Allulose 3-epimerase mutant enzyme and its use

Abstract

The present invention relates to an enzyme protein having a mutated amino acid sequence and having the activity of converting fructose to allulose, a recombinant microorganism containing a gene encoding the enzyme protein, a composition for producing allulose containing the same, and a method for producing allulose using the same.

Description

[Technical Field]

[0001] The present invention provides an allulose 3-epimerase mutant enzyme protein, a recombinant microorganism and / or a composition for allulose production containing the same, and / or a method for producing allulose using them. [Background technology]

[0002] Glucose is an essential sugar because it is used as an energy source when ingested by the human body. However, excessive glucose intake can lead to adult diseases such as obesity and diabetes. Allulose (D-allulose, D-psicose) is a sugar material that has recently emerged as a way to overcome this.

[0003] Allulose is produced by epimerization of fructose (D-fructose), and has a structure that makes it difficult to be used as an energy source when ingested, resulting in low calorie content. In relation to this, it has the ability to suppress glucose absorption and prevent blood sugar levels from rising, making it an effective sugar for diabetes. It also inhibits the action of enzymes involved in fat accumulation, preventing fat accumulation, making it an effective sugar for obesity. In addition, it can be converted into allose, which has anti-cancer properties, and there are reports that it itself exhibits similar effects. This sugar material is expected to be used more widely as a sugar with health functions.

[0004] In addition, since it has 70% of the sweetness of sugar, it is a sugar that can maintain a suitable sweetness when eaten. Therefore, in recent years, it has been widely used as a functional sugar and is often used in low-calorie beverages.

[0005] Since the functionality of allulose was confirmed, research has been ongoing into methods for producing allulose by converting fructose. Research is also underway into methods for producing allulose using glucose, which is less expensive than fructose. Research is underway to convert fructose to allulose using allulose epimerases derived from Agrobacterium tumefaciense, Clostridium cellulolyticum, and Clostridium boleae, tagatose epimerases derived from Pseudomonas chichori and Rhodobacter sphaeroides, and enzymes derived from microorganisms such as Arthrobacter gloformis, Staphylococcus aureus, Mesorhizobium loti, and Methylonmonus sp. Recently, research is also being conducted into multi-enzyme conversion of glucose and starch to reduce the production cost of allulose.

[0006] Allulose is generally produced by epimerization of the hydroxyl group at the carbon-3 position of fructose. In enzymatic reactions, the conversion rate between fructose and the substrate is generally equilibrium at a ratio of 70:30. Therefore, the maximum conversion rate of allulose is approximately 30%.

[0007] However, many reports have confirmed that increasing the temperature of the conversion reaction increases the conversion rate of allulose. Therefore, if the thermostability of the enzyme is ensured and the stability of the enzyme is improved, it is possible to increase the conversion rate by increasing the temperature of the conversion reaction.

[0008] Therefore, this study aims to find enzyme mutants with improved thermostability by studying mutations that increase the thermostability of the enzyme, and to improve allulose productivity through increased activity and thermostability. Summary of the Invention [Problem to be solved by the invention]

[0009] One aspect of the present invention relates to an allulose 3-epimerase mutant enzyme protein having improved allulose conversion activity and thermostability.

[0010] Another aspect of the present invention relates to a nucleic acid sequence encoding the allulose 3-epimerase mutant enzyme protein.

[0011] Another aspect of the present invention relates to a recombinant microorganism comprising a nucleic acid sequence encoding the allulose 3-epimerase mutant enzyme protein.

[0012] Another aspect of the present invention relates to a composition for producing allulose using the allulose 3-epimerase mutant enzyme protein and / or recombinant microorganism.

[0013] Another aspect of the present invention relates to a method for producing allulose from a substrate using the allulose 3-epimerase mutant enzyme protein and / or a recombinant microorganism. [Means for solving the problem]

[0014] One aspect of the present invention provides an allulose 3-epimerase mutant enzyme protein in which some amino acids in the amino acid sequence of SEQ ID NO: 1 have been substituted.

[0015] Specifically, the mutant enzyme may be an allulose 3-epimerase mutant enzyme protein in which one or more amino acids selected from the group consisting of the amino acids at positions 23, 75, 115, 126, 255, 267, and 269 from the N-terminus in the amino acid sequence of SEQ ID NO: 1 are substituted with one or more amino acids selected from the group consisting of isoleucine (I), glutamic acid (E), glycine (G), valine (V), alanine (A), and lysine (K).

[0016] Another embodiment of the present invention provides a polynucleotide encoding the enzyme protein of the present invention. Specifically, the present invention provides a polynucleotide comprising a nucleic acid sequence encoding an amino acid sequence in which one or more amino acids selected from the group consisting of amino acids at positions 23, 75, 115, 126, 255, 267, and 269 from the N-terminus in the amino acid sequence of SEQ ID NO: 1 are substituted with one or more amino acids selected from the group consisting of isoleucine (I), glutamic acid (E), glycine (G), valine (V), alanine (A), and lysine (K).

[0017] Another embodiment of the present invention provides a recombinant vector comprising a polynucleotide of the present invention.

[0018] Another embodiment of the present invention provides a recombinant microorganism containing a gene encoding the enzyme protein of the present invention, which may be at least one microorganism selected from the group consisting of, but not limited to, Escherichia coli strains, Bacillus strains (e.g., Bacillus subtilis), Corynebacterium strains (e.g., Corynebacterium glutamicum), Saccharomyces strains (e.g., Saccharomyces cerevisiae), and Pichia strains (e.g., Pichia pastoris).

[0019] Another embodiment of the present invention provides a composition for producing allulose, comprising one or more selected from the group consisting of the enzyme protein of the present invention, cells of a recombinant microorganism expressing the enzyme protein, a cell lysate of the microorganism, a culture of the microorganism, and extracts thereof.

[0020] Another aspect of the present invention provides a method for producing allulose, which includes a step of reacting one or more members selected from the group consisting of the enzyme protein, a recombinant microorganism expressing the enzyme protein, a bacterial cell of the microorganism, a bacterial cell lysate of the microorganism, a culture of the microorganism, and an extract thereof, with a substrate.

[0021] The enzyme protein according to the present invention comprises an amino acid sequence of SEQ ID NO: 1 in which one or more amino acids selected from the group consisting of the 23rd, 75th, 115th, 126th, 255th, 267th, and 269th amino acids from the N-terminus are substituted with at least one amino acid selected from the group consisting of isoleucine (I), glutamic acid (E), glycine (G), valine (V), alanine (A), and lysine (K), and has improved allulose-converting activity and thermostability compared to the wild-type enzyme.

[0022] The recombinant microorganism containing a gene encoding the enzyme protein of the present invention has an amino acid sequence in which one or more amino acids selected from the group consisting of amino acids at positions 23, 75, 115, 126, 255, 267, and 269 from the N-terminus in the amino acid sequence of SEQ ID NO: 1 are substituted with at least one amino acid selected from the group consisting of isoleucine (I), glutamic acid (E), glycine (G), valine (V), alanine (A), and lysine (K), and has higher allulose conversion activity and higher thermostability than a microorganism (wild-type strain) containing a gene encoding an enzyme protein without amino acid mutations. [Effects of the Invention]

[0023] The present invention relates to an enzyme protein having allulose 3-epimerization activity in which specific amino acids are mutated, a recombinant microorganism containing the same, a composition for allulose production, or a method for producing allulose using the same, which has high fructose-to-allulose conversion activity and excellent thermostability. [Brief explanation of the drawings]

[0024] [Figure 1] FIG. 1 is a schematic diagram showing a recombinant vector in which the D-allulose 3-epimerase gene according to one embodiment of the present invention has been cloned. [Figure 2] FIG. 2 is a schematic diagram showing a recombinant vector in which the D-allulose 3-epimerase gene was cloned for enzyme purification. DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention will now be described in more detail.

[0026] One aspect of the present invention provides an allulose 3-epimerase mutant enzyme protein in which one or more amino acids selected from the group consisting of amino acids at positions 23, 75, 115, 126, 255, 267, and 269 from the N-terminus in the amino acid sequence of SEQ ID NO: 1 are substituted with one or more amino acids selected from the group consisting of isoleucine (I), glutamic acid (E), glycine (G), valine (V), and alanine (A).

[0027] Specifically, the substituted amino acid sequence may be one or more substitutions selected from the group consisting of alanine (A) at position 23 from the N-terminus with isoleucine (I), serine (S) at position 75 with glutamic acid (E), aspartic acid (D) at position 115 with glycine (G), arginine (R) at position 126 with valine (V), leucine (L) at position 255 with valine, glutamic acid at position 267 with lysine, and glycine at position 269 with alanine, but is not limited to these.

[0028] Furthermore, the substituted amino acid sequence may include, but is not limited to, one or more amino acid substitutions selected from the group consisting of a substitution of alanine at position 23 from the N-terminus with isoleucine, serine at position 75 with glutamic acid, aspartic acid at position 115 with glycine, asparagine at position 126 with valine, leucine at position 255 with valine, and glycine at position 269 with alanine in the amino acid sequence of SEQ ID NO: 1, and a substitution of glutamic acid at position 267 with lysine.

[0029] One aspect of the present invention provides an allulose 3-epimerase protein having an amino acid sequence that has 80% or more sequence identity, 85% or more sequence identity, 90% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.3% or more sequence identity with the amino acid sequence of SEQ ID NO: 1. Specifically, ... The allulose 3-epimerase protein, in which one or more amino acids selected from the group consisting of glycine (E), valine (V), and alanine (A) are substituted, may have 80% or more sequence identity, 85% or more sequence identity, 90% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.3% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, and the epimerase protein may exclude an epimerase protein having the amino acid sequence of SEQ ID NO: 1, i.e., a wild-type enzyme protein without a mutation.

[0030] In one aspect of the present invention, one or more amino acids selected from the group consisting of amino acids at positions 23, 75, 115, 126, 255, 267, and 269 from the N-terminus in the amino acid sequence of SEQ ID NO: 1 are isoleucine (I), glutamic acid (Glutamic Acid), or the like. The present invention provides an allulose 3-epimerase protein having an amino acid sequence having 80% or more sequence identity, 85% or more sequence identity, 90% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.3% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, wherein the epimerase protein does not include an epimerase protein having the amino acid sequence of SEQ ID NO: 1, i.e., a wild-type enzyme protein without a mutation.

[0031] In one aspect of the present invention, one or more amino acids selected from the group consisting of amino acids at positions 23, 75, 115, 126, 255, 267, and 269 from the N-terminus in the amino acid sequence of SEQ ID NO: 1 are isoleucine (I), glutamic acid (Glutamic Acid), or the like. The present invention provides an allulose 3-epimerase protein having an amino acid sequence having 80% or more sequence identity, 85% or more sequence identity, 90% or more sequence identity, 95% or more sequence identity, 96% or more sequence identity, 97% or more sequence identity, 98% or more sequence identity, 99% or more sequence identity, or 99.3% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, wherein the epimerase protein does not include an epimerase protein having the amino acid sequence of SEQ ID NO: 1, i.e., a wild-type enzyme protein without a mutation.

[0032] The allulose-converting activity of the enzyme protein of the present invention in the enzymatic reaction can be measured using bacterial cells expressing the enzyme, a culture medium in which the bacterial cells are cultured, and / or a supernatant obtained by centrifuging the culture medium. Specifically, the allulose-converting activity can be measured using bacterial cells obtained after reacting the bacterial cells with a substrate, a culture medium in which the bacterial cells are cultured, and / or a supernatant obtained by centrifuging the culture medium. The substrate can include, but is not limited to, one or more selected from the group consisting of fructose, allulose, tagatose, xylose, sorbose, ribulose, and ketose.

[0033] In the enzymatic reaction of the enzyme protein, the bacterial cells used to confirm the allulose conversion activity may be, but are not limited to, cells of Escherichia strains (e.g., Escherichia coli).

[0034] In a substrate conversion reaction using the mutant enzyme protein or a microbial cell that produces the mutant enzyme protein, the allulose conversion activity of the enzyme protein according to the present invention or the enzyme protein of the composition for allulose production according to the present invention is 105% or more, 110% or more, 115% or more, 120% or more, 125% or more, 130% or more, 135% or more, 140% or more, 145% or more, 150% or more, 155% or more, 160% or more, 105% to 200%, 105% to 180%, 105% to 170%, 105% to 165%, 110% to 200%, 110% to 180%, 110% to 17 ... 65%, 120%~200%, 120%~180%, 120%~170%, 120%~165%, 130%~200%, 130%~180%, 130%~170%, 130%~165%, 140%~200%, 140%~180%, 140%~170%, 140%~165%, 150%~200%, 150%~180%, 150%~1 The enzyme activity may be 70%, 150% to 165%, 160% to 200%, 160% to 180%, 160% to 170%, 160% to 165%, for example, 107.16%, 116.69%, 124.69%, 136.67%, 140%, 151.97%, 159.88%, 162.01%, 162.04%, or 167.27%. The enzyme activity using the enzyme-producing microbial cells or enzyme protein may be evaluated by carrying out a substrate conversion reaction at 70°C for 30 minutes using microbial cells expressing the enzyme protein, or by carrying out a substrate conversion reaction at 70°C for 30 minutes using a purified enzyme obtained by disrupting the enzyme-producing microbial cells.

[0035] The thermal stability of the enzyme protein of the present invention or the enzyme protein of the composition for allulose production of the present invention can be confirmed by, but is not limited to, comparing the allulose conversion activity before and after heat treatment of the bacterial cells expressing the enzyme protein, the culture medium in which the bacterial cells have been cultured, and / or the supernatant obtained by centrifuging the culture medium, or by measuring the half-life of the enzyme protein, which is the point at which the activity becomes 50% compared to 100% allulose conversion activity before heat treatment.

[0036] In the enzymatic reaction of the enzyme protein, the bacterial cells used to confirm the allulose conversion activity may be, but are not limited to, cells of Escherichia strains (e.g., Escherichia coli).

[0037] The thermal stability may be confirmed by heat treating a purified enzyme obtained by disrupting microbial cells expressing the enzyme protein or microbial cells producing the enzyme at 60°C or 70°C for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 7.5 hours, 8 hours, 9 hours, 10 hours, or 11 hours.

[0038] The half-life (hours) of the enzyme protein of the present invention or the enzyme protein of the composition for allulose production of the present invention, under heat treatment conditions at 60°C, is 130% or more, 150% or more, 200% or more, 250% or more, 300% or more, 350% or more, 400% or more, 130% to 500% based on 100% of the half-life of the epimerase protein expressed in a microorganism in which a wild-type enzyme protein comprising the amino acid sequence of SEQ ID NO: 1 is expressed. %, 130% to 450%, 130% to 420%, 200% to 500%, 200% to 450%, 200% to 420%, 300% to 500%, 300% to 450%, 300% to 420%, 350% to 500%, 350% to 450%, 350% to 420%, 380% to 500%, 380% to 450% or 380% to 420%, for example, 133% or 400%, but is not limited to these.

[0039] The half-life (hours) of the enzyme protein of the present invention or the enzyme protein of the composition for allulose production of the present invention, under heat treatment conditions at 60°C, may be 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 1.7 to 50 hours, 1.7 to 30 hours, 1.7 to 10 hours, 1.7 to 8 hours, 1.7 to 7 hours, 2 to 50 hours, 2 to 30 hours, 2 to 10 hours, 2 to 8 hours, 2 to 7 hours, 3 to 50 hours, 3 to 30 hours, 3 to 10 hours, 3 to 8 hours, 3 to 7 hours, 4 to 50 hours, 4 to 30 hours, 4 to 10 hours, 4 to 8 hours, 4 to 7 hours, 5 to 50 hours, 5 to 30 hours, 5 to 10 hours, 5 to 8 hours, 5 to 7 hours, 6 to 50 hours, 6 to 30 hours, 6 to 10 hours, 6 to 8 hours, or 6 to 7 hours, for example, but is not limited to, 2 hours or 6 hours.

[0040] The enzyme protein whose half-life under heat treatment conditions at 60°C has been confirmed may be an allulose 3-epimerase protein in which one or more amino acids selected from the group consisting of amino acids at positions 23, 75, 115, 126, 255, 267, and 269 from the N-terminus in the amino acid sequence of SEQ ID NO: 1 have been substituted with one or more amino acids selected from the group consisting of isoleucine (I), glutamic acid (E), glycine (G), valine (V), alanine (A), and lysine (K).

[0041] The half-life (hours) of the enzyme protein of the present invention or the enzyme protein of the composition for allulose production of the present invention, under heat treatment conditions at 70°C, can be 0.5 hours or more, 2 hours or more, 4 hours or more, 6 hours or more, 8 hours or more, 10 hours or more, 0.5 to 20 hours, 0.5 to 15 hours, 0.5 to 12 hours, 1 to 20 hours, 1 to 15 hours, 1 to 12 hours, 5 to 20 hours, 5 to 15 hours, 5 to 12 hours, 8 to 20 hours, 8 to 15 hours or 8 to 12 hours, for example, 0.67 hours or 10 hours, but is not limited to these.

[0042] The enzyme protein of the present invention or the enzyme protein of the composition for allulose production of the present invention may have an allulose conversion activity after heat treatment at 70°C for 7 hours of 60% to 100%, or 65% to 100%, for example, 65.57%, based on the allulose conversion activity of the enzyme protein before heat treatment (100%), but is not limited thereto.

[0043] The enzyme protein whose half-life was confirmed under the heat treatment condition of 70°C has one or more amino acids selected from the group consisting of amino acids at positions 23, 75, 115, 126, 255, 267, and 269 from the N-terminus in the amino acid sequence of SEQ ID NO: 1, which are isoleucine (I), glutamic acid (Glutamic acid), or the like. The allulose 3-epimerase protein may be an allulose 3-epimerase protein in which one or more amino acids selected from the group consisting of leucine (E), glycine (G), valine (V), alanine (A), and lysine (K) have been substituted. Specifically, the allulose 3-epimerase protein may include, in the amino acid sequence of SEQ ID NO: 1, one or more amino acid substitutions selected from the group consisting of: a substitution of alanine at position 23 from the N-terminus with isoleucine, a substitution of serine at position 75 with glutamic acid, a substitution of aspartic acid at position 115 with glycine, a substitution of asparagine at position 126 with valine, a substitution of leucine at position 255 with valine, and a substitution of glycine at position 269 with alanine, and a substitution of glutamic acid at position 267 with lysine.

[0044] Since the activity of the enzyme protein of the present invention is regulated by metal ions, the addition of metal ions in allulose production using the Mycobacterium strain can increase the conversion efficiency of fructose to allulose, i.e., the allulose production rate. Therefore, a composition for allulose production containing one or more selected from the group consisting of an enzyme protein, a recombinant microorganism expressing the enzyme protein, a bacterial cell of the microorganism, a bacterial cell lysate of the microorganism, a culture of the microorganism, and an extract thereof, or a composition for allulose production using a microorganism that produces the same, may further contain a metal ion. Furthermore, the method for producing allulose using the enzyme protein may further include a step of adding a metal ion.

[0045] The metal ions may be one or more selected from the group consisting of manganese ions, magnesium ions, nickel ions, cobalt ions, etc. For example, the metal ions may be manganese ions, cobalt ions, or a mixture thereof. When manganese ions, cobalt ions, or a mixture thereof are present as metal ions, the allulose conversion activity may be increased by 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, or 2.3-fold or more, for example, by 1.2 to 2.3-fold, compared to the case in which the metal ions are not present.

[0046] The amount of the metal ions added can be in the range of 0.5 mM to 5 mM, 0.5 mM to 4 mM, 0.5 mM to 3 mM, for example, 0.5 mM to 2 mM, taking into consideration the effect of improving the allulose production yield.

[0047] Another embodiment of the present invention provides a recombinant microorganism comprising a gene encoding the enzyme protein of the present invention.

[0048] The microorganism may be at least one microorganism selected from the group consisting of strains of the genus Escherichia coli, bacteria of the genus Bacillus (e.g., Bacillus subtilis), bacteria of the genus Corynebacterium (e.g., Corynebacterium glutamicum), bacteria of the genus Saccharomyces (e.g., Saccharomyces cerevisiae), and bacteria of the genus Pichia (e.g., Pichia pastoris), but is not limited thereto.

[0049] Another embodiment of the present invention provides a composition for producing allulose, comprising one or more selected from the group consisting of the enzyme protein of the present invention, a recombinant microorganism expressing the enzyme protein, cells of the microorganism, a cell lysate of the microorganism, a culture of the microorganism, and an extract thereof.

[0050] The culture contains the enzyme produced by the recombinant microorganism and may contain the microorganism or may be in a cell-free form without the microorganism. The disrupted product contains a disrupted product obtained by disrupting cells of the recombinant microorganism or a supernatant obtained by centrifuging the disrupted product, and the enzyme produced by the recombinant microorganism.

[0051] In this specification, unless otherwise specified, the recombinant microorganism used for producing allulose is used to mean one or more species selected from the group consisting of the bacterial cells of the microorganism, a culture of the strain, and a disrupted product of the strain.

[0052] Another embodiment of the present invention provides a method for producing allulose, comprising a step of reacting one or more selected from the group consisting of the enzyme protein of the present invention, a recombinant microorganism expressing the enzyme protein, cells of the microorganism, a cell lysate of the microorganism, a culture of the microorganism, and an extract thereof, with a substrate.

[0053] The substrate may be one selected from the group consisting of fructose, allulose, tagatose, xylose, sorbose, ribulose, and ketose, but is not limited thereto.

[0054] The allulose production method may further include a step of separating and / or purifying allulose from the reaction product. The culture may be, but is not limited to, a culture obtained by reacting one or more selected from the group consisting of the recombinant microorganism, the recombinant microorganism's cells, the recombinant microorganism's cells, a disrupted microorganism's cells, a culture of the microorganism, and an extract thereof with a substrate.

[0055] Specifically, the allulose production method according to the present invention can include a separation step of the allulose conversion reaction product, which includes ion purification and simulated moving bed (SMB) chromatography separation. Specifically, the allulose conversion reaction product is subjected to SMB chromatography separation to separate it into an allulose fraction having a higher allulose content than the conversion reaction product and a fructose raffinate. The allulose fraction can be processed into a liquid syrup through an allulose concentration step, or into allulose crystals through an allulose crystallization step. The allulose crystallization step includes a step of secondary ion purification of the allulose fraction obtained in the high-performance chromatography step, a step of concentrating the ion-purified allulose fraction, and a step of crystallizing allulose from the concentrate to obtain allulose crystals and an allulose crystallization mother liquor. [Example]

[0056] The present invention will now be described in more detail with reference to the following examples, but the scope of the present invention is not limited to these examples.

[0057] Example 1. Cloning of the D-allulose 3-epimerase gene Example 1-1. Preparation of enzyme gene D-allulose 3-epimerase (D-tagatose 3-epimerase) identified in Microbacterium foliorum (SY27B-MF; accession number: KCCM11774P) was expressed in Escherichia coli to confirm enzyme activity, and DNA sequences were synthesized to identify mutants with improved thermostability. The amino acid sequence of the protein was synthesized using codons optimized for E. coli to ensure optimal expression in E. coli.

[0058] The gene sequence obtained by codon optimization was synthesized by Integrated DNA Technologies (IDT, USA). The synthesized gene is called MDPE, and the information on the codon-optimized gene is shown in Table 1. The amino acid sequence of the MDPE gene corresponds to SEQ ID NO: 1, and the base sequence corresponds to SEQ ID NO: 2.

[0059] [Table 1]

[0060] Example 1-2. Production of recombinant microorganisms containing enzyme genes The MDPE polynucleotide synthesized by codon optimization for expression in E. coli in Example 1-1 was amplified by PCR (polymerase chain reaction). Specifically, a polymerase chain reaction (PCR) reaction solution containing 50 ng of the synthesized MDPE gene, 10 pmol of forward primer (nucleotide sequence of SEQ ID NO: 3) for each amino acid residue, 10 pmol of reverse primer (nucleotide sequence of SEQ ID NO: 4), 1 μl of fusion polymerase, 10 μl of reaction buffer (5X), and 1 μl of 10 mM dNTP was prepared and PCR amplification was performed. The sequences of the primers used are shown in Table 2 below.

[0061] [Table 2]

[0062] Specifically, using the PCR reaction solution, a PCR reaction was carried out using a GeneAmp PCR system 9700, including one cycle at 98°C (30 seconds), 18 cycles of [98°C (10 seconds), 55°C (30 seconds), 72°C (2 minutes 30 seconds)], and one cycle at 72°C (5 minutes), and after gene amplification, a large amount of gene was obtained.

[0063] The pUC19 vector (NEB (New England Biolabs)) was digested with the restriction enzymes HindIII and BamHI, and the amplified gene and pUC19 vector were then inserted into 2X HiFi DNA master mix (NEB) so that they were ligated into the restriction enzyme sites to prepare the pUC19 / allulose 3-epimerase recombinant vector (pUC19_MDPE). A schematic diagram of the recombinant vector is shown in Figure 1. The recombinant vector was transformed into Escherichia coli DH10b competent cells (TransGen, Trans10) by the heat shock method to produce the recombinant microorganism (DH10b pUC19_MDPE).

[0064] Example 2. Mutation of the D-allulose 3-epimerase gene Based on the analysis of the amino acid sequences between homologous genes and the analysis of the tertiary structure models of the active site and metal binding site, the selected amino acids were mutated and replaced with other amino acids. These recombinant enzyme mutants were then produced in E. coli, and the changes in the activity of allulose epimerase were analyzed.

[0065] Example 2-1. Site-directed mutagenesis (SDM) Among the epimerases, the sequences of the D-allulose 3-epimerases derived from sequence-verified Arthrobacter globiformis, Agrobacterium tumefaciense, Clostridium cellulolyticum, and Methylomonus sp. were analyzed to identify homologous sites, and the positions of mutations corresponding to the homologous sites were confirmed.

[0066] Specifically, a homology comparison of the epimerases confirmed that the amino acid at position 23 in the D-allulose-3-epimerases from Agrobacterium tumefaciense, Clostridium cellulolyticum, and Methylomonas was isoleucine (I), which was selected as the mutation site. Furthermore, a comparison with the D-allulose 3-epimerase from Arthrobacter globiformis confirmed that the amino acid at position 75 was glutamic acid (E), which was selected as the mutation site. The selected positions were confirmed to maintain the structural activity of the enzyme using the Protein Data Bank (PDB) database.

[0067] The substitution mutations in MDPE were generated using site-directed mutagenesis (SDM) with Quikchange® (Stratagene), which was adapted from previous research. Stratagene's Quikchange® site-directed mutagenesis method does not involve amplifying the gene corresponding to the enzyme portion by PCR and subcloning it into plasmid DNA, but rather amplifies the entire plasmid DNA by PCR and digests the template DNA with DpnI enzyme. Quikchange® site-directed mutagenesis has the advantage of being able to induce mutations more quickly.

[0068] pUC19_MDPE from Example 1-2 was used as the template DNA for replication for the MDPE substitution mutation in Example 1. A polymerase chain reaction (PCR) reaction solution containing 10 ng of pUC19_MDPE, 10 pmol of forward primer (nucleotide sequence of SEQ ID NO: 5 or 7) for each amino acid residue (I23, E75), 10 pmol of reverse primer (nucleotide sequence of SEQ ID NO: 6 or 8), 1 μl of Fusion polymerase, 10 μl of reaction buffer (5X), and 1 μl of 10 mM dNTP was prepared in a final volume of 50 μl, and PCR amplification was performed. The primer information used is shown in Table 3 below.

[0069] [Table 3]

[0070] Specifically, PCR was performed using the PCR reaction solution in a GeneAmp PCR system 9700, including one cycle at 98°C (30 seconds), 18 cycles of [98°C (10 seconds), 55°C (30 seconds), 72°C (2 minutes 30 seconds)], and one cycle at 72°C (5 minutes), and a large amount of gene was obtained after gene amplification.

[0071] 1 μl of DpnI (10 Units / μl, NEB) enzyme, which recognizes and degrades non-mutagenized template DNA, was added to the 50 μl PCR reaction solution and treated for 3 hours at 37° C. After this treatment, the PCR-treated gene pUC19_MDPE_A23I (A23I mutation) using the MDPE_A23I cloning primer and the PCR-treated gene pUC19_MDPE_S75E (S75E mutation) using the MDPE_S75E cloning primer were transformed into E. coli DH10B in a manner substantially similar to the method for producing the recombinant strain DH10b pUC19_MDPE in Example 1-2, to obtain DH10b pUC19_MDPE_A23I recombinant strain and DH10b pUC19_MDPE_S75E recombinant strain.

[0072] To confirm the enzyme mutations in the recombinant strains, the amino acid sequence was compared using the NCBI website (BLAST) tool and wild-type enzymes.

[0073] Example 2-2. Error-prone PCR-mediated mutation Mutants were generated through error-prone PCR, which involves introducing mutations during gene amplification. Specifically, to generate the mutants, 10 ng of template DNA (pUC19_MDPE), 10 pmol of forward primer (nucleotide sequence of SEQ ID NO: 9) for each amino acid residue, 10 pmol of reverse primer (nucleotide sequence of SEQ ID NO: 10), 1 μl of Taq polymerase (Clontech), 5 μl of reaction buffer (10X TITANIUM Taq Buffer), 1 μl of diversify dNTP Mix, 1 μl of dGTP (2 mM), and 4 μl of MnSO were mixed and PCR-grade water was added to prepare a polymerase chain reaction (PCR) reaction solution in a final volume of 50 μl. PCR amplification was performed, and the primer information used is shown in Table 3.

[0074] Specifically, the PCR reaction solution was used in a GeneAmp PCR system 9700, and the PCR reaction was cycled once at 94°C for 30 seconds, followed by 25 cycles of 94°C for 30 seconds and 68°C for 1 minute, followed by 1 minute at 68°C.

[0075] The gene amplified through mutagenesis was subjected to electrophoresis to identify a band at the desired position, and the identified gene band was extracted. The PCR-treated gene was transformed into E. coli DH10B in a manner substantially similar to the method for preparing the recombinant strain DH10b pUC19_MDPE in Example 1-2, thereby obtaining a recombinant microorganism.

[0076] Example 2-3. Screening of mutations using HTS (High-Throughput-Screening) The recombinant microorganisms transformed in Example 2-2 were isolated by spreading on LB ampicillin solid medium consisting of 10 g / L Bacto Tryptone (Difco), 5 g / L yeast extract (Difco), 10 g / L NaCl (Taisho Chemical), 100 mg / L Ampicillin (Sigma), and 20 g / L Bacto Agar. The isolated colonies were subjected to primary screening using HTS.

[0077] After the primary selection, 300 μL of LB-ampiciline liquid medium (composition: 10 g / L Bacto Tryptone (Difco), 5 g / L enzyme extract (Difco), 10 g / L NaCl (Taisho Chemical), and 100 mg / L Ampicillin (Sigma)) was inoculated into a 96-well (Greiner) multicolony incubator, and then cultured at 37°C with shaking at 200 rpm for 16 to 18 hours.

[0078] The enzymatic activity of the shake-cultured cells was confirmed by fructose dehydrogenase assay. 75 μL of the culture medium obtained by the shake-culture was transferred to a 96-well plate, and the same amount of 100 mM allulose substrate (100 mM McIlvaine buffer, pH 6.0) was added, followed by cell reaction at 60°C for 30 minutes.

[0079] 10 μL of the reaction mixture in which fructose was produced from allulose through the reaction was transferred to a new 96-well plate, and reaction mixture A (150 mM McIlvaine buffer, pH 4.5), reaction mixture B (0.1 M potassium ferricyanide, 0.1% Triton X-100), and reaction mixture C (0.1 M potassium ferricyanide, 0.1% Triton X-100, 0.05% BSA) were mixed in a volume ratio of 70:10:10, and 90 μL was added to the reaction mixture that had been converted to fructose.

[0080] After incubation at 37°C for 30 minutes, 50 μL of reaction solution D (1.25 mM iron(III) sulfate, 0.3% SDS, 8% phosphoric acid) was added and the reaction was stopped by incubation at 37°C for 20 minutes. After stopping the reaction, the absorbance was measured at 660 nm, and strains with higher absorbance than the recombinant strain DH10b pUC19_MDPE of Example 1-2, which had not undergone enzyme mutation, were selected.

[0081] Example 2-4. Identification of amino acid residues in mutant proteins Among the mutations obtained through the recombinant microorganism obtained in Example 2-1 and the error-prone PCR in Example 2-2, the mutations of the amino acid residues of the enzyme selected in Example 2-3 were confirmed.

[0082] Specifically, the plasmid was purified using a plasmid extraction kit (GeneAll), and the gene sequence of the subcloned epimerase portion of the plasmid was transcribed from the amino acid sequence of the gene sequence using a sequence interpretation service (Macrogen). The mutation site was confirmed by comparing with the wild-type amino acid sequence, and the primer information used is shown in Table 4 below.

[0083] [Table 4]

[0084] The recombinant strains were screened for the enzymes with the A23I, S75E, D115G, R126V, L255V, and G269A mutations, respectively, and the following strains were prepared: DH10b pUC19_MDPE_A23I (A23I mutation), DH10b pUC19_MDPE_S75E (S75E mutation), DH10b pUC19_MDPE_D115G (D115G mutation), DH10b pUC19_MDPE_R126V (R126V mutation), DH10b pUC19_MDPE_L255V (L255V mutation), and DH10b pUC19_MDPE_G269A (G269A mutation).

[0085] The positions of the mutated amino acids in the reaction were confirmed by generating a three-dimensional structure of the enzyme through a template search of the Protein Data Bank (PDB) using a homology model of the three-dimensional structure of the protein via SWIISS-MODEL (http: / / www.expasy.org / swissmod / SWIIS-MODEL.html), and the form of the three-dimensional structure was confirmed.

[0086] Example 3. Evaluation of activity of the first screened mutant enzyme Example 3-1. Confirmation of enzyme activity using a strain producing wild-type enzyme The DH10b pUC19_MDPE strain from Example 1 was cultured on LB-ampiciline solid medium containing 10 g / L Bacto Tryptone (Difco), 5 g / L enzyme extract (Difco), 10 g / L NaCl (Taisho Chemical), 100 mg / L Ampicillin (Sigma), and 20 g / L Bacto agar. One colony was picked from the solid culture and inoculated into 3 ml of LB-ampiciline liquid medium containing 10 g / L Bacto Tryptone (Difco), 5 g / L enzyme extract (Difco), 10 g / L NaCl (Taisho Chemical), and 100 mg / L Ampicillin (Sigma). The culture was then cultured at 37°C with shaking at 250 rpm for 16 to 18 hours.

[0087] After the bacterial cell culture, OD 600nm When the conversion rate changed from 2 to 3, a conversion reaction using microbial cells was carried out. Specifically, the conversion reaction using the cells of the microorganism producing the mutant enzyme was carried out at 70°C for 30 minutes using a reaction solution containing 1 mM manganese ions in 50 mM PIPES buffer (pH = 7.0) so that the concentration of the reaction substrate, fructose, was 400 g / L, and the cell concentration of the strain was 1 mg (dcw) / mL.

[0088] After the conversion reaction, the supernatant was collected by centrifugation (13,000 rpm, 15 minutes) and analyzed by high-performance liquid chromatography (HPLC). The HPLC analysis was performed using an Agilent 1280 RID (Refractive Index Detector) equipped with a Cosmosil Sugar-D column (Agilent, USA). The mobile phase solvent was 80% (v / v) acetonitrile, and the temperature was 30°C with a flow rate of 1.0 mL / min.

[0089] The results obtained by HPLC analysis of the supernatant of the microbial culture are shown in Table 5 below.

[0090] Example 3-2. Confirmation of enzyme activity using mutant enzyme-producing strains The strains in which the enzyme mutations in Example 2 were confirmed (DH10b pUC19_MDPE_A23I, DH10b pUC19_MDPE_S75E, DH10b pUC19_MDPE_D115G, DH10b pUC19_MDPE_R126V, DH10b pUC19_MDPE_L255V, or DH10b pUC19_MDPE_G269A) were grown in substantially the same manner as in Example 3-1, and the products obtained from the substrate conversion reactions were analyzed by HPLC. The results of the analysis are shown in Table 5 below.

[0091] In Table 5 below, the substrate conversion activity is expressed as a relative conversion activity based on 100% of the substrate conversion activity of the strain producing the wild-type enzyme.

[0092] [Table 5]

[0093] The substrate conversion activity of the enzyme was confirmed using strains producing the enzyme. As a result, the recombinant strains expressing the A23I, S75E, and R126V mutant enzymes showed an activity increased by more than 150% compared to the recombinant strain expressing the wild-type enzyme, and the recombinant strains expressing the D115G, L255V, or G269A mutant enzymes showed an activity increased by approximately 107 to 125% compared to the recombinant strain expressing the wild-type enzyme.

[0094] Example 4. Evaluation of thermostability of strains producing the first selected mutant enzymes To test the thermostability of the strains producing the selected mutant enzymes, strains that were confirmed to have improved activity among the strains confirmed to have enzyme mutations in Example 3-2 were subjected to high-temperature heat treatment. Specifically, this experiment was conducted to determine the degree to which enzyme activity was maintained depending on the high-temperature treatment time. Only strains expressing the mutant enzymes were collected, suspended in 50 mM PIPES buffer (pH = 7.0), and heat-treated at 60°C.

[0095] After the heat treatment, the conversion activity was measured by adding fructose substrate and manganese ions to the cells and carrying out a reaction using essentially the same procedure as in Example 3-2. The conversion activity measured over time was converted into a half-life, and the thermostability of the mutant strain was determined based on 100% activity before the heat treatment reaction. The half-life at which the activity of the mutant strain reached 50% was measured.

[0096] The half-life of the mutant enzymes was 1.5 hours for the wild-type enzyme-expressing cells, 6 hours for DH10b pUC19_MDPE_A23I, and 2 hours for DH10b pUC19_MDPE_G269A. Furthermore, the relative half-life of the mutant enzymes was 4 times longer than that of the wild-type enzyme-producing cells (1.5 hours), and the relative half-life of the mutant enzymes was 1.33 times longer than that of the wild-type enzyme-producing cells.

[0097] Example 5. Selection of secondary mutations and analysis of enzyme properties Example 5-1. Screening for secondary mutations Mutagenesis was carried out using A23I, which was selected in Examples 3 and 4 for its excellent enzymatic activity and thermostability, as a template DNA via gene amplification (error-prone PCR) for secondary mutagenesis. This process introduced additional mutations to the primary mutation, A23I, allowing the effects of double mutation to be confirmed.

[0098] Specifically, a recombinant vector (pUC19_MDPE_A23I) in which the A23I mutation had occurred was prepared using pUC19_MDPE and the MDPE_A23I_Cloning primer in substantially the same manner as in Example 2-1.

[0099] Using the prepared pUC19_MDPE_A23I, enzyme mutations were induced in a manner substantially similar to the error-prone PCR process of Example 2-2, and the resulting mutants were subcloned into pUC19. The subcloned mutants were subjected to HTS in Example 2-3 to select mutants with increased activity, and a strain (DH10b pUC19_MDPE_A23I / E267K) in which A23I and E267K mutations were confirmed was obtained in a manner substantially similar to Example 2-4.

[0100] Example 5-2. Confirmation of enzyme activity using the second-screened mutant enzyme-producing strain The wild-type enzyme-producing strain DH10b pUC19_MDPE in Example 1 and the strains DH10b pUC19_MDPE_A23I and DH10b pUC19_MDPE_A23I / E267K in which enzyme mutations were confirmed were examined for their conversion activities in a manner substantially similar to that described in Example 3-2. The results are shown in Table 6 below.

[0101] In Table 6 below, the substrate conversion activity is expressed as a relative activity, with the substrate conversion activity of the strain producing the wild-type enzyme being 100% as the standard.

[0102] [Table 6]

[0103] Analysis of the enzyme's conversion activity using a bacterial cell reaction of the enzyme-producing microorganism revealed that the mutant enzyme with the A23I and E267K double mutations had the same or higher enzymatic activity as the mutant enzyme with only the A23I mutation.

[0104] Example 5-3. Confirmation of enzyme thermostability using the secondary selected mutant enzyme-producing strain To confirm the thermostability of the strains producing the secondary screened mutant enzymes, excluding the strain producing the wild-type enzyme with low thermostability, DH10b pUC19_MDPE_A23I and DH10b pUC19_MDPE_A23I / E267K were subjected to a heat treatment at 70°C in substantially the same manner as in Example 4, and the enzyme activity was analyzed, and the half-life was calculated to evaluate the thermostability.

[0105] As a result of measuring the half-life, the half-life of DH10b pUC19_MDPE_A23I was 0.67 hours, and the half-life of DH10b pUC19_MDPE_A23I / E267 was 10 hours, indicating that the strain producing the A23I / E267K double mutant enzyme has better thermostability.

[0106] Example 6. Confirmation of activity and thermostability of purified enzyme Example 6-1. Construction of recombinant strains for enzyme purification To prepare a recombinant strain for enzyme purification, the plasmid pUC19_MDPE_A23I / E267K was purified from the DH10b pUC19_MDPE_A23I / E267K recombinant strain of Example 5-1 in substantially the same manner as in Example 2-4 to prepare the plasmid pUC19_MDPE_A23I / E267K.

[0107] A gene amplification (polymerase chain reaction, PCR) reaction solution containing 10 ng each of the prepared pUC19_MDPE_A23I / E267K, pUC19_MDPE of Example 1-2, and pUC19_MDPE_A23I of Example 2-1, 10 pmol of a forward primer (base sequence of SEQ ID NO: 13) for each amino acid residue, 10 pmol of a reverse primer (base sequence of SEQ ID NO: 14), 1 μl of fusion polymerase, 10 μl of reaction buffer (5X), and 1 μl of 10 mM dNTP was prepared for each template, and PCR was performed. Information on the primers used is shown in Table 7 below.

[0108] [Table 7]

[0109] The PCR reaction solution was used in a GeneAmp PCR system 9700, and PCR reactions were carried out including one cycle at 98°C (30 seconds), 30 cycles of 98°C (10 seconds), 55°C (30 seconds), and 72°C (30 seconds), followed by one cycle at 72°C (3 minutes). The gene product obtained and the pET21a (Novagene) plasmid were subcloned and inserted into a fragment obtained by treating with NdeI and XhoI enzymes.

[0110] Specifically, the gene was amplified to enable expression under the strong T7 promoter, and the pET-21a vector was digested with the restriction enzymes NdeI and XhoI1 to fuse it with a His-Tag for expression. The resulting vector was then ligated using the In-Fusion Snap Assembly Kit (Takara Bio Inc.) to create the recombinant vector pET-21a / allulose 3-epimerase (pET21a_MDPE), a diagram of which is shown in Figure 2. The resulting recombinant vector was transformed into E. coli BL21(DE3) by the heat shock method to produce the recombinant strain (BL21 pET21a_MDPE).

[0111] In addition, pET21a_MDPE was used instead of pUC19_MDPE, and PCR was performed in substantially the same manner as in Example 2. The PCR-prepared gene was then transformed into E. coli BL21(DE3) by the heat shock method to prepare a recombinant strain.

[0112] The amino acid residues of the enzyme in the obtained recombinant strains were confirmed in a manner substantially similar to that in Example 2, and a strain in which the A23I mutation was confirmed (BL21 pET21a_MDPE_A23I) and a strain in which the A21I and E267K mutations were confirmed in a manner substantially similar to that in Example 5 (BL21 pET21a_MDPE_A23I / E267K) were prepared.

[0113] Example 6-2. Purification of enzyme 3 mL of each of the BL21 pET21a_MDPE, BL21 pET21a_MDPE_A23I, or BL21 pET21a_MDPE_A23I / E267K strains was inoculated into LB-ampiciline liquid medium (Bacto Tryptone (Difco) 10 g / L, enzyme extract (Difco) 5 g / L, NaCl (Taisho Chemical) 10 g / L, Ampicillin (Sigma) 100 mg / L) as a colony, followed by shaking culture at 37°C and 250 rpm until the optical density at 600 nm (OD) reached 1.5. The culture was then inoculated into 50 mL of the same liquid medium as the LB-ampiciline liquid medium and shaken cultured at 37°C and 250 rpm.

[0114] When the absorbance at 600 nm of the shake-cultured culture reached 0.5, 0.1 mM IPTG was added to induce overexpression of the target enzyme. From this point on, the culture conditions were changed to 20°C and 180 rpm and maintained for 16 hours. The cells were then centrifuged at 4000 rpm for 10 minutes to recover the cells.

[0115] 50 mL of the collected bacterial solution was dissolved in 1 mL of chilled 50 mM PIPES (pH 7.0) buffer, and then disrupted five times for 20 seconds using a bead beater. The disrupted cells were centrifuged at 13,000 rpm at 4°C for 10 minutes, and the supernatant was collected.

[0116] A Bio-Rad PolyPrep chromatography column was packed with 1 ml of Ni-NTA resin, and 10 ml of lysis buffer (50 mM sodium phosphate, 10 mM imidazole, pH 8.0), equivalent to 10 times the resin volume, was applied to equilibrate the resin with the buffer. The collected supernatant was then applied to the column. Subsequently, 10 ml of Wash Solution I (same composition as the lysis buffer) containing 50 mM sodium phosphate and 10 mM imidazole, pH 8.0, was applied, followed by 10 ml of Wash Solution II containing 50 mM sodium phosphate and 20 mM imidazole, pH 8.0. After applying Wash Solutions I and II, the column was eluted with 3 ml of 50 mM sodium phosphate and 200 mM imidazole, pH 8.0, equivalent to 3 times the resin volume, to obtain the protein eluate.

[0117] The resulting protein eluate was placed in an Amicon centricon and centrifuged at 4,000 rpm for 20 minutes to concentrate the protein. The concentrate was then filled with 20 times the volume of 50 mM PIPES (pH 7.0) and centrifuged at 4,000 rpm four times to exchange the buffer with 50 mM PIPES (pH 7.0), thereby purifying the enzyme.

[0118] Example 6-3. Confirmation of purified enzyme activity The His-tag purified enzymes of BL21 pET21a_MDPE, BL21 pET21a_MDPE_A23I, or BL21 pET21a_MDPE_A23I / E267K prepared in Example 6-2 were prepared, and a reaction solution containing 1 mM manganese ions in 50 mM PIPES buffer (pH 7.0) was used so that the concentration of the reaction substrate fructose was 50 mM, and the concentration of the purified enzymes was adjusted to 0.008 mg / mL, and the reaction was carried out at 70°C for 30 minutes.

[0119] After the enzymatic reaction, the mixture was boiled for 5 minutes and centrifuged (13,000 rpm, 15 minutes) to collect the supernatant, which was then analyzed by high-performance liquid chromatography (HPLC). The liquid chromatography analysis was performed using an HPLC (Agilent, USA) equipped with a Cosmosil Sugar-D column and a Refractive Index Detector (RID, Agilent 1280 RID). The mobile phase solvent was 80% (v / v) acetonitrile, and the temperature was 30°C, with a flow rate of 1.0 mL / min. The enzyme activity analyzed by HPLC is shown in Table 8 below.

[0120] [Table 8]

[0121] The enzyme activity was confirmed in a conversion reaction carried out using the purified enzyme. Similar to the results of analyzing the enzyme activity using the bacterial cell reaction, the activity was increased compared to the wild-type enzyme.

[0122] Example 6-4. Confirmation of thermostability of purified enzyme The purified enzyme from BL21 pET21a_MDPE or BL21 pET21a_MDPE_A23I / E267K in Example 6-2 was heat-treated at 70°C for 30 minutes, 1 hour, 90 minutes, 3 hours, 5 hours, or 7 hours, and the remaining enzyme activity was analyzed by the HPLC analysis method in Example 6-3, and the results are shown in Table 9. The values ​​in Table 9 indicate the relative activity (%) of the enzyme after heat treatment to 100% enzyme activity before heat treatment.

[0123] [Table 9]

[0124] Due to its excellent thermal stability, the A23I / E267K mutant enzyme showed no significant half-life within the heat treatment time (0-7 hours). The wild-type enzyme had a half-life of approximately 1 hour and 40 minutes, while the A23I / E267K mutant enzyme maintained over 60% of its activity even after 7 hours of heat treatment. Substituting this data into the standard curve equation, the half-life of the A23I / E267K mutant enzyme was calculated to be approximately 9 hours. Experiments to confirm enzyme activity and thermal stability confirmed that the mutant enzyme can be used for commercial allulose production.

Claims

1. An allulose 3-epimerase protein having a sequence that has 80% or more sequence identity with a sequence in which one or more amino acids selected from the group consisting of amino acids at positions 23, 75, 115, 126, 255, 267, and 269 from the N-terminus in the amino acid sequence of SEQ ID NO: 1 are substituted with one or more amino acids selected from the group consisting of isoleucine (I), glutamic acid (E), glycine (G), valine (V), alanine (A), and lysine (K), excluding enzyme proteins having the amino acid sequence of SEQ ID NO:

1.

2. The allulose 3-epimerase protein of claim 1, which comprises one or more substitutions selected from the group consisting of a substitution of alanine at the 23rd position from the N-terminus with isoleucine, a substitution of serine at the 75th position with glutamic acid, a substitution of aspartic acid at the 115th position with glycine, a substitution of asparagine at the 126th position with valine, a substitution of leucine at the 255th position with valine, a substitution of glutamic acid at the 267th position with lysine, and a substitution of glycine at the 269th position with alanine in the amino acid sequence of SEQ ID NO:

1.

3. The allulose 3-epimerase protein according to claim 1, wherein the allulose 3-epimerase protein has a conversion activity of 130% to 200% based on 100% of the allulose conversion activity of an enzyme having the amino acid sequence of SEQ ID NO:

1.

4. The allulose 3-epimerase protein of claim 1, which comprises, in the amino acid sequence of SEQ ID NO: 1, one or more amino acid substitutions selected from the group consisting of a substitution of alanine at position 23 from the N-terminus with isoleucine, a substitution of serine at position 75 with glutamic acid, a substitution of aspartic acid at position 115 with glycine, a substitution of asparagine at position 126 with valine, a substitution of leucine at position 255 with valine, and a substitution of glycine at position 269 with alanine, and a substitution of glutamic acid at position 267 with lysine.

5. The allulose 3-epimerase protein according to claim 4, which has an allulose conversion activity of 130% to 200% based on 100% of the allulose conversion activity of an enzyme having the amino acid sequence of SEQ ID NO:

1.

6. The allulose 3-epimerase protein according to claim 4, wherein the allulose conversion activity after heat treatment at 70°C for 7 hours is 60 to 100%, based on the allulose conversion activity of the allulose 3-epimerase protein before heat treatment (100%).

7. A polynucleotide encoding the allulose 3-epimerase protein according to any one of claims 1 to 6.

8. A recombinant microorganism comprising a gene encoding the allulose 3-epimerase protein according to any one of claims 1 to 6.

9. 9. The recombinant microorganism according to claim 8, wherein the microorganism is at least one microorganism selected from the group consisting of strains of the genus Escherichia, strains of the genus Bacillus, strains of the genus Corynebacterium, strains of the genus Saccharomyces, and strains of the genus Pichia.

10. A composition for producing allulose, comprising at least one selected from the group consisting of the allulose 3-epimerase protein according to any one of claims 1 to 6, a recombinant microorganism expressing the allulose 3-epimerase protein, cells of the microorganism, a disrupted cell product of the microorganism, a culture of the microorganism, and an extract thereof.

11. The composition for allulose production according to claim 10, wherein the half-life of the allulose 3-epimerase protein is 130% to 500% based on 100% of the half-life of a wild-type enzyme protein having the amino acid sequence of SEQ ID NO: 1 under heat treatment conditions at 60°C.

12. The composition for allulose production according to claim 10, wherein the half-life of the allulose 3-epimerase protein is 1.7 to 50 hours under heat treatment conditions at 60°C.

13. The composition for allulose production according to claim 10, wherein the half-life of the allulose 3-epimerase protein is 0.5 to 20 hours under heat treatment conditions at 70°C.

14. The composition for producing allulose according to claim 10, further comprising one or more ions selected from the group consisting of manganese ions and cobalt ions.

15. A method for producing allulose, comprising a step of reacting at least one selected from the group consisting of the allulose 3-epimerase protein according to any one of claims 1 to 6, a recombinant microorganism expressing the allulose 3-epimerase protein, cells of the microorganism, disrupted cells of the microorganism, a culture of the microorganism, and an extract thereof, with a substrate.

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