Fructose-6-phosphate 3-epimerase and its uses

Abstract

The present invention relates to a fructose-6-phosphate epimerization enzyme protein, a nucleic acid molecule encoding the enzyme protein, a recombinant vector and transformed microorganism containing the nucleic acid molecule, and a composition for producing allulose using these.

Description

Technical Field

[0001] The present invention relates to a phosphorylated sugar epimerase protein, more specifically, a 3-epimerase protein of fructose-6-phophate, a nucleic acid molecule encoding the enzyme protein, a recombinant vector and a transformed strain containing the nucleic acid molecule, and a composition for producing allose using the strain.

Background Art

[0002] The epimerase of phosphorylated sugar is an epimerase bound to carbon in various phosphorylated sugars, and the ketohexose-6-phosphate epimerase can epimerize C3 or C4. The ketohexose may be one or more ketohexoses selected from the group consisting of fructose, allose, sorbose, and tagatose.

[0003] The epimerase of fructose-6-phosphate includes a 3-epimerase and a 4-epimerase. Specifically, allose-3-epimerase (D-allose-3-epimerase, EC5.1.3.30) epimerizes fructose (D-fructose) by 3-epimerization (epimerization at the 3rd carbon) to produce allose-6-phosphate.

[0004] When producing allose from fructose using the enzyme, there is a certain level of reaction equilibrium between fructose used as a substrate and allose as a product, and the efficiency is very low. Therefore, when attempting to produce high-purity allose through a single reaction using the enzyme, an additional step of separating and removing fructose from the final reaction solution is required.

[0005] When attempting to industrially produce allulose using fructose as a raw material, the enzymes used must meet industrial production conditions, particularly high thermal stability, and possess the highest possible conversion rate. Furthermore, since sugar is used as a substrate, browning of the sugar easily occurs under alkaline conditions, so it is necessary to satisfy conversion reaction conditions that prevent sugar browning as much as possible.

[0006] Therefore, in order to use it industrially, there is a pressing need for an enzyme that can produce phosphorylated fructose from fructose as a raw material, and a method for producing phosphorylated fructose using this enzyme, which satisfies at least one of the following conditions: suitable substrate conversion rate, thermal stability of the enzyme, and enzyme reaction conditions. [Overview of the project] [Problems that the invention aims to solve]

[0007] An example of the present invention relates to a fructose-6-phosphate 3-epimerization enzyme protein, a nucleic acid molecule encoding the enzyme protein, a recombinant vector containing the nucleic acid molecule, and a transformed microorganism.

[0008] Another example of the present invention relates to a method for producing allulose-6-phosphate using fructose-6-phosphate, comprising one or more selected from the group consisting of a fructose-6-phosphate 3-epimerase protein, cells of a microorganism expressing the enzyme, lysates of the cells, cultures of the microorganism, supernatant of the culture of the microorganism, or extracts thereof, and a composition for producing allulose-6-phosphate.

[0009] An additional example of the present invention relates to a composition for producing a ketohexose, such as allulose, using fructose-6-phosphate, and a method for producing allulose, comprising one or more selected from the group consisting of a fructose-6-phosphate 3-epimerase protein, a microbial cell expressing the enzyme, a lysate of the microbial cell, a culture of the microbial cell, the supernatant of the microbial culture, or extracts thereof. [Means for solving the problem]

[0010] To achieve the objectives of the present invention, an example of the present invention relates to a fructose-6-phosphate 3-epimerase protein containing an amino acid sequence having a sequence homology of 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more with the amino acid sequence of SEQ ID NO: 1. For example, it is obvious that any amino acid sequence having such homology and exhibiting efficacy corresponding to the protein consisting of the amino acid sequence of SEQ ID NO: 1 is included within the scope of the present invention, even if it has an amino acid sequence in which some sequences are deleted, modified, substituted, or added.

[0011] A specific example of the present invention is a fructose-6-phosphate 3-epimerase comprising an amino acid sequence having a sequence homology of 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more with the amino acid sequence of SEQ ID NO: 1. The fructose-6-phosphate 3-epimerase may be encoded by the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having at least 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more homology with the nucleotide sequence of SEQ ID NO: 2.

[0012] As amino acid sequences exhibiting homology to the aforementioned numerical values, any fructose-6-phosphate 3-epimerase protein that is substantially identical to or corresponding to the enzyme can be included without limitation. Furthermore, as such homologous sequences, any protein variants in which a portion of the sequence is deleted, modified, substituted, or added are also included within the scope of the present invention, as long as they substantially exhibit fructose-6-phosphate 3-epimerase function.

[0013] In the foregoing, the terms “homology” or “identical” refer to the degree to which a given amino acid sequence or nucleotide sequence matches, and may be expressed as a percentage. In this specification, homologous sequences having the same or similar activity as a given amino acid sequence or nucleotide sequence are expressed as “% homology”.

[0014] The fructose-6-phosphate 3-epimerase according to the present invention catalyzes the 3-epimation reaction of fructose-6-phosphate, and specifically can carry out the 3-epimation reaction of fructose-6-phosphate to convert it to allulose-6-phosphate.

[0015] The fructose-6-phosphate 3-epimerase protein according to the present invention may be an enzyme derived from Clostridium lundense, specifically from Clostridium lundense DSM 17049.

[0016] The reaction temperature range for the Clostridium lundense-derived fructose-6-phosphate 3-epimerase may be 40-70°C, 45-75°C, 45-77°C, 50-70°C, or 50-75°C. The optimal temperature may be, for example, the result of a reaction proceeding for 5 minutes under pH 7.0 conditions, but is not limited thereto. The optimal temperature condition for fructose-6-phosphate 3-epimerase is 60°C, and it exhibits more than 50% of its maximum enzyme activity over a wide temperature range of 40-70°C conditions.

[0017] The reaction pH range of the Clostridium lundense-derived fructose-6-phosphate 3-epimerase can be pH 6-8, pH 6-7.5, pH 6.5-8, pH 6.5-7.5, pH 7-8, or pH 7-7.5, with maximum activity at pH 7.0-7.5 and over 80% of maximum enzyme activity in the pH 6.0-8.0 range.

[0018] The maximum allulose production of the Clostridium lundense-derived fructose-6-phosphate 3-epimerase may be 16% or more by weight, 18% or more by weight, 20% or more by weight, 25% or more by weight, 27% or more by weight, 30% or more by weight, or 32% or more by weight. Specifically, the maximum allulose production of the enzyme may be measured by adding 0.1 mg / ml of the enzyme to a solution of 20 g / L of fructose-6-phosphate and carrying out the reaction, or more specifically, by adding 0.1 mg / ml of the enzyme to a solution of 20 g / L of fructose-6-phosphate and carrying out the enzymatic reaction under pH 7.0 and 50°C conditions.

[0019] The maximum allulose conversion rate can be calculated using the following formula 1. The duration of the enzymatic reaction to determine the maximum allulose conversion rate may be 8 hours or more, 10 hours or more, 12 hours or more, 14 hours or more, or 16 hours or more, with an upper limit of 18 hours or less, or 20 hours or less, and the specific reaction time may be within a range combining the lower and upper limits, for example, 16 to 20 hours. [Formula 1] Maximum allulose conversion rate (%) = (Amount of allulose produced (g / L) / Amount of fructose-6-phosphate administered (g / L)) * (Molecular weight of allulose / Molecular weight of fructose-6-phosphate) * 100

[0020] The allulose production ratio (by weight) among the sugars of the product of the enzymatic reaction can be calculated using the following formula. The enzymatic reaction time for determining the allulose production ratio can be 2 to 6 hours, 3 to 6 hours, or 4 to 6 hours, for example, 4 to 6 hours. [Formula 2] The aforementioned allulose production ratio (weight %) = Allulose production amount / (Fructose production amount + Allulose production amount) * 100

[0021] The fructose-6-phosphate 3-epimerase protein according to the present invention can have its enzymatic activity increased or decreased by metal ions. Specifically, the enzymatic activity of the fructose-6-phosphate 3-epimerase protein is increased by Mn, Co, and Ni ions, showing, for example, 1.1 times or more, 1.2 times or more, 1.3 times or more, or 1.5 times or more activity compared to the enzymatic activity under conditions without metal ions, and in particular, Mn and Co ions show 2 times or more, or 3 times or more activity, specifically 2 to 5 times or less, 2 to 4 times or less, 3 to 5 times or less, or 3 to 5 times or less activity compared to conditions without metal ions. The fructose-6-phosphate 3-epimerase protein has the property that its activity is decreased by Ca, Cu, Fe, or Ze ions. Therefore, it is preferable that the conditions for producing allulose using the fructose-6-phosphate 3-epimerase protein do not contain at least one metal ion selected from the group consisting of Ca, Cu, Fe, and Ze ions.

[0022] It is obvious that any amino acid sequence having partial deletions, alterations, substitutions, or additions is included within the scope of the present invention, as long as the fructose-6-phosphate 3-epimerase according to an example of the present invention contains an amino acid sequence having a sequence homology of 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more with the amino acid sequence of SEQ ID NO: 1, and exhibits efficacy corresponding to that of a protein consisting of the amino acid sequence of SEQ ID NO: 1.

[0023] The Clostridium lundense-derived fructose-6-phosphate 3-epimerase protein was analyzed for amino acid sequence identity with ribulose-phosphate 3-epimerase (RuFP3E: amino acid sequence - SEQ ID NO: 5) from Ruminococcus sp. AF14-10, ribulose-phosphate 3-epimerase (CDFP3E: amino acid sequence - SEQ ID NO: 7) from Clostridium sp. DL-VIII, and ribulose-phosphate 3-epimerase (PkFP3E: amino acid sequence - SEQ ID NO: 9) from Paenibacillus kribbensis. The results showed that the identity with RuFP3E was 59.05%, with CDFP3E was 63%, and with PkFP3E was 60.96%, and the sequence homology with the amino acid sequence of SEQ ID NO: 1 was less than 70%.

[0024] Also, as a result of HPLC analyzing the conversion activity from fructose 6-phosphate to allose 6-phosphate, an allose peak was confirmed for the ClFP3E enzyme having the amino acid sequence of SEQ ID NO: 1, but no allose peak was confirmed for the RuFP3E, CDFP3E, and PkFP3E. Therefore, since the other candidate group enzymes except the ClFP3E enzyme had no activity against F6P, it was confirmed that when treated with a phosphatase, only fructose in a form where phosphate was removed from F6P, which is the first substrate in the reaction process, was generated (Figs. 7a, 7b).

[0025] As an additional example of the present invention, a nucleic acid molecule encoding the fructose-6-phosphate 3-epimerase of the present invention is provided. A specific example of the nucleic acid encoding the fructose-6-phosphate 3-epimerase according to the present invention can include the nucleotide sequence of SEQ ID NO: 2, or a nucleotide sequence having at least 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more homology with the nucleotide sequence of SEQ ID NO: 2.

[0026] The present invention also provides, in another aspect, a vector or a transformant containing a nucleic acid encoding the fructose-6-phosphate 3-epimerase of the present invention.

[0027] As used herein, the term "transformation" means introducing a vector containing a nucleic acid encoding a target protein into a host cell so that the protein encoded by the nucleic acid can be expressed in the host cell. As long as the transformed nucleic acid can be expressed in the host cell, it can include all of these regardless of whether it is inserted into the chromosome of the host cell or located outside the chromosome. Further, the nucleic acid includes DNA and RNA encoding the target protein. The nucleic acid can be introduced in any form as long as it can be introduced into the host cell and expressed. For example, the nucleic acid can be introduced into the host cell in the form of an expression cassette, which is a gene construct containing all the elements necessary for self-expression. The expression cassette can usually include a promoter operably linked to the nucleic acid, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette can be in the form of an expression vector capable of self-replication. Further, the nucleic acid can be introduced into the host cell in its own form and operably linked to the sequences necessary for expression in the host cell, but is not limited thereto.

[0028] Also, the term "operably linked" as used above means that the promoter sequence that initiates and mediates the transcription of the nucleic acid encoding the target protein of the present invention is functionally linked to the gene sequence.

[0029] The method for transforming the vector of the present invention includes any method for introducing nucleic acids into cells and can be carried out by selecting a suitable standard technique as known in the art using host cells. Examples include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cation liposome method, and lithium acetate-DMSO method.

[0030] As the host cell, it is preferable to use a host cell that has high DNA introduction efficiency and high expression efficiency of the introduced DNA, such as E. coli, but is not limited to this.

[0031] In yet another embodiment, the present invention provides a ketohexose, such as an allulose production composition, comprising one or more selected from the group consisting of the fructose-6-phosphate 3-epimerase protein according to the present invention, a microorganism expressing the enzyme protein, a transformed microorganism expressing the enzyme protein, the cells of the microorganism, the cell lysates of the microorganism, the culture of the microorganism, the culture supernatant of the microorganism, a concentrate of the culture supernatant of the microorganism, and powders thereof.

[0032] The culture contains an enzyme produced from a microorganism that produces fructose-6-phosphate 3-epimerase, and may be in a cell-free form that includes or does not include the strain. The lysate means a lysate obtained by crushing the cells of a microorganism that produces fructose-6-phosphate 3-epimerase, or the supernatant obtained by centrifugation of the lysate, and contains an enzyme produced from a microorganism that produces polyphosphate-dependent glucose phosphorylation enzyme.

[0033] The culture of the strain contains the enzyme produced by a microorganism that produces the fructose-6-phosphate 3-epimerase, and may be in a cell-free form that includes or does not include the microbial cells. In this specification, unless otherwise specified, the fructose-6-phosphate 3-epimerase-producing microorganism used means one or more selected from the group consisting of the microbial cells of the strain, the culture of the strain, the lysates of the microbial cells, the supernatant of the lysates, and extracts thereof.

[0034] The method for producing ketohexose, such as allulose, according to the present invention utilizes enzymes obtained from microorganisms, making it environmentally friendly. It converts allulose production from fructose in a novel way through a simple enzymatic reaction, significantly reducing production costs while maximizing production efficiency.

[0035] The allulose production composition according to the present invention may additionally contain one or more metal ions selected from the group consisting of Mn, Co, and Ni ions. The concentration of the metal ions may be 0.5 mM to 20 mM, for example, 0.5 mM to 10 mM, 1.0 mM to 10 mM, 1.5 mM to 8.0 mM, 2.0 mM to 8.0 mM, 3.0 mM to 7.0 mM, 4.0 mM to 6.0 mM, or 0.2 mM to 10 mM.

[0036] When producing allulose using the allulose production composition described above, the reaction temperature and reaction pH conditions for the enzyme or enzyme-producing microorganism are as described above for the reaction temperature and reaction pH conditions for the enzyme.

[0037] The allulose production composition may include one or more selected from the group consisting of a hexokinase enzyme that converts fructose to fructose-6-phosphate, a transformed microorganism expressing the enzyme protein, the cells of the microorganism, lysates of the microorganism, cultures of the microorganism, supernatant of the culture of the microorganism, concentrates of the supernatant of the culture of the microorganism, and powders thereof.

[0038] The fructose-6-phosphate is preferably obtained by treating fructose or a fructose-containing substance with hexokinase, but it is also included within the scope of protection of the present invention if it is provided by other chemical synthesis methods.

[0039] The fructose-6-phosphate can be produced from glucose-6-phosphate, and the allulose production composition may additionally contain a glucose-6-phosphate isomerase that isomerizes glucose-6-phosphate to fructose-6-phosphate.

[0040] The glucose-6-phosphate can be produced by directly phosphorylating glucose or by conversion from glucose-1-phosphate. The glucose may be glucose obtained by treating starch or a starch hydrolysate, such as dextrin, with glucose-producing amylase, and the glucose-1-phosphate may be obtained by treating the glucose with a phosphorylation enzyme. The ketohexose production composition may additionally include an enzyme system for producing glucose-6-phosphate.

[0041] Specifically, the enzymes included in the allulose production composition of the present invention and the substrates used for allulose production are not limited. The allulose production composition of the present invention may, but is not limited to, (a) (i) starch, maltodextrin, sucrose or a combination thereof, glucose, glucose-1-phosphate, glucose-6-phosphate, or fructose-6-phosphate; (ii) phosphate; (iii) allulose-6-phosphate dephosphorylation enzyme; (iv) glucose-6-phosphate isomerase; (v) phosphoglucomutase or glucose phosphorylation enzyme; and / or (vi) α-glucan phosphorylase, starch phosphorylase, maltodextrin phosphorylase, sucrose phosphorylase, α-amylase, pullulanase, isoamylase, glucoamylase or sucrase; or (b) a microorganism expressing the enzymes in item (a) or a culture of a microorganism expressing the enzymes in item (a) above.

[0042] Specifically, the starch / maltodextrin phosphorylase (EC2.4.1.1) and α-glucan phosphorylase of the present invention may include any protein that has the activity to transfer phosphate to glucose to produce glucose-1-phosphate from starch or maltodextrin.

[0043] The sucrose phosphorylase (EC2.4.1.7) of the present invention may include any protein that has the activity to transfer phosphate to glucose to produce glucose-1-phosphate from sucrose.

[0044] The starch liquefaction enzymes of the present invention, namely α-amylase (EC3.2.1.1), pullulanase (EC3.2.1.41), glucoamylase (EC3.2.1.3), and isoamylase, may include any protein that has the activity to convert starch or maltodextrin into glucose.

[0045] The sucrase (EC3.2.1.26) of the present invention may include any protein that has the activity to convert sucrose to glucose. The phosphoglucomutase (EC5.4.2.2) applicable to the present invention may include any protein that has the activity to convert glucose-1-phosphate to glucose-6-phosphate. The glucose kinase (glucokinase) may include any protein that has the activity to transfer phosphate to glucose and convert it to glucose-6-phosphate. Specifically, the glucose kinase may be a polyphosphate-dependent glucose kinase.

[0046] The glucose-6-phosphate isomerase of the present invention may include any protein that has the activity to convert glucose-6-phosphate to fructose-6-phosphate.

[0047] The allulose-6-phosphate phosphatase of the present invention may include any protein that has the activity to convert allulose-6-phosphate to allulose. More specifically, the allulose-6-phosphate phosphatase may be a protein that has the activity to irreversibly convert allulose-6-phosphate to allulose. The allulose production composition may, but is not limited to, additionally include a phytase that carries out the dephosphorylation reaction with allulose-6-phosphate, such as allulose-6-phosphate phosphatase. The ketohexose production composition may further include allulose-6-phosphate phosphatase, a microorganism expressing the allulose-6-phosphate phosphatase, or a culture of a microorganism expressing the allulose-6-phosphate phosphatase.

[0048] The reaction temperature and pH conditions for using the ketohexose production composition described above to produce a ketohexose, such as allulose-6-phosphate, with an enzyme or a microorganism that produces the enzyme, are as described above for the reaction temperature and pH conditions of the enzyme.

[0049] The above method may additionally include a step of producing fructose-6-phosphate from fructose or a fructose-containing substance using hexokinase, and / or an additional step of removing phosphate by contacting a dephosphorylating enzyme with a microorganism expressing it, or a culture of said microorganism.

[0050] The step of removing the phosphate group can be carried out to produce allulose using allulose-6-phosphate phosphatase, a microorganism expressing allulose-6-phosphate phosphatase, or a culture of a microorganism expressing allulose-6-phosphate phosphatase. The phosphate group of allulose-6-phosphate can also be removed by other enzymes or chemical methods.

[0051] One example of the present invention provides a method for producing allulose, which includes the step of contacting fructose-6-phosphate 3-epimerase, a microorganism expressing the fructose-6-phosphate 3-epimerase, or a culture of the microorganism expressing the fructose-6-phosphate 3-epimerase to convert fructose-6-phosphate to allulose-6-phosphate.

[0052] The manufacturing method of the present invention may further include a step of converting allulose-6-phosphate to allulose by contacting allulose-6-phosphate with allulose-6-phosphate, an allulose-6-phosphate phosphatase, a microorganism expressing the allulose-6-phosphate phosphatase, or a culture of a microorganism expressing the allulose-6-phosphate phosphatase.

[0053] Furthermore, the manufacturing method of the present invention may additionally include a step of converting glucose-6-phosphate to fructose-6-phosphate by contacting glucose-6-phosphate with glucose-6-phosphate isomerase, a microorganism expressing glucose-6-phosphate isomerase, or a culture of a microorganism expressing glucose-6-phosphate isomerase, before the step of converting fructose-6-phosphate to allulose-6-phosphate.

[0054] Furthermore, the manufacturing method of the present invention may additionally include a step of converting glucose-1-phosphate to glucose-6-phosphate by contacting glucose-1-phosphate with phosphoglucocomutase, a microorganism expressing the phosphoglucocomutase, or a culture of a microorganism expressing the phosphoglucocomutase, before the step of converting glucose-6-phosphate to fructose-6-phosphate.

[0055] The manufacturing method of the present invention may additionally include a step of converting glucose to glucose-6-phosphate by contacting glucose with a glucose phosphorylation enzyme, a microorganism expressing the glucose phosphorylation enzyme or a culture of a microorganism expressing the glucose phosphorylation enzyme, and phosphate, prior to the step of converting glucose-6-phosphate to fructose-6-phosphate.

[0056] The production method of the present invention may additionally include a step of converting starch, maltodextrin, sucrose, or a combination thereof to glucose-1-phosphate by contacting starch, maltodextrin, sucrose, or a combination thereof with α-glucan phosphorylase, starch phosphorylase, maltodextrin phosphorylase, or sucrose phosphorylase; a microorganism expressing the phosphorylase; or a culture of a microorganism expressing the phosphorylase, and phosphate, prior to the step of converting glucose-1-phosphate to glucose-6-phosphate.

[0057] The production method of the present invention may additionally include a step of converting starch, maltodextrin, sucrose, or a combination thereof to glucose by contacting starch, maltodextrin, sucrose, or a combination thereof with α-amylase, pullulanase, glucoamylase, sucrase, or isoamylase; a microorganism expressing the amylase, pullulanase, or sucrase; or a microbial culture with the amylase, pullulanase, or sucrase.

[0058] The manufacturing method of the present invention may further include a step of converting glucose to starch, maltodextrin, or sucrose by contacting glucose with 4-α-glucanotrasphalasase, a microorganism expressing the 4-α-glucanotrasphalasase, or a culture of a microorganism expressing the 4-α-glucanotrasphalasase.

[0059] Allulose produced in this manner can be added to functional foods and pharmaceuticals for useful purposes. [Effects of the Invention]

[0060] The fructose-6-phosphate 3-epimerase according to the present invention satisfies at least one of the following characteristics: high enzyme conversion rate, acidic or neutral reaction pH conditions, and high thermal stability. Therefore, it can be usefully utilized in the industrial-scale production of ketohexoses using fructose-6-phosphate 3-epimerase. [Brief explanation of the drawing]

[0061] [Figure 1] This is an electrophoretic image confirming the expression and purification of a fructose-6-phosphate 3-epimerase protein according to an example of the present invention. [Figure 2] The results of a bio-LC analysis of a fructose-6-phosphate 3-epimerase protein according to an example of the present invention are shown. [Figure 3] This is the result of dephosphorylating the phosphorylated sugars in the reaction solution via an enzymatic reaction with allulose-6-phosphate phosphatase according to an example of the present invention, followed by analysis by LC. [Figure 4] This graph shows the results of analyzing the temperature characteristics of a fructose-6-phosphate 3-epimerase protein according to an example of the present invention. [Figure 5] This graph shows the results of analyzing the pH characteristics of a fructose-6-phosphate 3-epimerase protein according to an example of the present invention. [Figure 6]This graph shows the effect of metal ions on the 3-epimerase protein of fructose-6-phosphate, according to an example of the present invention. [Figure 7a] This shows the HPLC analysis results of the reaction products obtained after allulose production using three known types of ribulose-phosphate 3-epimerase. [Figure 7b] This shows the HPLC analysis results of the reaction products obtained after allulose production using three known types of ribulose-phosphate 3-epimerase. [Modes for carrying out the invention] [Examples]

[0062] The present invention will be described in more detail with reference to the following embodiments, but the scope of the rights is not intended to be limited to the following embodiments.

[0063] Example 1: Production of fructose-6-phosphate 3-epimerase Candidate enzymes expected to function as fructose-6-phosphate 3-epimerases were screened, and the enzyme expected to show the best effect was obtained by requesting gene synthesis via IDT gene synthesis to obtain a polynucleotide (SEQ ID NO: 2) encoding the amino acid sequence (SEQ ID NO: 1) of the enzyme (ClFP3E) derived from the Clostridium lundense DSM 17049 strain. Based on the synthesized ClFP3E DNA base sequence of SEQ ID NO: 2, primers were devised, and the base sequence of the gene was amplified by PCR. The forward and reverse primer sequences used for PCR amplification are as follows.

[0064] [Table 1]

[0065] The ClFP3E gene obtained in large quantities was introduced into the pET21a vector using restriction enzymes NdeI and XhoI to produce pET21_ClFP3E, which was then used to transform *E. coli* strain ER2566. Recombinant *E. coli* for enzyme protein expression were secured as colonies on agar plates prepared in LB medium containing 50 μg / ml ampicillin. After seed culture in 4 ml of LB medium, main culture was performed in 100 ml of LB medium. The culture conditions were 37°C at 200 rpm until the absorbance value at 600 nm reached 0.6, after which 0.1 mM IPTG was added to induce expression of the target protein. After induction, the strain was cultured at 25°C for approximately 16 hours, and then the cells were collected by centrifugation. The collected cells were suspended in lysis buffer (50 mM sodium phosphate buffer (pH 7.0), 300 mM NaCl, 10 mM imidazole), and the cells were disrupted using a beadbeater. Overexpression of the target protein ClFP3E was confirmed from the disrupted cell solution by SDS-PAGE gel analysis. The results of the overexpression analysis of the target protein ClFP3E are shown in Figure 1. The molecular weight of ClFP3E confirmed by SDS-PAGE gel analysis was approximately 28 kDa. After removing the cell pellet, only the conditioned medium was obtained and bound to a Ni-NTA column (Ni-NTA superflow, Qiagen). Proteins that did not bind to the column were removed using a washing buffer (50 mM sodium phosphate buffer (pH 7.0), 300 mM NaCl, 20 mM imidazole). In the final step, the target protein was eluted using an elution buffer (50 mM sodium phosphate buffer (pH 7.0), 300 mM NaCl, 200 mM imidazole). The finally secured protein was converted to 50 mM sodium phosphate buffer (pH 7.0) and stored for subsequent use.

[0066] Example 2: Evaluation of enzyme conversion activity The purified ClFP3E enzyme obtained in Example 1 (0.1 mg / ml) was added to a solution prepared by dissolving 20 g / L of fructose-6-phosphate in 50 mM sodium phosphate buffer (pH 7.0), and the enzymatic reaction was carried out at 50°C. Analysis of the enzyme reaction solution involved comparing it with the substrate via Bio-LC analysis to identify newly generated substances. However, due to the lack of an allulose-6-phosphate standard, accurate identification was impossible. Therefore, the solution was further treated with an allulose-6-phosphate (A6PP) decphosphorylating enzyme, and the generated allulose was finally identified. Bio-LC analysis confirmed that during the ClFP3E enzyme reaction, there was a decrease in F6P and the generation of a new peak, presumably A6P. The results of the Bio-LC analysis are shown in Figure 2 below. The following are the results of LC analysis after dephosphorylating the phosphorylated sugars in the reaction solution via the A6PP enzyme reaction. The analysis was performed using an Aminex HPX-87C column at 80°C with a flow rate of 0.6 ml / min, and the results are shown in Figure 3. The analysis confirmed the presence of fructose and allulose. The final conversion rate of ClFP3E was calculated to be 34.3% by quantifying the amount of allulose produced as the final product of the reaction solution after dephosphorylation via the allulose-6-phosphate phosphatase (A6PP) enzymatic reaction. The reaction product in this example is the final conversion rate of ClFP3E obtained after 16 hours of enzymatic reaction, and the maximum allulose conversion rate is calculated by the following formula. [Formula 1] Maximum allulose conversion rate (%) = (Amount of allulose produced (g / L) / Amount of fructose-6-phosphate administered (g / L)) * (Molecular weight of allulose / Molecular weight of fructose-6-phosphate) * 100

[0067] Example 3: Analysis of the temperature characteristics of the enzyme To investigate the effect of temperature on ClFP3E enzyme activity, 10 g / L of fructose-6-phosphate was dissolved in 50 mM sodium phosphate buffer (pH 7.0), and then 0.01 mg / ml of purified ClFP3E protein was added. The reaction was then carried out for 5 minutes under various temperature conditions between 40 and 80°C. Subsequently, A6PP enzyme was added to dephosphorylate the entire reaction composition, and the amount of allulose produced was quantitatively analyzed by HPLC. The relative activity of the enzyme at the reaction temperature is shown in Figure 4, with the activity at 60°C, where the highest activity was measured, as the baseline.

[0068] The experimental results confirmed that the optimal temperature for ClFP3E is 60°C, and that it exhibits more than 50% of its maximum enzyme activity across a wide temperature range of 40-70°C.

[0069] Example 4: Analysis of the pH characteristics of the enzyme To investigate the effect of pH on ClFP3E enzyme activity, 10 g / L of fructose-6-phosphate was dissolved in a buffer solution between pH 5.0 and 8.5 (pH 5.0-6.5, sodium citrate / pH 6.5-8.5, Tris-HCl). Then, 0.01 mg / ml of purified ClFP3E protein was added, and the enzymatic reaction was carried out at 60°C for 5 minutes. Subsequently, A6PP enzyme was added to dephosphorylate the entire reaction composition, and the amount of allulose produced was quantitatively analyzed by HPLC. The results of the enzyme activity at different reaction pH levels are shown in Figure 5 as relative activity, with pH 7.5 being the baseline for the most active pH.

[0070] The experimental results showed that maximum activity was confirmed under pH 7.0-7.5 conditions, and that the enzyme exhibited more than 80% of its maximum activity in the pH range of 6.0-8.0.

[0071] Example 5: Analysis of the effect of metal ions on enzymes To confirm the activity of ClFP3E depending on the type of metal ion added during the reaction, 10 g / L of fructose-6-phosphate was dissolved in 50 mM sodium phosphate buffer (pH 7.0), and then 5 mM of each metal ion (MgCl2, MnCl2, CaCl2, CoCl2, CuCl2, NiSO4, FeSO4, ZeSO4) was added. 0.01 mg / ml of ClFP3E-producing protein was added to the reaction buffer containing each metal ion, and the reaction was carried out at 60°C for 5 minutes. Subsequently, A6PP enzyme was added to dephosphorylate all reaction compositions, and the amount of allulose produced was quantitatively analyzed by HPLC. The relative activity of the enzymes for each type of metal ion is shown in Figure 6, with the experimental group without metal ions as the baseline.

[0072] The experimental results showed that when MnCl2 and CoCl2 were added, the activity was more than three times higher than under conditions without metal ions. It was found that the ClFP3E enzyme uses manganese and cobalt as cofactors. Nickel also increased its activity. CaCl2, CuCl2, FeSO4, and ZeSO4 decreased the enzyme's activity.

[0073] Comparative Example 1: Allulose production analysis using ribulose-phosphate 3-epimerase Polynucleotides of ribulose-phosphate 3-epimerase from Ruminococcus sp. AF14-10 (RuFP3E: amino acid sequence - SEQ ID NO. 5 and nucleic acid sequence - SEQ ID NO. 6), ribulose-phosphate 3-epimerase from Clostridium sp. DL-VIII (CDFP3E: amino acid sequence - SEQ ID NO. 7 and nucleic acid sequence - SEQ ID NO. 8), and ribulose-phosphate 3-epimerase from Paenibacillus kribbensis (PkFP3E: amino acid sequence - SEQ ID NO. 9 and nucleic acid sequence - SEQ ID NO. 10) were obtained by requesting gene synthesis. Analysis of the amino acid sequence homology (SEQ ID NO: 1) of fructose-6-phosphate 3-epimerase (ClFP3E) derived from the Clostridium lundense DSM 17049 strain revealed that it had 59.05% amino acid sequence identity with RuFP3E, which has the amino acid sequence of SEQ ID NO: 5; 63% amino acid sequence identity with CDFP3E, which has the amino acid sequence of SEQ ID NO: 7; and 60.96% amino acid sequence identity with PkFP3E, which has the amino acid sequence of SEQ ID NO: 9. Based on the DNA sequence of the synthesized ClFP3E of Sequence ID No. 2, a large quantity of the gene was obtained in substantially the same manner as in Example 1. The obtained gene was introduced into E. coli and expressed in substantially the same manner as in Example 1, after which the target protein was eluted. The final secured protein was converted in 50 mM sodium phosphate buffer (pH 7.0) and stored for subsequent use. To analyze the conversion activity from fructose-6-phosphate to allulose-6-phosphate, an enzymatic reaction was carried out using the obtained enzyme with fructose-6-phosphate as a substrate, in the same manner as in Example 2. After the enzymatic reaction, a dephosphorylation reaction was carried out using allulose-6-phosphate phosphatase (A6PP) enzyme, and the reaction product was measured by the amount of allulose produced using high-performance liquid chromatography (HPLC). The HPLC analysis conditions utilized an Agilent 1260 RID (Refractive Index Detector) on an Agilent HPLC (USA) equipped with an Aminex HPX-87C column (BIO-RAD). Water was used as the mobile phase solvent, and the analysis was performed at a temperature of 80°C and a flow rate of 0.6 ml / min. The results of the HPLC analysis are shown in Figures 7a and 7b. The reaction product in this example is the allulose conversion rate of the enzyme obtained after 4 hours of enzymatic reaction. This was an experiment to screen candidate enzymes for FP3E, and the reaction solution was analyzed after stopping the reaction at the intermediate step of allulose production. In Figures 7a and 7b, the allulose conversion rate of ClFP3E was confirmed to be 16%, and the reaction proceeded at approximately 50% of the maximum conversion rate obtained in Example 2. Of the total ClFP3E product in this example, the allulose production ratio was 75% by weight, and the allulose production ratio is calculated by the following formula. [Formula 2] The aforementioned allulose production ratio (weight %) = amount of allulose produced / (amount of fructose produced + amount of allulose produced) × 100

[0074] As shown in Figures 7a and 7b, no allulose peak was observed in the other candidate enzymes except for the ClFP3E enzyme in Example 1. Therefore, since the other candidate enzymes besides ClFP3E did not have activity against F6P, it was confirmed that when treated with a phosphatase, only fructose, in which phosphate has been removed from F6P, the initial substrate in the reaction step, was produced.

Claims

1. One or more selected from the group consisting of a fructose-6-phosphate epimerizing enzyme protein having the amino acid sequence of SEQ ID NO: 1 and converting fructose-6-phosphate to allulose-6-phosphate, a microorganism expressing the enzyme protein, a transformed microorganism expressing the enzyme protein, the cells of the microorganism, the cell lysates of the microorganism, the culture of the microorganism, the culture supernatant of the microorganism, a concentrate of the culture supernatant of the microorganism, and powders thereof, and A composition for allulose production containing one or more metal ions selected from the group consisting of manganese ions and nickel ions.

2. The allulose production composition according to claim 1, wherein the enzyme is encoded by the nucleotide sequence of Sequence ID No.

2.

3. The allulose production composition according to claim 1, wherein the enzyme is derived from Clostridium lumens and has an enzymatic reaction temperature of 40 to 70°C and an enzymatic reaction pH of 6 to 8.

4. The allulose production composition according to claim 1, wherein the enzyme's activity is increased by manganese ions, cobalt ions, or nickel ions.

5. The composition for allulose production according to any one of claims 1 to 4, further comprising an allulose-6-phosphate dephosphorylation enzyme, a microorganism expressing the same, or a culture of the said microorganism.

6. The composition for allulose production according to any one of claims 1 to 5, further comprising an isomerase that converts glucose-6-phosphate to fructose-6-phosphate, a microorganism expressing the same, or a culture of the said microorganism.

7. The aforementioned composition, (a) (i) starch, maltodextrin, sucrose or a combination thereof; (ii) phosphate; (iii) allulose-6-phosphate dephosphorylation enzyme; (iv) glucose-6-phosphate isomerase; (v) phosphoglucomutase or glucose phosphorylation enzyme; and (vi) α-glucanophosphorylase, starch phosphorylase, maltodextrin phosphorylase, sucrose phosphorylase, α-amylase, pullulanase, isoamylase, glucoamylase or sucrase; or (b) further comprising a microorganism expressing the enzyme of item (a) above or a culture of said microorganism, A composition for allulose production according to any one of claims 1 to 6.

8. A method for producing allulose, comprising the step of converting fructose-6-phosphate to allulose-6-phosphate using the allulose production composition described in any one of claims 1 to 7.

9. The method according to claim 8, further comprising the step of converting allulose-6-phosphate to allulose by contacting allulose-6-phosphate with phytase, a microorganism expressing phytase, or a culture of the microorganism.

10. The method according to claim 8, wherein the step of converting fructose-6-phosphate to allulose-6-phosphate is carried out at a reaction temperature of 40 to 70°C and a reaction pH of 6 to 8.

11. The method according to claim 8, further comprising the step of producing fructose-6-phosphate from fructose or a fructose-containing substance using hexokinase.

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