66V, the catalytic activity of DAEase-I66V towards D-fructose was investigated by HPLC in different optimal buffers (morpholineethanesulfonic acid (MES) pH 5.5-6.5, saline NaCl pH 6.5-8.0, Tris HCl pH 8.0-9.0, and PBS pH 9.0-10.0). The enzyme solutions were inactivated by heating in boiling water at 100°C for 10 min. The enzyme activity reaches its maximum at pH 8.0. When the pH is lower than 8.0 or higher than 8.0, the enzyme activity is lost, manifested as a decrease in D-psicose production. Therefore, pH 8.0 was selected as the optimal reaction pH for the mutant DAEase-I66V. When the pH is lower than 6.5, the enzyme activity is less than 80% after 120 minutes. The enzyme activity reaches its highest at around pH 8.0. When the pH is higher than 9.0, the enzyme activity is less than 20% after 120 minutes. Therefore, DAEase-I66V can maintain good enzyme activity stability at a pH of around 8.0.

[0077] Example 4 Comparison of Kcat / Km between original DAEase and mutant DAEase-I66V

[0078] The enzyme reaction rate follows the Michaelis-Menten equation. In this experiment, as many substrate concentrations as possible were selected for enzyme activity determination. The Vmax and Km of DAEase (2hk0) and the mutant strain DAEase-I66V were calculated using the Hill nonlinear regression curve model of the Origin software. According to the conclusions given by the curve model, the Vmax of the original DAEase is approximately 8.0±0.45, and the Vmax value of DAEase-I66V is approximately 7.9±0.24. At the same time, the Km of the original DAEase is 21.95±3.5mM, and the Km of DAEase-I66V is approximately 18.3±2mM. Therefore, the Kcat / Km values ​​of the original strain and DAEase-I66V were calculated to be 7.1±1mM, respectively. -1 s -1 and 8.2 ± 0.88 mM -1 s -1 The Kcat / Km of DAEase-I66V was higher than that of the original DAEase, indicating that the catalytic efficiency of the mutant enzyme, as well as the affinity and catalytic ability of the enzyme for the substrate, were higher than those of the original strain.

[0079] Example 5 Catalytic Synthesis of D-psicose

[0080] The D-psicose-3-epimerase mutant DAEase-I66V gene sequence was amplified by reverse PCR using constructed mutant primers. The linearized band was then circularized using a one-step cloning method. Positive colonies were then plated and picked. The constructed and verified mutant plasmid was then introduced into competent E. coli BL21(DE3) cells to generate recombinant E. coli.

[0081] The recombinant Escherichia coli strain was cultured in LB medium (5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl) at 37°C for 12 h to obtain a seed solution; the seed solution was inoculated with an enriched fermentation medium (the enriched medium was still LB) (inoculation amount was 1%, 37°C), and after growing to the logarithmic growth phase, an inducer 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG, C9H 18 O5S), IPTG concentration of 0.1 M, induction temperature of 18 ° C, induction time of 12 h, the recombinant strain was induced to produce D-psicose-3-epimerase DAEase-I66V.

[0082] Fructose was used as the substrate, and crude D-psicose-3-epimerase produced by recombinant Escherichia coli was added for an enzymatic reaction. The enzyme-catalyzed reaction system was: 50 mmol / L Tris-HCl buffer, 5 U / mL D-psicose-3-epimerase mutant DAEase-I66V, 400 g / L fructose, pH 7.5-8.0, and reaction time at 37°C for 120 min. D-psicose was obtained with a D-psicose yield of 88.2 g / L and a D-fructose conversion rate of 33%.

[0083] Example 6 Catalytic Synthesis of D-psicose

[0084] Fructose was used as the substrate, and the crude D-psicose-3-epimerase mutant DAEase-I66V produced by recombinant Escherichia coli and the unmutated D-psicose-3-epimerase were added for enzyme catalysis. The enzyme catalysis reaction system was: 50mmol / L Tris-HCl buffer, 5U / mL The D-psicose-3-epimerase mutant DAEase-I66V / unmutated D-psicose-3-epimerase were reacted with 400 g / L fructose and 0.125 mol / L sodium borate at a pH of 7.5-8.0 at 37°C for 120 min to obtain D-psicose. The D-psicose yield of the D-psicose-3-epimerase mutant DAEase-I66V reached 128.2 g / L, and the D-fructose conversion rate was 62%. The D-psicose yield of the unmutated D-psicose-3-epimerase reached 79 g / L, and the D-fructose conversion rate was 22%.

[0085] Borates form complexes with fructose, interacting with enzyme systems and altering the equilibrium of reactions involving cis-diol carbohydrates based on differences in binding affinity for the sugar. Sodium tetraborate was used in this experiment. Adding 0.125 mol / L of sodium tetraborate and reacting for 2 hours under optimal conditions revealed that the mutant achieved a maximum of 128.2 g / L of D-psicose using 400 g / L of fructose as a substrate, with a fructose conversion rate of 62%.

[0086] SEQ ID NO: 1

[0087] MKHGIYYSYWEHEWSAKFGPYIEKVAKLGFDIIEVAAHHINEYSDAELATIRKSAKDNGII

[0088] LTAGIGPSKTKNLSSEDAAVRAAGKAFFERTLSNVAKLDIHTIGGALHSYWPIDYSQPVDK

[0089] AGDYARGVEGINGIADFANDLGINLCIEVLNRFENHVLNTAAEGVAFVKDVGKNNVKVM

[0090] LDTFHMNIEEDSFGDAIRTAGPLLGHFHTGESNRRVPGKGRMPWHEIGLALRDINYTGAVI

[0091] MEPFVKTGGTIGSDIKVWRDLLSGGADIAKMDEDARNALAFSRFVLGGLESGKETAAAKF

[0092] ERQHMDSSTSAA

[0093] SEQ ID NO: 2

[0094] MKHGIYYSYWEHEWSAKFGPYIEKVAKLGFDIIEVAAHHINEYSDAELATIRKSAKDNGII

[0095] LTAGVGPSKTKNLSSEDAAVRAAGKAFFERTLSNVAKLDIHTIGGALHSYWPIDYSQPVDK

[0096] AGDYARGVEGINGIADFANDLGINLCIEVLNRFENHVLNTAAEGVAFVKDVGKNNVKVM

[0097] LDTFHMNIEEDSFGDAIRTAGPLLGHFHTGESNRRVPGKGRMPWHEIGLALRDINYTGAVI

[0098] MEPFVKTGGTIGSDIKVWRDLSGGADIAKMDEDARNALAFSRFVLGGLESGKETAAAKF

[0099] ERQHMDSSTSAA

[0100] SEQ ID NO:3:

[0101] ATGAAACACGGAATATACTATTCATATTGGGAACACGAATGGTCTGCTAAGTTCGGTCC

[0102] GTATATCGAGAAAGTCGCGAAATTAGGCTTTGATATCATCGAGGTTGCCGCGCATCATAT

[0103] TAACGAGTATTCCGATGCAGAGCTGGCGACCATTCGCAAAAGTGCGAAGGACAACGG

[0104] TATCATCCTGACGGCTGGCATTGGCCCGAGCAAAACGAAAAACCTGTCGAGCGAAGA

[0105] CGCGGCAGTTCGTGCGGCGGGTAAGGCCTTCTTCGAACGCACCCTGAGCAATGTTGCC

[0106] AAGTTGGACATCCACACCATTGGCGGCGCTTTGCACAGCTACTGGCCGATTGATTACTC

[0107] CCAGCCGGTAGATAAAGCAGGCGACTACGCACGTGGTGTGGAAGGTATTAACGGCATC

[0108] GCGGACTTTGCGAATGACCTGGGCATTAACCTCTGCATTGAGGTCCTGAACCGCTTCG

[0109] AGAACCACGTTTTGAATACCGCAGCAGAGGGTGTCGCGTTCGTGAAGGACGTTGGTA

[0110] AGAACAATGTGAAGGTGATGCTGGATACCTTCCACATGAATATTGAGGAAGACAGCTT

[0111] TGGTGACGCGATCCGCACTGCCGGTCCACTGTTGGGCCATTTTCACACGGGTGAATCT

[0112] AACCGTCGTGTTCCGGGTAAAGGTCGTATGCCGTGGCATGAGATCGGCCTGGCTCTGA

[0113] GAGATATCAACTATACCGGTGCGGTGATCATGGAACCGTTTGTGAAGACCGGAGGCAC

[0114] CATCGGCTCCGATATCAAAGTGTGGCGTGACCTTAGCGGTGGCGCGGATATAGCGAAA

[0115] ATGGATGAAGACGCCCGTAATGCTCTGGCGTTTAGCCGTTTCGTTCTGGGTGGGCTCGA

[0116] GTCTGGTAAAGAAACCGCTGCTGCGAAATTTGAACGCCAGCACATGGACTCGTCTACT

[0117] AGCGCAGCTTAA

[0118] SEQ ID NO:4:

[0119] ATGAAACACGGAATATACTATTCATATTGGGAACACGAATGGTCTGCTAAGTTCGGTCC

[0120] GTATATCGAGAAAGTCGCGAAATTAGGCTTTGATATCATCGAGGTTGCCGCGCATCATAT

[0121] TAACGAGTATTCCGATGCAGAGCTGGCGACCATTCGCAAAAGTGCGAAGGACAACGG

[0122] TATCATCCTGACGGCTGGCGTTGGCCCGAGCAAAACGAAAAACCTGTCGAGCGAAGA

[0123] CGCGGCAGTTCGTGCGGCGGGTAAGGCCTTCTTCGAACGCACCCTGAGCAATGTTGCC

[0124] AAGTTGGACATCCACACCATTGGCGGCGCTTTGCACAGCTACTGGCCGATTGATTACTC

[0125] CCAGCCGGTAGATAAAGCAGGCGACTACGCACGTGGTGTGGAAGGTATTAACGGCATC

[0126] GCGGACTTTGCGAATGACCTGGGCATTAACCTCTGCATTGAGGTCCTGAACCGCTTCG

[0127] AGAACCACGTTTTGAATACCGCAGCAGAGGGTGTCGCGTTCGTGAAGGACGTTGGTA

[0128] AGAACAATGTGAAGGTGATGCTGGATACCTTCCACATGAATATTGAGGAAGACAGCTT

[0129] TGGTGACGCGATCCGCACTGCCGGTCCACTGTTGGGCCATTTTCACACGGGTGAATCT

[0130] AACCGTCGTGTTCCGGGTAAAGGTCGTATGCCGTGGCATGAGATCGGCCTGGCTCTGA

[0131] GAGATATCAACTATACCGGTGCGGTGATCATGGAACCGTTTGTGAAGACCGGAGGCAC

[0132] CATCGGCTCCGATATCAAAGTGTGGCGTGACCTTAGCGGTGGCGCGGATATAGCGAAA

[0133] ATGGATGAAGACGCCCGTAATGCTCTGGCGTTTAGCCGTTTCGTTCTGGGTGGGCTCGA

[0134] GTCTGGTAAAGAAACCGCTGCTGCGAAATTTGAACGCCAGCACATGGACTCGTCTACT

[0135] AGCGCAGCTTAA

Claims

1. A D-psicose-3-epimerase mutant, characterized in that: The mutant is derived from Agrobacterium tumefaciens as shown in SEQ ID NO: 1 Agrobacterium tumefaciens The amino acid at position 66 of the amino acid sequence of D-psicose-3-epimerase is mutated from isoleucine I to valine V. The amino acid sequence of the mutant is shown in SEQ ID NO:

2. 2 . A polynucleotide encoding the D-psicose-3-epimerase mutant according to claim 1 . 3 . A recombinant vector comprising a gene encoding the D-psicose-3-epimerase mutant of claim 1 . A recombinant microorganism comprising the recombinant vector according to claim 3, for producing the D-psicose-3-epimerase mutant according to claim 1.

5. The recombinant microorganism according to claim 4, characterized in that: The microorganism includes Escherichia coli or Bacillus subtilis.

6. A method for producing a D-psicose-3-epimerase mutant using the recombinant microorganism according to claim 4, characterized in that: The method comprises: (a) cultivating the recombinant microorganism according to claim 4; and (b) recovering the D-psicose-3-epimerase mutant.

7. Use of the D-psicose-3-epimerase mutant according to claim 1 or the recombinant microorganism according to any one of claims 4 or 5 in producing D-psicose.

8. The use according to claim 7, characterized in that The D-psicose-3-epimerase mutant according to claim 1 is provided to D-fructose to cause an enzyme-catalyzed reaction, and D-psicose, the product of the enzyme-catalyzed reaction, is purified.

9. The use according to claim 8, characterized in that The enzyme-catalyzed reaction system is: 50-80 mmol / L Tris-HCl buffer, 5-10 U / mL D-psicose-3-epimerase mutant, 400 g / L fructose, and pH 7.5-8.

0.

10. The use according to claim 8, characterized in that The enzyme-catalyzed reaction temperature is 35~37°C and the reaction time is 1~2 hours.

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