Psicose 3-epimeric mutant enzyme and application thereof

Abstract

The present invention relates to a zymoprotein comprising a mutated amino acid sequence and having an activity of converting fructose into psicose, a recombinant microorganism comprising a gene encoding the zymoprotein, a composition for producing psicose comprising the zymoprotein, and a method for producing psicose using the zymoprotein.

Description

Technical Field

[0001] The present invention provides a psicose 3-epimerase mutant protein, a recombinant microorganism containing the enzyme protein, and / or a composition for producing psicose, and / or a method for producing psicose using the enzyme protein. Background Art

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

[0003] Psicose is produced by the epimerization of fructose (D-fructose), and its structure makes it difficult to be used as an energy source when ingested, so it has a lower calorie content. In this regard, it has the function of inhibiting glucose absorption and preventing blood sugar from rising, so it can be used as an effective sugar for diabetic diseases. In addition, it prevents fat accumulation by inhibiting the action of enzymes involved in fat accumulation, so it also has an effective sugar function for obesity. Furthermore, a material that can be converted into allose with anti-cancer effects can be prepared, and it has been reported that it also has a similar effect itself, so it is expected to be more widely used as a sugar with health care functions.

[0004] In addition, since its sweetness is 70% of that of sucrose, it is a sugar that can maintain an appropriate sweetness when consumed. Therefore, in recent years, it has been widely used as a functional sugar and is widely used in low-calorie beverages.

[0005] Since the function of psicose has been confirmed, research on methods for preparing psicose by fructose conversion has been ongoing. In addition, methods for preparing psicose using glucose, which has a lower cost than fructose, are currently being studied. Regarding the conversion from fructose to psicose, allose epimerases derived from Agrobacterium tumefaciens ( Agrobacterium tumefaciense ), Clostridium cellulolyticum ( Clostridium cellulolyticum ), and Clostridium bolteae ( Clostridium boleae ), tagatose epimerases derived from Pseudomonas cichorii ( Pseudomonas chichori ), Rhodobacter sphaeroides ( Rhodobacter sphaeroides ), and enzymes from microorganisms such as Arthrobacter globiformis ( Arthrobacter gloformis ), Staphylococcus aureus ( Staphylococcus aureus ), Mesorhizobium loti ( Mesorhizobium loti ), and Methylomonas ( Methylonmonus sp. ) are used for conversion research. Recently, in order to reduce the production cost of psicose, production by multi-enzyme conversion using glucose or starch has also been studied.

[0006] The production of allulose is usually achieved by the epimerization of the hydroxyl residue at the C-3 position of fructose. In an enzymatic reaction, the conversion rates of fructose and the substrate are usually balanced at a ratio of 70:30. Therefore, the conversion rate of allulose is generally at most 30%.

[0007] However, many reports confirm that increasing the temperature of the conversion reaction can increase the conversion rate of allulose. Therefore, while ensuring the thermal stability of the enzyme and enhancing its stability, the conversion rate can also be increased by raising the temperature of the conversion reaction.

[0008] Therefore, the purpose of this study is to search for enzyme mutants with improved thermal stability by studying mutations that enhance the thermal stability of the enzyme, and to increase the productivity of allulose through activity and thermal stability. Summary of the Invention

[0009] (I) Technical Problems to be Solved

[0010] One example of the present invention relates to an allulose 3-epimerase mutant protein with enhanced allulose conversion activity and thermal stability.

[0011] Another example of the present invention relates to a nucleic acid sequence encoding the mutant enzyme protein.

[0012] Another example of the present invention relates to a recombinant microorganism containing a nucleic acid sequence encoding the mutant enzyme protein.

[0013] Another example of the present invention relates to a composition for producing allulose using the mutant enzyme protein and / or the recombinant microorganism.

[0014] Another example of the present invention relates to a method for producing allulose from a substrate using the mutant enzyme protein and / or the recombinant microorganism.

[0015] (II) Technical Solutions

[0016] One example of the present invention provides an allulose 3-epimerase mutant protein in which some amino acids in the amino acid sequence of SEQ ID NO: 1 are replaced.

[0017] Specifically, the mutant enzyme can be one or more amino acids selected from the 23rd, 75th, 115th, 126th, 255th, 267th, and 269th amino acids starting from the N-terminus of the amino acid sequence of SEQ ID NO: 1.

[0018] An allulose 3-epimerase protein substituted with one or more amino acids selected from isoleucine (I), glutamic acid (E), glycine (G), valine (V), alanine (A), and lysine (K).

[0019] Another example of the present invention provides a polynucleotide encoding the enzyme protein of the present invention,

[0020] Specifically, it contains a nucleic acid sequence encoding one or more amino acids selected from the 23rd, 75th, 115th, 126th, 255th, 267th, and 269th amino acids counted from the N-terminus of the amino acid sequence of SEQ ID NO: 1,

[0021] A polynucleotide of a nucleic acid sequence of an amino acid sequence substituted with one or more amino acids selected from isoleucine (I), glutamic acid (E), glycine (G), valine (V), alanine (A), and lysine (K).

[0022] Another example of the present invention provides a recombinant vector containing the polynucleotide of the present invention.

[0023] Another example of the present invention provides a recombinant microorganism containing a gene encoding the enzyme protein of the present invention. The microorganism includes one or more, but not limited to, strains selected from the following groups: Escherichia coli ( Escherichia coli ), Bacillus ( Bacillus ), strains (for example, Bacillus subtilis ), Corynebacterium strains (for example, Corynebacterium glutamicum ( Corynebacterium glutamicum ), yeast ( Saccharomyces ), strains (for example, Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), and Pichia strains ( Pichia ), strains (for example, Pichia pastoris ( Pichia pastoris ).

[0024] Another example of the present invention provides a composition for producing allulose, which contains one or more of the enzyme protein of the present invention, the cells of a recombinant microorganism expressing the enzyme protein, the cell lysate of the microorganism, the culture of the microorganism, and its extract.

[0025] Another example of the present invention provides a method for producing allulose, which comprises a step of reacting one or more selected from the enzyme protein, recombinant microorganism expressing the enzyme protein, microbial cells of the microorganism, cell lysates of the microorganism, cultures of the microorganism, and extracts thereof with a substrate.

[0026] The enzyme protein according to the present invention comprises one or more amino acids selected from the 23rd, 75th, 115th, 126th, 255th, 267th, and 269th amino acids starting from the N-terminus of the amino acid sequence of SEQ ID NO: 1,

[0027] a sequence substituted with one or more amino acids selected from isoleucine (I), glutamic acid (E), glycine (G), valine (V), alanine (A), and lysine (K) has enhanced allulose conversion activity and thermal stability compared to the wild-type enzyme.

[0028] The recombinant microorganism according to the present invention comprising a gene encoding the enzyme protein comprises one or more amino acids selected from the 23rd, 75th, 115th, 126th, 255th, 267th, and 269th amino acids starting from the N-terminus of the amino acid sequence of SEQ ID NO: 1,

[0029] a sequence substituted with one or more amino acids selected from isoleucine (I), glutamic acid (E), glycine (G), valine (V), alanine (A), and lysine (K) has high allulose conversion activity and high thermal stability compared to a microorganism (wild-type strain) comprising a gene encoding an enzyme protein without amino acid mutation.

[0030] Hereinafter, the present invention will be described in more detail.

[0031] One example of the present invention provides an allulose 3-epimerase protein selected from one or more amino acids selected from the 23rd, 75th, 115th, 126th, 255th, 267th, and 269th amino acids starting from the N-terminus of the amino acid sequence of SEQ ID NO: 1, and substituted with one or more amino acids selected from isoleucine (I), glutamic acid (E), glycine (G), valine (V), and alanine (A).

[0032] Specifically, the replaced amino acid sequence may be one or more replacements selected from the following group, but is not limited thereto: the 23rd alanine (Alanine; A) from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is replaced by isoleucine (Isoleucine; I), the 75th serine (Serine; S) is replaced by glutamic acid (Glutamic Acid; E), the 115th aspartic acid (Aspartic Acid; D) is replaced by glycine (Glycine; G), the 126th asparagine (Arginine; R) is replaced by valine (Valine; V), the 255th leucine (Leucine; L) is replaced by valine, the 267th glutamic acid is replaced by lysine, and the 269th glycine is replaced by alanine.

[0033] In addition, the replaced amino acid sequence may be one or more replacements including the following group, but is not limited thereto: selected from the 23rd alanine from the N-terminus of the amino acid sequence of SEQ ID NO: 1 being replaced by isoleucine, the 75th serine being replaced by glutamic acid, the 115th aspartic acid being replaced by glycine, the 126th asparagine being replaced by valine, the 255th leucine being replaced by valine, and the 269th glycine being replaced by alanine,

[0034] and the 267th glutamic acid being replaced by lysine.

[0035] An example of the present invention provides a psicose 3-epimerase protein having a sequence homology of more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or more than 99.3% with the amino acid sequence of SEQ ID NO: 1.

[0036] Specifically, the psicose 3-epimerase protein provided by the present invention, in which one or more amino acids selected from the 23rd, 75th, 115th, 126th, 255th, 267th, and 269th amino acids from the N-terminus of the amino acid sequence of SEQ ID NO: 1 are replaced by one or more amino acids selected from isoleucine (I), glutamic acid (E), glycine (G), valine (V), and alanine (A), may have a sequence homology of more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or more than 99.3% with the amino acid sequence of SEQ ID NO: 1. The enzyme protein may not include the enzyme protein having the amino acid sequence of SEQ ID NO: 1, that is, the wild-type enzyme protein without mutation.

[0037] An example of the present invention provides a psicose 3-epimerase protein having a sequence in which one or more amino acids selected from the 23rd, 75th, 115th, 126th, 255th, 267th, and 269th amino acids from the N-terminus of the amino acid sequence of SEQ ID NO: 1 are replaced by one or more amino acids selected from isoleucine (I), glutamic acid (E), glycine (G), valine (V), and alanine (A), or having a sequence homology of more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or more than 99.3% with the amino acid sequence of SEQ ID NO: 1. The enzyme protein may not include the enzyme protein having the amino acid sequence of SEQ ID NO: 1, that is, the wild-type enzyme protein without mutation.

[0038] In the enzymatic reaction of the enzyme protein of the present invention, the allulose conversion activity can be measured using the bacterial cells expressing the enzyme, the culture broth for culturing the bacterial cells, and / or the supernatant obtained by centrifuging the culture broth. Specifically, it can be measured using the bacterial cells obtained after reacting the bacterial cells with the substrate, the culture broth for culturing the bacterial cells, and / or the supernatant obtained by centrifuging the culture broth. The substrate may include one or more selected from fructose, allulose, tagatose, xylose, sorbose, ribulose, and ketose, but is not limited thereto.

[0039] In the enzymatic reaction of the enzyme protein, the cells used for confirming the allulose conversion activity may be Escherichia coli (e.g., Escherichia coli ( Escherichia coli )) strains, but are not limited thereto.

[0040] In the substrate conversion reaction using the mutant enzyme protein or the microbial cells producing the mutant enzyme protein, based on 100% of the allulose conversion activity of the wild-type enzyme protein comprising the amino acid sequence of SEQ ID NO: 1, the allulose conversion activity of the enzyme protein according to the present invention or the composition for producing allulose according to the present invention may be 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 170%, 110% to 165%, 120% to 200%, 120% to 180%, 120% to 170%, 120% to 165%, 130% to 200%, 130% to 180%, 130% to 170%, 130% to 165%, 140% to 200%, 140% to 180%, 140% to 170%, 140% to 165%, 150% to 200%, 150% to 180%, 150% to 170%, 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% of the activity. The evaluation of the enzyme activity of the microbial cells or enzyme protein producing the enzyme may be completed by performing a substrate conversion reaction for 30 minutes at 70°C using the microbial cells expressing the enzyme protein, or by performing a substrate conversion reaction for 30 minutes at 70°C using the purified enzyme obtained by disrupting the microbial cells producing the enzyme.

[0041] The thermal stability of the enzyme protein according to the present invention or the enzyme protein of the composition for producing allulose according to the present invention may be confirmed by comparing the allulose conversion activity before and after heat treatment of the cells expressing the enzyme protein, the culture solution for culturing the cells and / or the supernatant obtained by centrifuging the culture solution, or by comparing with 100% of the allulose conversion activity before heat treatment and measuring the half-life of the enzyme protein when the activity reaches 50%, but is not limited thereto.

[0042] In the enzymatic reaction of the enzyme protein, the microbial cells used for confirming the allulose conversion activity may be Escherichia coli (e.g., Escherichia coli ( Escherichia coli )) strains, but are not limited thereto.

[0043] For the confirmation of the thermal stability, it can be achieved by subjecting the microbial cells expressing the enzyme protein or the purified enzyme obtained by disrupting the microbial cells producing the enzyme to heat treatment 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.

[0044] The half-life (hours (hr)) of the enzyme protein of the present invention or the enzyme protein of the composition for producing allulose of the present invention, under the heat treatment condition of 60 °C, based on 100% of the half-life of the enzyme protein expressed by the microorganism expressing the wild-type enzyme protein containing the amino acid sequence of SEQ ID NO: 1, can be 130% or more, 150% or more, 200% or more, 250% or more, 300% or more, 350% or more, 400% or more, 130% to 500%, 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%, 380% to 420%, for example, 133% or 400%, but is not limited thereto.

[0045] The half-life (hours (hr)) of the enzyme protein of the present invention or the enzyme protein of the composition for producing allulose of the present invention, under the heat treatment condition of 60 °C, can 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, 6 to 7 hours, for example, 2 hours or 6 hours, but is not limited thereto.

[0046] The enzyme protein for confirming the half-life under the heat treatment condition of 60 °C can be selected from

[0047] From the N-terminus of the amino acid sequence of SEQ ID NO: 1

[0048] One or more amino acids among the 23rd, 75th, 115th, 126th, 255th, 267th, and 269th amino acids

[0049] The psicose 3-epimerase protein substituted with one or more amino acids selected from isoleucine (I), glutamic acid (E), glycine (G), valine (V), alanine (A), and lysine (K).

[0050] The half-life (hours (hr)) of the enzyme protein of the present invention or the enzyme protein of the composition for producing psicose of the present invention under the heat treatment condition 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, 8 to 12 hours. For example, it can be 0.67 hours or 10 hours, but not limited thereto.

[0051] After the enzyme protein of the present invention or the enzyme protein of the composition for producing psicose of the present invention undergoes a heat treatment reaction at 70 °C for 7 hours, the psicose conversion activity can be 60% to 100% or 65% to 100% based on 100% of the psicose conversion activity of the enzyme protein before the heat treatment reaction. For example, it can have an activity of 65.57%, but not limited thereto.

[0052] The enzyme protein for which the half-life is confirmed under the heat treatment condition at 70 °C can be selected from

[0053] Starting from the N-terminus of the amino acid sequence of SEQ ID NO: 1

[0054] One or more amino acids among the 23rd, 75th, 115th, 126th, 255th, 267th, and 269th amino acids

[0055] The psicose 3-epimerase protein substituted with one or more amino acids selected from isoleucine (I), glutamic acid (E), glycine (G), valine (V), alanine (A), and lysine (K). Specifically,

[0056] It may include one or more amino acid substitutions selected from the substitution of alanine at the 23rd position from the N-terminus of the amino acid sequence of SEQ ID NO: 1 with isoleucine, the substitution of serine at the 75th position with glutamic acid, the substitution of aspartic acid at the 115th position with glycine, the substitution of asparagine at the 126th position with valine, the substitution of leucine at the 255th position with valine, and the substitution of glycine at the 269th position with alanine, and

[0057] the psicose 3-epimerase protein with glutamic acid at the 267th position substituted with lysine.

[0058] The enzyme protein of the present invention can be regulated for its activation by metal ions. Therefore, when producing psicose using the Microbacterium strain, adding metal ions can improve the conversion efficiency from fructose to psicose, that is, the psicose productivity. Therefore, a composition for producing psicose, which includes one or more selected from the enzyme protein, the recombinant microorganism expressing the enzyme protein, the cell body of the microorganism, the cell lysate of the microorganism, the culture of the microorganism, and its extract, or a composition for producing psicose using the microorganism producing the enzyme protein, may further include metal ions. In addition, the method for producing psicose using the enzyme protein may further include the step of adding metal ions.

[0059] The metal ions can be one or more selected from manganese ions, magnesium ions, nickel ions, and cobalt ions. In one example, the metal ions can be manganese ions, cobalt ions, or a mixture thereof. When manganese ions, cobalt ions, or a mixture thereof exist as metal ions, compared with the case where no metal ions are present, the psicose conversion activity can increase by 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.1 times, 2.2 times, or 2.3 times or more. For example, it can increase by 1.2 to 2.3 times.

[0060] Considering the effect of improving the yield of psicose, the addition amount of the metal ions can be 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.

[0061] Another example of the present invention provides a recombinant microorganism containing a gene encoding the enzyme protein of the present invention.

[0062] The microorganism can include one or more selected from the following groups, but is not limited thereto: Escherichia coli ( Escherichia coli ), Bacillus ( Bacillus ), strains (for example, Bacillus subtilis ), Corynebacterium ( Corynebacterium ), strains (for example, Corynebacterium glutamicum ( Corynebacterium glutamicum ), Saccharomyces ( Saccharomyces), strains (e.g., Saccharomyces cerevisiae ( Saccharomyces cerevisiae )) and Pichia strains ( Pichia ), e.g., Pichia pastoris ( Pichia pastoris ).

[0063] Another example of the present invention provides a composition for producing allulose, which comprises one or more of the enzyme protein of the present invention, a recombinant microorganism expressing the enzyme protein, the cell mass of the microorganism, the cell lysate of the microorganism, the culture of the microorganism, and its extract.

[0064] The culture contains the enzyme produced from the recombinant microorganism, and can be in the form containing the microorganism or in a cell-free form without the microorganism. In addition, the lysate contains the lysate obtained by disrupting the cell mass of the recombinant microorganism or the supernatant obtained by centrifuging the lysate, and the enzyme produced from the recombinant microorganism.

[0065] In this specification, unless otherwise specified, the recombinant microorganism for producing allulose refers to one or more selected from the cell mass of the microorganism, the culture of the strain, and the lysate of the strain.

[0066] Another example of the present invention provides a method for producing allulose, which comprises a step of reacting one or more selected from the enzyme protein of the present invention, a recombinant microorganism expressing the enzyme protein, the cell mass of the microorganism, the cell lysate of the microorganism, the culture of the microorganism, and its extract with a substrate.

[0067] The substrate may comprise one or more selected from fructose, allulose, tagatose, xylose, sorbose, ribulose, and ketose, but is not limited thereto.

[0068] The method for producing allulose may further comprise a step of separating and / or purifying allulose from the reaction product. The culture may be a culture in which one or more selected from the recombinant microorganism, the cell mass of the recombinant microorganism, the cell mass of the microorganism, the cell lysate of the microorganism, the culture of the microorganism, and its extract react with the substrate, but is not limited thereto.

[0069] Specifically, the method for producing allulose according to the present invention can carry out the separation process of the allulose reaction product, which includes ion purification and simulated moving bed (SMB) chromatography separation of the allulose conversion reaction product. In a specific example, the allulose conversion reaction product is subjected to simulated moving bed (SMB) chromatography separation to separate an allulose component with an allulose content higher than that of the conversion reaction product and a fructose raffinate. The allulose component can be commercialized as a liquid syrup through an allulose concentration process or can be commercialized as allulose crystals through an allulose crystallization process. The allulose crystallization process includes the steps of performing a second ion purification on the allulose component obtained in the high-purity separation process, concentrating the ion-purified allulose component, and crystallizing allulose from the concentrate to obtain allulose crystals and an allulose crystallization mother liquor.

[0070] (III) Beneficial effects

[0071] The present invention relates to an enzyme protein with allulose 3-epimerase activity having specific amino acid mutations, a recombinant microorganism containing the enzyme protein, a composition for producing allulose, or a method for producing allulose using the enzyme protein, which has high conversion activity from fructose to allulose and excellent thermal stability. Brief description of the drawings

[0072] Figure 1 Schematic diagram showing a recombinant vector in which the D-allulose 3-epimerase gene is cloned into a vector according to an example of the present invention.

[0073] Figure 2 Schematic diagram showing a recombinant vector in which the D-allulose 3-epimerase gene for enzyme purification is cloned into a vector. Detailed description of the specific embodiments

[0074] The present invention will be described in more detail by the following examples, and is not intended to limit the scope of the rights by the following examples.

[0075] Example 1 Cloning of the D-allulose 3-epimerase gene

[0076] Example 1-1. Preparation of the enzyme gene

[0077] Microbacterium foliorum ( Microbacterium foliorumThe D-tagatose 3-epimerase identified in (SY27B-MF;Accession No.: KCCM11774P) was expressed in Escherichia coli to confirm the enzyme activity, and a DNA sequence was synthesized to search for variants with enhanced high thermal stability. To ensure correct expression in Escherichia coli, the amino acid sequence of the protein was synthesized using codons optimized for Escherichia coli.

[0078] The gene sequence obtained by codon optimization was commissioned to Integrated DNA Technologies, Inc. (IDT, USA) for synthesis. The synthesized gene was named MDPE, and the corresponding gene information after codon optimization is shown in Table 1 below. The amino acid sequence of the MDPE gene corresponds to the sequence of SEQ ID NO: 1, and the base sequence corresponds to the sequence of SEQ ID NO: 2.

[0079] Table 1

[0080]

[0081] Example 1-2. Preparation of recombinant microorganisms containing the enzyme gene

[0082] The MDPE polynucleotide synthesized in Example 1-1. by codon optimization for expression in Escherichia coli was amplified by PCR (Polymerase Chain Reaction). Specifically, a gene amplification (Polymerase Chain Reaction, PCR) reaction solution with a final volume of 50 μl was prepared, which contained 50 ng of the above-synthesized MDPE gene, 10 pmol of the forward primer (SEQ ID NO: 3 base sequence) for each amino acid residue, 10 pmol of the reverse primer (SEQ ID NO: 4 base sequence), 1 μl of Phusion polymerase, 10 μl of reaction buffer (5X), and 1 μl of 10 mM dNTP. PCR amplification was carried out, and the sequence information of the primers used is shown in Table 2 below.

[0083] Table 2

[0084]

[0085] Specifically, using the above PCR reaction solution, a PCR reaction was carried out in a GeneAmp PCR System 9700, including 1 cycle at 98°C (30 seconds), 18 cycles of [98°C (10 seconds), 55°C (30 seconds), 72°C (2 minutes 30 seconds)], and 1 cycle at 72°C (5 minutes) to ensure a large amount of gene after gene amplification.

[0086] After cutting the pUC19 vector (New England Biolabs (NEB)) with the restriction enzymes HindIII and BamHI, the amplified gene was ligated with the pUC19 vector using 2X HiFi DNA master mix (NEB) and inserted into the restriction enzyme site to construct the pUC19 / allulose 3-epimerase recombinant vector (pUC19_MDPE). The schematic diagram of the constructed recombinant vector is as shown in Figure 1 shown. The constructed recombinant vector was transformed into Escherichia coli DH10b competent cells (TransGen, Trans10) by heat shock to prepare recombinant microorganisms (DH10b pUC19_MDPE).

[0087] Example 2 Induction of mutations in the D-allulose 3-epimerase gene

[0088] Based on the amino acid sequence analysis between homologous genes, the active site, and the tertiary structure model analysis of the metal binding site, the selected amino acid substitutions were mutated to other amino acids. After producing these recombinant mutant enzymes in Escherichia coli, the changes in the activity of allulose epimerase were analyzed.

[0089] Example 2-1. Site-directed mutagenesis (SDM)

[0090] The sequences of D-allulose 3-epimerase from Arthrobacter globiformis ( Arthrobacter globiformis ), Agrobacterium tumefaciens ( Agrobacterium tumefaciense ), Clostridium cellulolyticum ( Clostridium cellulolyticum ), and Methylomonas spp. ( Methylomonus sp ) in the confirmed sequence of the epimerase were analyzed to identify the homologous regions, and the mutation sites were determined based on the homologous regions.

[0091] Specifically, by comparing the homology of the epimerase, it was confirmed that the 23rd amino acid in the D-allulose 3-epimerase from Agrobacterium tumefaciens ( Agrobacterium tumefaciense ), Clostridium cellulolyticum ( Clostridium cellulolyticum ), and Methylomonas spp. ( Methylomonus sp ) is isoleucine, so it was selected as the mutation site. Compared with the D-allulose 3-epimerase from Arthrobacter globiformis, it was confirmed that the 75th amino acid is glutamate, so it was selected as the mutation site. The Protein Data Bank was used to confirm the selected sites to maintain the structural activity of the enzyme.

[0092] The substitution mutations of the MDPE were generated by site-directed mutagenesis (SDM) using Quikchange® (Stratagene) applied in previous studies. The Quikchange® site-directed mutagenesis method of Stratagene amplifies the entire plasmid DNA by PCR and degrades the template DNA with DpnI enzyme, rather than amplifying the corresponding gene of the enzyme part by PCR and subcloning it into the plasmid DNA. The Quikchange® site-directed mutagenesis method has the advantage of being able to induce mutations faster.

[0093] Using the pUC19_MDPE of Example 1-2. as the template DNA, the replication of the MDPE substitution mutation of Example 1 was carried out. A gene amplification (polymerase chain reaction, PCR) reaction solution with a final volume of 50 μl was prepared, which contained 10 ng of pUC19_MDPE, 10 pmol of the forward primer for each amino acid residue (I23, E75) (base sequence of SEQ ID NO: 5 or 7), 10 pmol of the reverse primer (base sequence of SEQ ID NO: 6 or 8), 1 μl of Phusion polymerase, 10 μl of reaction buffer (5X), and 1 μl of 10 mM dNTP, and PCR amplification was carried out. The primer information used is shown in Table 3 below.

[0094] Table 3

[0095]

[0096] Specifically, using the said PCR reaction solution, a PCR reaction was carried out in a GeneAmp PCR system 9700, including 1 cycle at 98 °C (30 seconds), 18 cycles of [98 °C (10 seconds), 55 °C (30 seconds), 72 °C (2 minutes 30 seconds)], and 1 cycle at 72 °C (5 minutes) to ensure a large number of genes after gene amplification.

[0097] 1 μl of DpnI (10 units / μl, NEB) enzyme that recognizes and degrades the non-mutagenized template DNA was added to the reaction solution with a volume of 50 μl after PCR and treated at 37 °C for 3 hours. After the treatment, according to the method basically the same as the preparation method of the recombinant strain DH10b pUC19_MDPE of Example 1-2., the gene pUC19_MDPE_A23I (A23I mutation) treated by PCR using the MDPE_A23I_cloning primer and the gene pUC19_MDPE_S75E (S75E mutation) treated by PCR using the MDPE_S75E_cloning primer were respectively transformed into Escherichia coli HD10b, thereby obtaining the recombinant strains DH10b pUC19_MDPE_A23I and DH10b pUC19_MDPE_S75E.

[0098] To identify the enzyme mutations in the recombinant strains prepared above, comparison was made using the NCBI website (BLAST) tool and sequence alignment with the amino acid sequence of the wild-type enzyme.

[0099] Example 2-2. Mutation by Error Prone PCR

[0100] Mutants were prepared by error prone PCR that induces mutations during gene amplification. Specifically, to prepare mutants, 10 ng of template DNA (pUC19_MDPE), 10 pmol of the forward primer for each amino acid residue (base sequence of SEQ ID NO: 9), 10 pmol of the reverse primer (base 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 mixture, 1 μl of dGTP (2 mM), and 4 μl of MnSO4 were added, and PCR-grade water was added to make the final volume of the gene amplification (polymerase chain reaction, PCR) reaction solution 50 μl. PCR amplification was carried out, and the primer information used is shown in Table 3 below.

[0101] Specifically, using the said PCR reaction solution, after 1 cycle of 94 °C for 30 seconds in a GeneAmp PCR system 9700, 25 cycles of 94 °C for 30 seconds and 68 °C for 1 minute were repeated, and then the reaction was carried out at 68 °C for 1 minute.

[0102] The mutagenically amplified gene was electrophoresed to ensure the band at the desired site, and the ensured gene band was extracted. According to the method substantially the same as the preparation method of the recombinant strain DH10b pUC19_MDPE in Example 1-2., the PCR-treated gene was transformed into Escherichia coli DH10b to ensure recombinant microorganisms.

[0103] Example 2-3. Mutation Screening Using HTS (High-Throughput-Screening)

[0104] The transformed recombinant microorganisms in Example 2-2 were spread on an LB ampicillin solid medium composed of 10 g / L of Bacto tryptone (Difco), 5 g / L of yeast extract (Difco), 10 g / L of sodium chloride (Taisho Chemical), 100 mg / L of ampicillin (Sigma), and 20 g / L of Bactor agar for culture. The ensured colonies were first screened using HTS.

[0105] After the first screening, 300 μL of LB-ampicillin liquid medium (composed of 10 g / L Bacto tryptone (Difco), 5 g / L yeast extract (Difco), 10 g / L sodium chloride (Taisho Chemical), and 100 mg / L ampicillin (Sigma)) was inoculated into a 96-well plate (Greiner) multi-colony incubator and shaken at 37°C and 200 rpm for 16 to 18 hours.

[0106] The enzyme activity of the shaken culture cells was confirmed by the fructose-dehydrogenase assay. In this method, 75 μL of the culture solution obtained from the above shaking culture was transferred to a 96-well plate, and an equal volume of 100 mM psicose substrate (100 mM Mcilvaine buffer pH 6.0) was added, and the cell reaction was carried out at 60°C for 30 minutes.

[0107] 10 μL of the reaction solution in which psicose was converted to fructose was transferred to a new 96-well plate and mixed with reaction solution A (150 mM Mcilvain buffer pH 4.5), reaction solution B (0.1 M potassium ferricyanide, 0.1% Triton X-100), and reaction solution C (0.1 M potassium ferricyanide, 0.1% Triton, 0.05% BSA) in a volume ratio of 70:10:10, and then 90 μL was added to the reaction solution converted to fructose.

[0108] Then, after incubation at 37°C for 30 minutes, 50 μL of reaction solution D (1.25 mM Iron(III) sulfate hydrate, 0.3% SDS, 8% phosphoric acid) was added, and the reaction was terminated by incubation at 37°C for 20 minutes. After the reaction was terminated, the absorbance was measured at 660 nm and compared with the recombinant strain DH10b pUC19_MDPE without enzyme mutation in Examples 1-2., and the strains with high absorbance were screened out.

[0109] Example 2-4. Confirmation of mutant amino acid residues of mutant proteins

[0110] Among the recombinant microorganisms obtained in Example 2-1. above and the mutants obtained by error-prone PCR in Example 2-2. above, the amino acid residue mutations of the enzyme screened in Example 2-3. above were confirmed.

[0111] Specifically, after obtaining the purified plasmid using a plasmid preparation kit (GENALL), the gene sequence of the subcloned epimerase part of the plasmid was transcribed into the amino acid sequence of the gene sequence through a sequence analysis service (Macrogen). By comparing with the amino acid sequence of the wild type, the mutation sites were confirmed. The primer information used is shown in Table 4 below.

[0112] Table 4

[0113]

[0114] Among the enzymes in the recombinant strains, strains containing enzymes with confirmed A23I, S75E, D115G, R126V, L255V, and G269A mutations were first screened out, and 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) were prepared.

[0115] Using the SWIISS-MODEL (http: / / www.expasy.org / swissmod / SWIIS-MODEL.html), the three-dimensional structure of the enzyme was generated through template search in the Protein data bank (PDB) using the protein three-dimensional structure homology model, the positions of the amino acids mutated through the above reaction were confirmed, and the morphology of the three-dimensional structure was confirmed.

[0116] Example 3 Activity Evaluation of the First-Screened Mutant Enzymes

[0117] Example 3-1. Confirming the Enzyme Activity Using the Strain Producing the Wild-Type Enzyme

[0118] The DH10b pUC19_MDPE strain of Example 1 above was spread on an LB ampicillin solid medium composed of 10 g / L of Bacto tryptone (Difco), 5 g / L of yeast extract (Difco), 10 g / L of sodium chloride (Taisho Chemical), 100 mg / L of ampicillin (Sigma), and 20 g / L of Bactor agar for culture. One colony was picked from the culture result of the solid medium and inoculated into 3 ml of LB-ampicillin liquid medium (composed of 10 g / L of Bacto tryptone (Difco), 5 g / L of yeast extract (Difco), 10 g / L of sodium chloride (Taisho Chemical), and 100 mg / L of ampicillin (Sigma)), and shaken culture was carried out at 37°C and 250 rpm for 16 to 18 hours.

[0119] After the above-mentioned bacterial cell culture was completed, when the OD 600nm reached 2 to 3, a transformation reaction was carried out using the microbial cells. Specifically, for the transformation reaction using the microbial cells producing the mutant enzyme, a reaction solution containing 50 mM PIPES buffer (pH 7.0) with 1 mM manganese ions was used, such that the fructose concentration as the reaction substrate was 400 g / L, the cell concentration of the strain was 1 mg (dcw) / mL, and the reaction was carried out at 70°C for 30 minutes.

[0120] After the above-mentioned transformation reaction was completed, the supernatant was recovered by centrifugation (13,000 rpm, 15 minutes) and analyzed by high-performance liquid chromatography (HPLC). HPLC analysis was carried out using an HPLC (Agilent, USA) equipped with a Cosmosil SugarD column and an RID (refractive index detector, Agilent 1280 RID). The mobile phase solvent was 80% (v / v) acetonitrile, the temperature was 30°C, and the flow rate was 1.0 mL / minute.

[0121] The results obtained by HPLC analysis of the supernatant of the above microbial culture solution are shown in Table 5 below.

[0122] Example 3-2. Confirming the enzyme activity using the mutant enzyme-producing strain

[0123] For the strains with enzyme mutations that confirmed the above Example 2 (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), the strains were cultured in a method substantially the same as that of the above Example 3-1, subjected to a substrate conversion reaction, and the obtained products were analyzed by HPLC analysis. The analysis results are shown in Table 5 below.

[0124] In Table 5 below, the substrate conversion activity is expressed as the relative conversion activity based on 100% of the substrate conversion activity of the strain producing the wild-type enzyme.

[0125] Table 5

[0126]

[0127] Using the strains producing the enzyme to confirm the substrate conversion activity of the enzyme, the results showed that the recombinant strains expressing the A23I, S75E, and R126V mutant enzymes showed more than 150% increase in activity compared with the recombinant strains expressing the wild-type enzyme, and the recombinant strains expressing the D115G, L255V, or G269A mutant enzymes showed about 107% to 125% increase in conversion activity compared with the recombinant strains expressing the wild-type enzyme.

[0128] Example 4 Thermal Stability Evaluation of the Mutant Enzyme Strains from the First Screening

[0129] To test the thermal stability of the strains producing the mutant enzymes from the first screening, the strains with confirmed enzyme mutations and increased activity in the above Example 3-2 were subjected to high-temperature heat treatment. Specifically, in the experiment to determine the degree of enzyme activity maintenance according to the high-temperature treatment time, only the strains expressing the mutant enzyme were recovered, suspended in 50 mM PIPES buffer (pH 7.0), and heat-treated at 60 °C.

[0130] After the heat treatment, fructose substrate and manganese ions were added in a process substantially the same as that of the above Example 3-2 to carry out the cell reaction, and the conversion activity was measured. The half-life of the activity was calculated based on the conversion activity measured over time. Based on 100% of the activity before the heat treatment reaction, the thermal stability level of the mutant strains was confirmed, and the half-life when the activity of the mutant strains reached 50% was measured.

[0131] The results of measuring the half-life showed that the half-life of the bacterial cells expressing the wild-type enzyme was 1.5 hours, that of DH10b pUC19_MDPE_A23I was 6 hours, and that of DH10b pUC19_MDPE_G269A was 2 hours. In addition, expressed as a relative percentage based on the half-life (1.5 hours) of the bacterial cells producing the wild-type enzyme as 1, the relative half-life of DH10b pUC19_MDPE_A23I was 4 times that of the wild-type enzyme, and the relative half-life of pUC19_MDPE_G269A was 1.33 times that of the wild-type enzyme. Thus, it was confirmed that the thermal stability of the bacterial cells expressing the mutant enzyme was superior to that of the bacterial cells expressing the wild-type enzyme.

[0132] Example 5 Second Mutant Screening and Enzyme Characterization Analysis

[0133] Example 5-1. Second Mutation Screening

[0134] Using A23I with excellent enzyme activity and thermal stability screened in the above Examples 3 and 4 as the template DNA, mutagenesis was carried out by secondary mutagenic gene amplification (error-prone PCR). During this process, further mutations occurred in the first mutant A23I, and thus the effect of double mutations could be confirmed.

[0135] Specifically, according to the method substantially the same as that in Example 2-1., using pUC19_MDPE and MDPE_A23I_cloning primers, a recombinant vector (pUC19_MDPE_A23I) with the A23I mutation was constructed.

[0136] Using the constructed pUC19_MDPE_A23I, enzyme mutations were induced according to the method substantially the same as the error-prone PCR process in Example 2-2., and subcloned into pUC19. Mutant bacterial cells with increased activity were screened from the subcloned mutant bacterial cells by HTS in Example 2-3., and strains with A23I and E267K mutations (DH10b pUC19_MDPE_A23I / E267K) were obtained according to the method substantially the same as that in Example 2-4.

[0137] Example 5-2. Confirming Enzyme Activity Using the Mutant Enzyme-Producing Strains Screened in the Second Round

[0138] For 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, the transformation activities of the strains were confirmed by the method substantially the same as that in Example 3-2., and the results are shown in Table 6 below.

[0139] In Table 6 below, the substrate conversion activity is expressed as the relative activity based on 100% of the substrate conversion activity of the strain producing the wild-type enzyme.

[0140] Table 6

[0141]

[0142] The conversion activity of the enzyme was analyzed using the cell reaction of the enzyme-producing microorganism. As a result, it was confirmed that the mutant enzyme with the double mutations of A23I and E267K had the same or higher enzyme activity compared to the mutant enzyme with only the A23I mutation.

[0143] Example 5-3. Confirming the Thermal Stability of the Mutant Enzyme Produced by the Strains Selected in the Second Screening

[0144] To confirm the thermal stability of the mutant enzyme strains selected in the second screening, wild-type enzyme strains with low thermal stability were removed. In a method substantially the same as that of Example 4, DH10b pUC19_MDPE_A23I and DH10b pUC19_MDPE_A23I / E267K were heat-treated at 70°C, the degree of enzyme activity was analyzed, the half-life was calculated, and the thermal stability was evaluated.

[0145] The results of measuring the half-life showed that 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. Thus, it was confirmed that the strain producing the A23I / E267K double mutant enzyme had more excellent thermal stability.

[0146] Example 6 Confirming the Activity and Thermal Stability of the Purified Enzyme

[0147] Example 6-1. Preparing a Recombinant Strain for Enzyme Purification

[0148] To prepare a recombinant strain for enzyme purification, in a method substantially the same as that of Example 2-4, the plasmid pUC19_MDPE_A23I / E267K purified from the DH10b pUC19_MDPE_A23I / E267K recombinant strain of Example 5-1 was prepared.

[0149] For each template, prepare a gene amplification (PCR) reaction solution with a final volume of 50 μl containing 10 ng each of the prepared pUC19_MDPE_A23I / E267K, pUC19_MDPE of the above Examples 1-2, and pUC19_MDPE_A23I of the above Example 2-1, 10 pmol of the forward primer (base sequence of SEQ ID NO: 13) for each amino acid residue, 10 pmol of the reverse primer (base sequence of SEQ ID NO: 14), 1 μl of Phusion polymerase, 10 μl of reaction buffer (5X), and 1 μl of 10 mM dNTP. Perform PCR, and the primer information used is shown in Table 7 below.

[0150] Table 7

[0151]

[0152] Using the above PCR reaction solution, perform a PCR reaction in a GeneAmp PCR System 9700, including 1 cycle at 98°C (30 seconds), 30 cycles of [98°C (10 seconds), 55°C (30 seconds), 72°C (30 seconds)], and 1 cycle at 72°C (3 minutes). Subclone the obtained gene product and insert it into the pET21a (Novagene) plasmid obtained by treatment with NdeI and XhoI enzymes.

[0153] Specifically, in order to be able to express under the strong T7 promoter, fuse the amplified gene with His-Tag, cut the pET21a vector with restriction enzymes NdeI and XhoI to enable expression, and then use the In-Fusion Snap Assembly kit (Takara) for ligation to construct the recombinant vector pET21a / allulose 3-epimerase (pET21a_MDPE). The schematic diagram of the constructed recombinant vector is as Figure 2 shown. Transform the constructed recombinant vector into Escherichia coli BL21(DE3) by the heat shock method, and prepare a recombinant strain (BL21 pET21a_MDPE).

[0154] In addition, use pET21a_MDPE instead of pUC19_MDPE, perform a PCR reaction according to the method substantially the same as that of Example 2 above, and then transform the PCR-amplified gene into Escherichia coli BL21(DE3) by the heat shock method to prepare a recombinant strain.

[0155] For the recombinant strains obtained above, according to the method substantially the same as that in Example 2, the amino acid residue mutations of the enzyme were confirmed, and a strain confirmed to have an A23I mutation (BL21 pET21a_MDPE_A23I) was prepared. According to the method substantially the same as that in Example 5 above, a strain confirmed to have A21I and E267K mutations (BL21 pET21a_MDPE_A23I / E267K) was prepared.

[0156] Example 6-2. Enzyme Purification

[0157] 3 ml of each of the strains BL21 pET21a_MDPE, BL21 pET21a_MDPE_A23I or BL21 pET21a_MDPE_A23I / E267K was inoculated into an LB-ampicillin liquid medium (composed of 10 g / L of Bacto tryptone (Difco), 5 g / L of yeast extract (Difco), 10 g / L of sodium chloride (Taisho Chemical), and 100 mg / L of ampicillin (Sigma)), and cultured with shaking at 37°C and 250 rpm until the absorbance (OD) at 600 nm reached 1.5. Then, the culture solution was inoculated into 50 ml of a liquid medium of the same type as the LB-ampicillin liquid medium, and cultured with shaking at 37°C and 250 rpm.

[0158] When the absorbance of the culture solution during shaking culture at 600 nm was 0.5, 0.1 mM IPTG was added to induce the overexpression of the target enzyme. From the time point of overexpression induction, the culture conditions were changed to 20°C and 180 rpm and maintained for 16 hours. Then, the cells were centrifuged at 4000 rpm for 10 minutes, and the cells were recovered.

[0159] After dissolving 50 mL of the recovered cells in 1 mL of cooled 50 mM PIPES pH 7.0 buffer, the cells were disrupted 5 times with a bead mill for 20 seconds each. The cell lysate was centrifuged at 13000 rpm at 4°C for 10 minutes, and only the supernatant was recovered.

[0160] Fill 1 ml of Ni-NTA resin into a Bio-RAD poly-prep chromatography column, and add 10 ml of lysis buffer (50 mM sodium phosphate, 10 mM imidazole, pH 8.0), which is 10 times the resin packing volume, to the column to equilibrate the resin with the buffer. Then add the recovered supernatant to the column. Subsequently, add 10 ml of Wash Buffer I (having the same composition as the lysis buffer) of 50 mM sodium phosphate and 10 mM imidazole, pH 8.0, and add 10 ml of Wash Buffer II of 50 mM sodium phosphate and 20 mM imidazole, pH 8.0. After adding Wash Buffer I and Wash Buffer II, elute with 3 ml of 50 mM sodium phosphate and 200 mM imidazole, pH 8.0 solution, which is 3 times the resin packing volume, to obtain a protein eluate.

[0161] Put the obtained protein eluate into an Amicon centricon and centrifuge at 4000 rpm for 20 minutes to concentrate the protein. Purify the enzyme by filling with 20 times the volume of 50 mM PIPES, pH 7.0, and repeating centrifugation 4 times at 4000 rpm, and changing the buffer to 50 mM PIPES, pH 7.0.

[0162] Example 6-3. Confirmation of purified enzyme activity

[0163] Prepare the His-tag purified enzymes of BL21 pET21a_MDPE, BL21 pET21a_MDPE_A23I, or BL21 pET21a_MDPE_A23I / E267K prepared in Example 6-2 above. Use a reaction solution containing 1 mM manganese ions in 50 mM PIPES buffer (pH 7.0) to make the fructose concentration of the reaction substrate reach 50 mM, and set the concentration of the purified enzyme to 0.008 mg / ml. React at 70 °C for 30 minutes.

[0164] After the enzyme reaction is completed, boil for 5 minutes, centrifuge (13000 rpm, 15 minutes) and recover the supernatant, and then analyze it by high performance liquid chromatography (HPLC). Perform the HPLC analysis using RID (refractive index detector, Agilent 1280 RID) of HPLC (Agilent, USA) equipped with a Cosmosil Sugar D column. The mobile phase solvent uses 80% (v / v) acetonitrile, the temperature is 30 °C, and the flow rate is 1.0 mL / minute. The enzyme activities analyzed by the HPLC are shown in Table 8 below.

[0165] Table 8

[0166]

[0167] The results of confirming enzyme activity through the conversion reaction using the purified enzyme showed an increase in enzyme activity compared to the wild-type enzyme, which was similar to the results of the enzyme activity analysis using the whole-cell reaction.

[0168] Example 6-4. Confirmation of the thermal stability of the purified enzyme

[0169] The purified enzyme of 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 then the remaining enzyme activity was analyzed by the HPLC analysis method of Example 6-3. The results are shown in Table 9 below. The values shown in Table 9 refer to the relative activity (%) of the enzyme after heat treatment compared to 100% of the enzyme activity before heat treatment.

[0170] Table 9

[0171]

[0172] The results of confirming the thermal stability showed that due to the excellent thermal stability of the A23I / E267K mutant enzyme, the half-life of enzyme activity could not be confirmed within the heat treatment time (7 hours). The half-life of the wild-type enzyme was confirmed to be approximately 1 hour and 40 minutes, and it was confirmed that the A23I / E267K mutant enzyme still maintained more than about 60% of its activity even after heat treatment for 7 hours. The result of calculating the half-life of the A23I / E267K mutant enzyme by substituting it into the calibration curve formula showed that it was confirmed to be approximately 9 hours. Through the enzyme activity and thermal stability confirmation experiments, it was confirmed that the mutant enzyme was applicable to the industrial production of allulose.

Claims

1. An allulose 3-epimerase protein, which is an enzyme protein not including the one with the amino acid sequence of SEQ ID NO: 1, wherein, The enzyme protein has a sequence with more than 80% sequence homology to a sequence in which one or more amino acids selected from the 23rd, 75th, 115th, 126th, 255th, 267th, and 269th amino acids from the N-terminus of the amino acid sequence of SEQ ID NO: 1 are replaced by one or more amino acids selected from isoleucine, glutamic acid, glycine, valine, alanine, and lysine.

2. The psicose 3-epimerase protein according to claim 1, wherein, The enzyme protein comprises one or more replacements selected from the group consisting of: Starting from the N-terminus of the amino acid sequence of SEQ ID NO: 1 The 23rd alanine is replaced by isoleucine, The 75th serine is replaced by glutamic acid, The 115th aspartic acid is replaced by glycine, The 126th asparagine is replaced by valine, The 255th leucine is replaced by valine, The 267th glutamic acid is replaced by lysine, and The 269th glycine is replaced by alanine.

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

4. The psicose 3-epimerase protein according to claim 1, wherein, The enzyme protein comprises one or more amino acid replacements selected from the replacement of the 23rd alanine by isoleucine, the 75th serine by glutamic acid, the 115th aspartic acid by glycine, the 126th asparagine by valine, the 255th leucine by valine, and the 269th glycine by alanine from the N-terminus of the amino acid sequence of SEQ ID NO: 1, and the replacement of the 267th glutamic acid by lysine.

5. The psicose 3-epimerase protein according to claim 4, wherein, Based on 100% of the allulose conversion activity of the enzyme comprising the amino acid sequence of SEQ ID NO: 1, the enzyme protein has a conversion activity of 130% to 200%.

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

7. A polynucleotide encoding the enzyme protein according to any one of claims 1 to 6.

8. A recombinant microorganism comprising a gene encoding the enzyme protein according to any one of claims 1 to 6.

9. The recombinant microorganism according to claim 8, wherein, The microorganism is one or more microorganisms selected from the group consisting of Escherichia ( Escherichia coli ), Bacillus ( Bacillus ), Corynebacterium ( Corynebacterium ), Saccharomyces ( Saccharomyces ), and Pichia ( Pichia ) strains.

10. A composition for producing allulose, comprising one or more selected from the enzyme protein according to any one of claims 1 to 6, a recombinant microorganism expressing the enzyme protein, the cells of the microorganism, the cell lysate of the microorganism, the culture of the microorganism, and its extract.

11. The composition for producing allulose according to claim 10, wherein, Under the heat treatment condition of 60 °C, the half-life of the enzyme protein is 130% to 500% based on 100% of the half-life of the wild-type enzyme protein comprising the amino acid sequence of SEQ ID NO:

1.

12. The composition for producing allulose according to claim 10, wherein, Under the heat treatment condition of 60 °C, the half-life of the enzyme protein is 1.7 to 50 hours.

13. The composition for producing allulose according to claim 10, wherein, Under the heat treatment condition of 70 °C, the half-life of the enzyme protein is 0.5 to 20 hours.

14. The composition for producing allulose according to claim 10, wherein, The composition further comprises one or more selected from manganese ions and cobalt ions.

15. A method for producing allulose, which comprises a step of reacting one or more selected from the enzyme proteins described in any one of claims 1 to 6, recombinant microorganisms expressing the enzyme proteins, cell bodies of the microorganisms, cell lysates of the microorganisms, cultures of the microorganisms, and extracts thereof with a substrate.

Images