High-temperature-stability D-psicose 3-epimerase mutant enzyme and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and discloses a D-psicose 3-epimerase mutant enzyme with high temperature stability and application of the D-psicose 3-epimerase mutant enzyme. The mutant enzyme of the D-psicose 3-epimerase is obtained by replacing the 87th site of threonine in the amino acid sequence of the D-psicose 3-epimerase derived from Cabalronia insemination with isoleucine and / or replacing the 242nd site of threonine in the amino acid sequence of the D-psicose 3-epimerase with proline. The mutant enzyme of the D-psicose 3-epimerase is a mutant enzyme of the D-psicose 3-epimerase derived from Cabalronia insemination. The catalytic activity of the mutant enzyme can be improved by 100% or above compared with that of wild DAEase, meanwhile, the thermal stability is also remarkably improved, and the mutant enzyme has good industrial application potential.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a high-temperature-stability D-allulose 3-epimerase mutant enzyme and its applications. Background Technology

[0002] D-allulose is a rare sugar found in very low amounts in nature, and is the C3 epimer of D-fructose. It is considered an ideal substitute for sucrose, possessing 70% of the relative sweetness of sucrose while providing only 0.4 kcal / g, a 90% reduction compared to sucrose. D-allulose has been reported to regulate various physiological functions, such as anti-diabetic, anti-obesity, antioxidant, and anti-inflammatory effects. Currently, the safety of D-allulose has been confirmed by the U.S. Food and Drug Administration (FDA), allowing its use as a low-calorie sweetener in the food industry.

[0003] Due to its low abundance in nature and complex chemical synthesis, the bioproduction of D-allulose has become a research hotspot. Among them, D-allulose 3-epimerase (DAEase, EC 5.1.3.30) is the key enzyme in the production of D-allulose, which can directly catalyze the reversible epimerization reaction of the hydroxyl group at the C3 position of D-fructose to synthesize D-allulose.

[0004] As a key biocatalyst for D-allulose production, DAEase has become a research hotspot in recent years. Related studies mainly focus on: DAEase discovery and characterization, heterologous expression, crystal structure, molecular modification, and its application in D-allulose production. However, wild-type D-allulose 3-epimerases often suffer from low catalytic activity and poor thermal stability, failing to meet the requirements of industrial production processes. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a highly active and thermostable mutant of D-allulose 3-epimerase and its application in allulose production.

[0006] In a first aspect of the present invention, a mutant enzyme of D-allulose 3-epimerase is provided, which is obtained by replacing the 87th threonine in the amino acid sequence of D-allulose 3-epimerase derived from Cabaleronia insecticola with isoleucine and / or replacing the 242nd threonine with proline.

[0007] Furthermore, the amino acid sequence of the D-allulose 3-epimerase is shown in SEQ ID NO:1.

[0008] Furthermore, except for amino acids at positions 87 and / or 242, the mutant enzyme has amino acids that are identical or substantially identical to the sequence shown in SEQ ID NO:1; even further, "substantially identical" means that at most 50 amino acids (e.g., 1-20, 1-10, 9, 8, 7, 6, 5, 4, 3, 2, 1) are different, including amino acid substitution, deletion, or addition, and the mutant enzyme still has the activity of catalyzing the synthesis of allulose.

[0009] In some embodiments of the present invention, the mutant enzyme is obtained by replacing the threonine at position 87 of the amino acid sequence of D-allulose 3-epimerase derived from Cabaleronia insecticola with isoleucine, and its amino acid sequence is shown in SEQ ID NO:2.

[0010] In some embodiments of the present invention, the mutant enzyme is obtained by replacing the 242nd threonine in the amino acid sequence of D-allulose 3-epimerase derived from Cabaleronia insecticola with proline, and its amino acid sequence is shown in SEQ ID NO:3.

[0011] In some embodiments of the present invention, the mutant enzyme is obtained by replacing the threonine at position 87 of the amino acid sequence of D-allulose 3-epimerase derived from Cabaleronia insecticola with isoleucine and the threonine at position 242 with proline, as shown in SEQ ID NO:4.

[0012] In a second aspect of the invention, a nucleotide encoding the mutant enzyme described in the first aspect is provided.

[0013] In some embodiments of the present invention, the mutant enzyme is obtained by replacing the threonine at position 87 of the amino acid sequence of D-alulose 3-epimerase derived from Cabaleronia insecticola with isoleucine, and its encoded nucleotide sequence is shown in SEQ ID NO:6.

[0014] In some embodiments of the present invention, the mutant enzyme is obtained by replacing the 242nd threonine in the amino acid sequence of D-alulose 3-epimerase derived from Cabaleronia insecticola with proline, and its encoded nucleotide sequence is shown in SEQ ID NO:7.

[0015] In some embodiments of the present invention, the mutant enzyme is obtained by replacing the threonine at position 87 of the amino acid sequence of D-allulose 3-epimerase derived from Cabaleronia insecticola with isoleucine and the threonine at position 242 with proline, and its encoded nucleotide sequence is shown in SEQ ID NO:8.

[0016] In a third aspect of the invention, a recombinant vector is provided, which contains the nucleotides described in the second aspect.

[0017] In some embodiments of the present invention, the vector is a plasmid, such as the pET, pCW, pUC, pPIC9k, pMA5 series plasmids or combinations thereof.

[0018] In a fourth aspect of the invention, a host cell is provided, which contains the vector described in the third aspect or the nucleotides described in the second aspect integrated into its genome.

[0019] Furthermore, the host cell can be a prokaryotic cell or a eukaryotic cell; even further, the prokaryotic cell includes, for example, Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, lactic acid bacteria, etc., especially Escherichia coli; even further, the eukaryotic cell includes, for example, Pichia pastoris, Saccharomyces cerevisiae, Aspergillus niger, Streptomyces, etc., especially Pichia pastoris.

[0020] As a preferred embodiment of the host cell of the present invention, the host cell is selected from one of the following: Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Pichia pastoris, Saccharomyces cerevisiae, and lactic acid bacteria.

[0021] In some embodiments of the present invention, the host cell is Escherichia coli, such as E. coli BL21(DE3).

[0022] In a fifth aspect of the invention, a method for preparing the mutant enzyme described in the first aspect is provided, comprising the steps of culturing the host cell described in the fourth aspect; or, transforming the recombinant vector described in the third aspect into the host cell and culturing the resulting host cell; or, inserting the nucleotide into the vector described in the second aspect, transforming the resulting recombinant vector into the host cell, and culturing the resulting host cell.

[0023] In some embodiments of the present invention, the host cell is Escherichia coli, and the culture includes shaking culture in a liquid culture medium (e.g., LB medium) at 35-40°C (e.g., 35, 36, 37, 38, 39, 40°C).

[0024] Furthermore, the culture also includes a step of inducing enzyme production, for example by adding isopropyl-β-D-thiogalactoside (IPTG); and even further, the induction of enzyme production is carried out at 15-20°C (e.g., 15, 16, 17, 18, 19, 20°C).

[0025] Furthermore, the preparation method further includes the step of isolating bacterial cells from the culture medium, for example, by centrifuging the culture medium and collecting the bacterial cells. Even further, the isolation of bacterial cells is carried out at 1-10℃ (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10℃).

[0026] Furthermore, the preparation method further includes a step of isolating the mutant enzyme, for example, by disrupting the bacterial cells (e.g., by ultrasonic disruption), followed by centrifugation (e.g., through a 0.45 μm aqueous filter membrane) to remove impurities and obtain a crude enzyme solution. Even further, the isolation of the mutant enzyme is carried out at 1-10°C (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10°C).

[0027] Furthermore, the preparation method also includes a step of purifying the mutant enzyme, for example, by passing the crude enzyme solution through Ni 2+ Affinity chromatography column purification.

[0028] In a sixth aspect of the invention, an enzyme preparation is provided, comprising the mutant enzyme described in the first aspect.

[0029] Furthermore, the enzyme preparation also includes one or more of the following components: filler (such as starch), stabilizer (such as glycerol), diluent (such as lactose), anti-caking agent (such as silica), and buffer.

[0030] Furthermore, the enzyme preparation is a liquid preparation or a solid preparation (e.g., a lyophilized preparation).

[0031] In a seventh aspect of the invention, the use of the mutant enzyme described in the first aspect in the preparation of D-allulose is provided.

[0032] In an eighth aspect of the present invention, a method for preparing D-allulose is provided, comprising the following steps: contacting the mutant enzyme described in the first aspect or the enzyme preparation described in the sixth aspect with a reaction substrate to carry out a catalytic reaction.

[0033] Furthermore, the reaction substrate includes fructose; even further, the reaction substrate is selected from: crystalline fructose, fructose solution, high fructose syrup, or combinations thereof.

[0034] Furthermore, the reaction system also includes a solvent, such as a buffer solution, for example, a Glycine-NaOH buffer solution.

[0035] Furthermore, the pH of the reaction system is alkaline (e.g., 7.5, 8, 8.5, 9, 9.5, 10, 10.5), particularly 8-10 and 8.5-9.5.

[0036] Furthermore, the reaction temperature is 50-70°C (e.g., 50, 55, 56, 58, 60, 62, 64, 65, 66, 68, 70°C), particularly 60-70°C.

[0037] Furthermore, the reaction time is 0.5-10 hours (e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours), particularly 0.5-6 hours, 1-5 hours.

[0038] Furthermore, the method also includes a step of inactivating the enzyme (to terminate the reaction), for example by high-temperature treatment.

[0039] Furthermore, the method also includes the step of separating and purifying D-allulose.

[0040] In some embodiments of the present invention, the method includes the following steps: (1) The mutant enzyme described in the first aspect or the enzyme preparation described in the sixth aspect is brought into contact with the reaction substrate to carry out a catalytic reaction; (2) Inactivate the enzymes in the reaction solution (e.g., by boiling); (3) Isolate and purify D-allulose.

[0041] Through extensive analysis and experiments, this invention yields a mutant enzyme of D-allulose 3-epimerase, whose catalytic activity is more than 100% higher than that of wild-type DAEase, while its thermal stability is also significantly improved, demonstrating good potential for industrial application. Attached Figure Description

[0042] Figure 1 The image shows the predicted three-dimensional structure of the wild-type DAEase protein.

[0043] Figure 2 The image shows potential mutation sites for wild-type DAEase.

[0044] Figure 3 The image shows SDS-PAGE electrophoresis images of wild-type DAEase and mutants T87I, T242P, and T87I / T242P.

[0045] Figure 4 The figure shows the relationship between the relative residual enzyme activity of wild-type DAEase and mutant and time in the temperature stability experiment.

[0046] Figure 5The results show the equilibrium transformation rates of wild-type DAEase and mutant. Detailed Implementation

[0047] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0048] All publications, patents, and published patent specifications cited in this article are incorporated herein in their entirety through citation.

[0049] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0050] The reagents and culture media used in the following examples were prepared as follows: 50mM Glycine-NaOH buffer (pH 9.0): 3.7535g glycine, 7.741g NaOH solid, add deionized water to a final volume of 1L.

[0051] LB medium: 10g tryptone, 5g yeast extract, 10g NaCl, add deionized water to a final volume of 1L, autoclave at 121℃ for 20min.

[0052] Example 1: Bioinformatics prediction of D-allulose 3-epimerase First, the wild-type protein sequence of DAEase (derived from Cabaleronia insecticola, its amino acid sequence is shown in SEQ ID NO:1) was submitted to AlphaFold2 for protein structure prediction. The results are as follows: Figure 1 As shown. The design of enzyme structure stability requires avoiding conserved sites. To avoid the impact of mutations on protein structure, web-based tools are used to predict conserved sites in the protein structure. Software prediction generates point mutations that improve thermal stability. Amino acids with high RMSD values ​​in molecular dynamics simulations are potential mutation sites. Figure 2 Based on the amino acid sequence database, mutation points were initially screened. Subsequently, computational biology software was used to calculate the change in Gibbs free energy of the enzyme structure of point mutations. Mutation sites were screened based on the threshold of the change. Combined with the prediction results of the deep learning model, the single mutants T87I and T242P and the double mutant T87I / T242P were finally screened (their amino acid sequences are shown in SEQ ID NO:2-4, respectively).

[0053] Example 2: Construction, expression, and purification of D-allulose 3-epimerase mutant The encoding nucleotide sequences of wild-type DAEase and the DAEase mutants T87I, T242P, and T87I / T242P obtained in Example 1 are shown in SEQ ID NO:5-8, respectively. All of them were synthesized by Kangwei Century Co., Ltd. and inserted into the NdeI / EcoRI restriction sites of plasmid pET-28a(+). The resulting recombinant plasmids were then chemically transformed into E. coli BL21(DE3) competent cells to obtain the corresponding recombinant strains.

[0054] Recombinant E. coli BL21(DE3) / pET-28a-DAEase (wild-type, WT) and E. coli BL21(DE3) / pET-28a-DAEase were used. T87I (Mutant, T87I), E. coli BL21(DE3) / pET-28a-DAEase T242P (Mutant, T242P) and E. coli BL21(DE3) / pET-28a-DAEase T87I / T242P (Mutant, T87I / T242P) Single colony divided into four zones by streaking.

[0055] Single colonies were picked and inoculated into 5 mL of LB liquid medium (Kan, 50 μg / mL) and incubated overnight at 37°C and 220 rpm. The overnight culture was then transferred at a 2% inoculation rate to a 500 mL shake flask containing 100 mL of LB medium (Kan, 50 μg / mL). The culture was allowed to mature until the bacterial concentration reached OD500. 600 When the concentration reaches 0.8-1.0, add IPTG to a final concentration of 0.1 mM and induce enzyme production at 16℃ and 220 rpm for 20-24 h.

[0056] The bacterial culture was centrifuged at 8000 rpm and 4℃ for 10 min to collect the bacterial cells. The bacterial cells were resuspended in Binding Buffer (25 mM Tris-HCl, 150 mM NaCl, pH 8.0) and placed on ice for ultrasonic disruption. After disruption, the cells were centrifuged at 4℃ and 12000 rpm for 20 min. Impurities were removed by passing the solution through a 0.45 μm aqueous filter membrane to obtain the crude enzyme solution.

[0057] Through Ni 2+The specific steps for purifying proteins using affinity chromatography are as follows: ① Equilibrate the column with 20 mL Binding Buffer; ② Load crude enzyme solution onto the nickel-bound column; ③ Elute contaminating proteins sequentially with 20 mL Binding Buffer and 20 mL of imidazole solutions of different concentrations (20-60 mM); ④ Elute the target protein with 20 mL of high-concentration (250 mM) imidazole solution and collect it; ⑤ Concentrate and desalt the target protein using an ultrafiltration tube (10 kDa) at low temperature (4000 g), and determine the protein concentration using the Bradford method. SDS-PAGE electrophoresis images of D-allulose 3-epimerase and mutants T87I, T242P, and T87I / T242P are shown below. Figure 3 As shown.

[0058] Example 3: Determination of relative enzyme activity of mutants In a 100 g / L D-fructose reaction solution prepared with 500 μL pH 9.0 Glycine-NaOH buffer (containing 5 mMn), 2+ Add 0.02 mg / mL of purified enzyme, react at 65℃ for 5 min, and then boil at 100℃ for 10 min to terminate the reaction. Centrifuge, take the supernatant, dilute to a suitable concentration, filter through a 0.22 μm filter membrane, detect by HPLC, and calculate the relative enzyme activity. With the enzyme activity level of wild-type DAEase as 100%, the relative activities of each mutant are shown in Table 1 below.

[0059] Table 1 Comparison of relative enzyme activities of wild-type DAEase and mutant at 65℃

[0060] Example 4: Determination of the temperature stability of the mutant The enzyme solution was heat-treated at 65°C for different times, and then added to a 100 g / L D-fructose reaction system prepared with 500 μL of pH 9.0 Glycine-NaOH buffer (containing 5 mM Mn). 2+ The enzyme dosage was 0.02 mg / mL, and the reaction was allowed to proceed accurately for 5 min. The reaction was then terminated by boiling at 100℃ for 10 min. After centrifugation, the supernatant was diluted to a suitable concentration, filtered through a 0.22 μm filter membrane, and analyzed by HPLC to calculate the residual enzyme activity. The enzyme activity measured in the untreated enzyme solution was recorded as 100% relative enzyme activity. The relationship between the relative residual enzyme activity and time for each mutant is shown in the figure. Figure 4 As shown in Table 2, the half-life of wild-type DAEase and each mutant at this temperature was obtained from the curve.

[0061] Table 2 Thermal stability of wild-type DAEase and its mutants at 65℃

[0062] It can be seen that at 65℃, the half-lives of mutants T87I, T242P, and T87I / T242P were extended from 100.5 min in the wild type to 433.2, 385.1, and 210.0 min, respectively, representing increases of 4.31, 3.83, and 2.09 times. This better temperature stability allows the enzyme to lose less activity during prolonged reactions, which is beneficial for later industrial production.

[0063] Example 5: Determination of the equilibrium transformation rate of mutants In a 500 g / L D-fructose reaction solution (containing 5 mMn2+) prepared in pH 9.0 Glycine-NaOH buffer, 0.02 mg / mL of purified enzyme was added. The reaction was carried out at 65 °C for 3 h, and then terminated by boiling at 100 °C for 10 min. After centrifugation, the supernatant was diluted to a suitable concentration, filtered through a 0.22 μm filter membrane, and analyzed by HPLC. The conversion rate was calculated. The equilibrium conversion rate of wild-type DAEase was 28.9%, while the equilibrium conversion rates of mutants T87I, T242P, and T87I / T242P were 29.1%, 29.6%, and 31.3%, respectively. Figure 5 The yield is higher than that of the wild type, and it has great application potential.

[0064] The sequences involved in this invention are as follows: Wild-type DAEase amino acid sequence (SEQ ID NO:1): MNKVGMFYTYWSTEWLVDFPAVAKRISGLGFDMMEISLSEFHNLPDAKKRELKTVADDLGLTVMCCIGLKPEYDFASPEQSVRDAGTEYVKHLLDDCHMLGAPVFAGLTFCAWPQSPPPG MKDKRPYVERAVDSVRRVIKVAEGYGIIYALEVVNRFEQWLANDAREALAFCDAVDNPWCKVQLDTFHMNIEENSFRDAILACRGRLGHFHLGEANRLPPGEGRLPWDEIFGALKEIDYDG T IVMEPFMRPGGSVSRAVGVWRDMSNGATDEQMDERARRSLNFVRGHLA The DAEase amino acid sequence with the mutation at position 87 (SEQ ID NO:2): MNKVGMFYTYWSTEWLVDFPAVAKRISGLGFDMMEISLSEFHNLPDAKKRELKTVADDLGLTVMCCIGLKPEYDFASPEQSVRDAG IEYVKHLLDDCHMLGAPVFAGLTFCAWPQSPPPGMKDKRPYVERAVDSVRRVIKVAEGYGIIYALEVVNRFEQWLANDAREALAFCDAVDNPWCKVQLDTFHMNIEENSFRDAILACRGRLGHFHLGEANRLPPGEGRLPWDEIFGALKEIDYDG T IVMEPFMRPGGSVSRAVGVWRDMSNGATDEQMDERARRSLNFVRGHLA Amino acid sequence of DAEase with 242 - site mutation (SEQ ID NO:3): MNKVGMFYTYWSTEWLVDFPAVAKRISGLGFDMMEISLSEFHNLPDAKKRELKTVADDLGLTVMCCIGLKPEYDFASPEQSVRDAG T EYVKHLLDDCHMLGAPVFAGLTFCAWPQSPPPGMKDKRPYVERAVDSVRRVIKVAEGYGIIYALEVVNRFEQWLANDAREALAFCDAVDNPWCKVQLDTFHMNIEENSFRDAILACRGRLGHFHLGEANRLPPGEGRLPWDEIFGALKEIDYDG P IVMEPFMRPGGSVSRAVGVWRDMSNGATDEQMDERARRSLNFVRGHLA Amino acid sequence of DAEase with 87 - site and 242 - site mutations (SEQ ID NO:4): MNKVGMFYTYWSTEWLVDFPAVAKRISGLGFDMMEISLSEFHNLPDAKKRELKTVADDLGLTVMCCIGLKPEYDFASPEQSVRDAG I EYVKHLLDDCHMLGAPVFAGLTFCAWPQSPPPGMKDKRPYVERAVDSVRRVIKVAEGYGIIYALEVVNRFEQWLANDAREALAFCDAVDNPWCKVQLDTFHMNIEENSFRDAILACRGRLGHFHLGEANRLPPGEGRLPWDEIFGALKEIDYDG P IVMEPFMRPGGSVSRAVGVWRDMSNGATDEQMDERARRSLNFVRGHLA The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications or equivalent substitutions made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0065] The foregoing embodiments and methods described in this invention may vary based on the capabilities, experience, and preferences of those skilled in the art.

[0066] Listing the steps of the method in a certain order in this invention does not constitute any restriction on the order of the method steps.

Claims

1. A mutant enzyme of D-allulose 3-epimerase, which is obtained by replacing the 87th threonine in the amino acid sequence of D-allulose 3-epimerase derived from Cabaleronia insecticola with isoleucine and / or replacing the 242nd threonine with proline. Preferably, the amino acid sequence of the D-allulose 3-epimerase is shown in SEQ ID NO:

1.

2. The mutant enzyme as described in claim 1, characterized in that, The mutant enzyme was obtained by replacing the 87th threonine residue with isoleucine in the amino acid sequence of D-allulose 3-epimerase derived from Cabaleronia insecticola, and its amino acid sequence is shown in SEQ ID NO:2; or, The mutant enzyme was obtained by replacing threonine at position 242 of the amino acid sequence of D-allulose 3-epimerase derived from Cabaleronia insecticola with proline, and its amino acid sequence is shown in SEQ ID NO:3; or, The mutant enzyme was obtained by replacing the threonine at position 87 with isoleucine and the threonine at position 242 with proline in the amino acid sequence of D-allulose 3-epimerase derived from Cabaleronia insecticola, and its amino acid sequence is shown in SEQ ID NO:

4.

3. A nucleotide encoding the mutant enzyme of claim 1 or 2; Preferably, the nucleotide sequence is as shown in SEQ ID NO:6, or as shown in SEQ ID NO:7, or as shown in SEQ ID NO:

8.

4. A recombinant vector containing the nucleotides of claim 3.

5. A host cell containing the vector of claim 4 or the nucleotide of claim 3 integrated into its genome; Preferably, the host cell is selected from one of the following: Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Pichia pastoris, Saccharomyces cerevisiae, and lactic acid bacteria; More preferably, the host cell is Escherichia coli.

6. An enzyme preparation comprising the mutant enzyme of claim 1 or 2; Preferably, the enzyme preparation is a liquid preparation or a solid preparation.

7. The use of the mutant enzyme of claim 1 or 2 in the preparation of D-allulose.

8. A method for preparing D-allulose, comprising the following steps: contacting the mutant enzyme of claim 1 or 2 or the enzyme preparation of claim 6 with a reaction substrate to carry out a catalytic reaction; Preferably, the reaction substrate includes fructose; more specifically, the reaction substrate is selected from crystalline fructose, fructose solution, high fructose syrup, or combinations thereof.

9. The preparation method according to claim 8, characterized in that, The reaction temperature is 50-70℃, preferably 60-70℃, and more preferably 65℃; Preferably, the pH of the reaction system is alkaline, preferably 8-10.

10. The preparation method according to claim 8 or 9, characterized in that, The method includes the following steps: (1) The mutant enzyme or enzyme preparation is brought into contact with the reaction substrate to carry out a catalytic reaction; (2) Inactivate the enzymes in the reaction solution; (3) Isolate and purify D-allulose.

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