WO2022139231A1 - Dephosphorylation enzyme protein having high substrate specificity and use thereof - Google Patents

Abstract

The present invention relates to a dephosphorylation enzyme protein and, more specifically, to: an allulose-6-phosphate dephosphorylation enzyme protein having high substrate specificity only for allulose-6-phosphate; a nucleic acid molecule encoding the enzyme protein; a recombinant vector and a transformed strain, comprising the nucleic acid molecule; and a composition for producing allulose using the strain.

Description

Dephosphorylation enzyme protein with high substrate specificity and uses thereof

The present invention relates to a dephosphorylation enzyme protein, more particularly, an allulose-6-phosphate dephosphorylation enzyme protein with high substrate specificity only for allulose-6-phosphate, a nucleic acid molecule encoding the enzyme protein, and a recombinant comprising the nucleic acid molecule It relates to a vector, a transformed strain, and a composition for producing allulose using the strain.

In the case of industrial production of allulose using fructose as a raw material, the enzyme used must have high industrial production conditions, particularly thermal stability, and the highest conversion rate. Also, since sugar is used as a substrate, the browning of sugar is reduced under alkaline conditions. Since it occurs easily in

Therefore, there is an urgent need for an enzyme that satisfies at least one suitable substrate conversion rate, thermal stability of the enzyme, and enzyme reaction conditions for industrial use, and produces phosphorylated fructose using fructose as a raw material, and a method for producing phosphorylated fructose using the same. .

Most dephosphorylation enzymes that exist in nature do not have high specificity for a specific substrate, so they dephosphorylate all of the reaction intermediates, glucose-1-phosphate, glucose-6-phosphate, and fructose-6-phosphate, at the same time to produce allulose in high yield. is difficult to produce.

In the present invention, it was attempted to increase allulose production efficiency by securing allulose-6-phosphate dephosphorylation enzyme with high substrate specificity only for allulose-6-phosphate.

An example of the present invention relates to a dephosphorylation enzyme protein having high substrate specificity, a nucleic acid molecule encoding the enzyme protein, a recombinant vector comprising the nucleic acid molecule, and a transformed microorganism.

Another example of the present invention provides an allulose-6-phosphate dephosphorylation enzyme having high substrate specificity, a microorganism expressing the enzyme protein, a transforming microorganism expressing the enzyme protein, a cell body of the microorganism, and the microorganism It relates to a method for producing allulose by dephosphorylating allulose-6-phosphate, comprising at least one selected from the group consisting of a cell lysate, a culture of the microorganism, and an extract thereof, and a composition for producing allulose .

A further example of the present invention is an allulose-6-phosphate dephosphorylation enzyme having high substrate specificity, a microorganism expressing the enzyme protein, a transforming microorganism expressing the enzyme protein, a cell body of the microorganism, a cell body of the microorganism It relates to a composition for producing allulose and a method for producing allulose, comprising at least one selected from the group consisting of a lysate, a culture of the microorganism, and an extract thereof.

In order to achieve the object of the present invention, an example of the present invention is 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more of the amino acid sequence of SEQ ID NO: 1 % or greater, 95% or greater, 97% or greater, or 99% or greater amino acid sequence. For example, if it is an amino acid sequence that has such sequence identity and exhibits efficacy corresponding to the protein consisting of the amino acid sequence of SEQ ID NO: 1, even if some sequences have deleted, modified, substituted or added amino acid sequences, the scope of the present invention It is self-evident that it is included within.

A specific example of the present invention is 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% sequence identity with the amino acid sequence of SEQ ID NO: 1 Provided is a fructose-6-phosphate 3-epimerase comprising an amino acid sequence of greater than or equal to 99% or greater. The fructose-6-phosphate 3-epimerase is at least 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more of the nucleotide sequence of SEQ ID NO: 2 or the nucleotide sequence of SEQ ID NO: 2 , may be encoded by a nucleotide sequence having 97% or more or 99% or more sequence identity.

As an amino acid sequence showing the sequence identity of the above numerical values, any dephosphorylation enzyme protein of allulose-6-phosphate substantially identical to or corresponding to the enzyme may be included without limitation. In addition, as long as the sequence having such sequence identity is an amino acid sequence that substantially exhibits an allulose-6-phosphate dephosphorylation enzyme function, protein variants in which some sequences are deleted, modified, substituted or added are also included within the scope of the present invention.

As used herein, the term “sequence homology” or “sequence identity” refers to the degree of agreement with a given amino acid sequence or nucleotide sequence, and may be expressed as a percentage. In the present specification, a homologous sequence having the same or similar activity to a given amino acid sequence or base sequence is expressed as "% homology".

The dephosphorylation enzyme of allulose-6-phosphate according to the present invention can catalyze the dephosphorylation reaction of 6-phosphate present in allulose 6-phosphate to convert it to allulose, but It has an irreversible enzymatic activity that does not catalyze the reverse reaction that converts loss to allulose-6-phosphate.

The enzyme protein according to the present invention has high specificity for an allulose-6-phosphate substrate, and has no or very low conversion properties for glucose-1-phosphate and glucose-6-phosphate. In the enzyme, the ratio of the conversion rate of allulose-6-phosphate to allulose to the conversion rate of glucose-6-phosphate or glucose-1-phosphate to glucose is 20 times or more, 50 times or more, or 100 times or more. and may have, for example, a numerical range of 20 to 150 times.

In addition, the enzyme according to the present invention has high substrate specificity for allulose-6-phosphate compared to fructose-6-phosphate, and specifically, a mixed substrate containing fructose-6-phosphate and allulose-6-phosphate or glucose When acting on a mixed substrate containing -1phosphate, glucose-6-phosphate, fructose-6-phosphate and allulose-6-phosphate, 60% by weight or more based on 100% by weight of the total dephosphorylated sugar composition of the reaction product , at least 65 wt%, at least 70 wt%, at least 75 wt%, at least 78 wt%, or at least 80 wt% allulose, which has a high substrate specificity for allulose-6-phosphate. In the enzyme according to the present invention, the ratio of the conversion rate of allulose-6-phosphate to allulose to the conversion rate of fructose-6-phosphate to fructose is 6 times or more, 7 times or more, 8 times or more, 9 times or more. , 10 times or more, 11 times or more, 12 times or more, 13 times or more, 14 times or more, or 15 times or more, for example, 6 to 20 times, 6 to 18 times, 6 to 15 times higher substrate conversion properties. can have The enzyme according to the present invention converts allulose-6-phosphate to allulose with respect to the total conversion rate of the conversion rate of fructose-6-phosphate to fructose and the conversion rate of glucose-1-phosphate and glucose-6-phosphate to glucose. The ratio of the conversion rate to convert is more than 2 times, more than 3 times, more than 4 times, more than 5 times, more than 6 times, more than 7 times, more than 8 times, more than 9 times, more than 10 times, more than 11 times, more than 12 times, 13 times or more, 14 times or more, or 15 times or more, for example, 6 to 20 times, 6 to 18 times, 6 to 15 times, higher substrate conversion properties. Specifically, the conversion rate and the measurement of the amount of allulose production relative to the total dephosphorylated saccharides of the reaction product may be a reaction at a temperature of 50° C. for 2 hours, pH 7.0, and an amount of enzyme 0.1 mg/ml.

Allulose-6-phosphate dephosphorylation enzyme protein according to the present invention may be an enzyme derived from a Clostridiales sp. strain, HAD family hydrolase enzyme.

The Clostridiales sp. strain-derived allulose-6-phosphate dephosphorylation enzyme (CloA6PP) has a high activity at 40 to 55 ° C, for example, 70% or more of the maximum enzyme activity, and specifically high activity at 45 to 55 ° C. For example, it has an activity of 80% or more of the maximum enzyme activity.

The CloA6PP enzyme has an activity of 70% or more, 75% or more, or 80% or more of the maximum enzyme activity in a wide pH range of pH 5.5 to 9.0.

CloA6PP enzyme according to the present invention may increase or decrease enzyme activity by metal ions. Specifically, the CloA6PP enzyme protein has a higher activity than the control without a metal ion when Mg, Mn, Co, Ca, and Ni are added, for example, 1.1 times or more compared to the enzyme activity under the condition without metal ions, 1.2 It exhibits more than fold, more than 1.3 fold, more than 1.5 fold, or more than 2 fold, and in particular, Mn ion has 11 fold or more activity. In particular, in particular, in the case of Mn ion, it may have an activity of 11 times or more, for example, it may have a numerical range of 2 to 20 times.

In addition, the CloA6PP enzyme may have reduced enzymatic activity by metal ions, for example, Cu, Fe, Zn, compared to the enzyme activity in the absence of metal ions, and has the lowest activity by Fe.

The CloA6PP enzyme according to the present invention has 22.17% amino acid sequence identity as a result of analyzing the amino acid sequence identity of allulose-6-phosphate dephosphorylation enzyme (TalA6PP) derived from Thermoleophilum album, and allulose-6-phosphate dephosphorylation enzyme derived from Acidobacteria bacterium. As a result of analyzing the amino acid sequence identity of the enzyme (AbaA6PP), it had 23.11% amino acid sequence identity, and the amino acid sequence identity with the CloA6PP enzyme was 30% or less.

CbaA6PP and ChbA6PP had no product confirmed by HPLC, so it was confirmed that there was no dephosphorylation enzyme activity of allulose-6-phosphate. In the reaction product to which TalA6PP and AbaA6PP were added, the activity of the additive enzyme was present, but the substrate specificity acting specifically for A6P was very poor, so it was confirmed that glucose, fructose, and allulose were all produced. On the other hand, CloA6PP has excellent both enzyme activity and substrate specificity, so it can be seen that only allulose is produced in excess. As a result of the enzymatic reaction using CloA6PP, the proportion of allulose in the total product was about 89.1%, and at the same time, a very small amount of fructose was converted, and no glucose was identified.

As a further example of the present invention, there is provided a nucleic acid molecule encoding the CloA6PP enzyme of the present invention. A specific example of the nucleic acid encoding the allulose-6-phosphate dephosphorylation enzyme according to the present invention, the allulose-6-phosphate dephosphorylation enzyme is the nucleotide sequence of SEQ ID NO: 2 or the nucleotide sequence of SEQ ID NO: 2 and a nucleotide sequence having at least 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more identity.

As another aspect, the present invention provides a vector or transformant comprising a nucleic acid encoding an allulose-6-phosphate dephosphorylation enzyme of the present invention.

As used herein, the term “transformation” refers to introducing a vector including a nucleic acid encoding a target protein into a host cell so that the protein encoding 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 may include all of them regardless of whether they are inserted into the chromosome of the host cell or located outside the chromosome. In addition, the nucleic acid includes DNA and RNA encoding the target protein. The nucleic acid may be introduced in any form as long as it can be expressed and introduced into a host cell. For example, the nucleic acid may be introduced into a host cell in the form of an expression cassette, which is a gene construct including all elements necessary for self-expression. The expression cassette may include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, which are usually operably linked to the nucleic acid. The expression cassette may be in the form of an expression vector capable of self-replication. In addition, the nucleic acid may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.

In addition, as used herein, the term “operably linked” means that a promoter sequence that initiates and mediates transcription of a nucleic acid encoding a target protein of the present invention and the gene sequence are functionally linked.

The method for transforming the vector of the present invention includes any method of introducing a nucleic acid into a cell, and may be performed by selecting a suitable standard technique as known in the art depending on the host cell. For example, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate -DMSO method, etc., but is not limited thereto.

As the host cell, it is preferable to use a host with high DNA introduction efficiency and high expression efficiency of the introduced DNA, and may be, for example, E. coli, but is not limited thereto.

As another aspect, the present invention provides a dephosphorylation enzyme of allulose-6-phosphate according to the present invention, a microorganism expressing the enzyme protein, a transformed microorganism expressing the enzyme protein, a cell of the microorganism, and the microorganism It provides a composition for producing allulose comprising at least one selected from the group consisting of a cell lysate of

The culture contains an enzyme produced from a microorganism producing an enzyme for dephosphorylation of allulose-6-phosphate, and may be in a cell-free form including the strain or not including the strain. The lysate means a lysate obtained by lysing the cells of a microorganism that produces allulose-6-phosphate dephosphorylation enzyme or a supernatant obtained by centrifuging the lysate to produce the allulose-6-phosphate dephosphorylation enzyme It includes enzymes produced from microorganisms that

The culture of the strain contains the enzyme produced from the microorganism producing the allulose-6-phosphate dephosphorylation enzyme, and may be in a cell-free form with or without the microorganism cells. In the present specification, unless otherwise stated, the microorganisms used to produce the allulose-6-phosphate dephosphorylation enzyme are the cells of the strain, the culture of the strain, the lysate of the cell, the supernatant of the lysate, and one or more selected from the group consisting of extracts thereof.

The allulose production method according to the present invention is environmentally friendly because it uses an enzyme obtained from a microorganism, converts the production of allulose from fructose to an unprecedented method from a simple enzymatic reaction, and can greatly reduce production costs while maximizing production effects. have.

The composition for producing allulose according to the present invention may further include one or more metal ions selected from the group consisting of Mg, Mn, Co, Ca, and Ni ions, preferably Mn ions. The concentration of the metal ion 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.

In the case of producing allulose using the composition for producing allulose, the reaction temperature and reaction pH conditions using the allulose-6-phosphate dephosphorylation enzyme or enzyme-producing microorganism are detailed in the reaction temperature and reaction pH conditions of the enzyme. It's like a bar.

The composition for producing allulose according to the present invention comprises a fructose-6-phosphate 3-epimerase enzyme that produces allulose 6-phosphate from fructose-6-phosphate, a transforming microorganism expressing the enzyme protein, and the microorganism It may include one or more selected from the group consisting of cells, a cell lysate of the microorganism, a culture of the microorganism, a culture supernatant of the microorganism, a concentrate of the culture supernatant of the microorganism, and powders thereof.

The fructose-6-phosphate 3-epimerase enzyme catalyzes the 3-epimerization reaction of fructose-6-phosphate, and specifically performs the 3-epimerization reaction of fructose-6-phosphate. Any enzyme capable of converting to allulose-6-phosphate may be used without limitation, for example, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more or 99 of the amino acid sequence of SEQ ID NO: 17. Having an amino acid sequence identity of % or more, fructose 6-phosphate converts fructose-6-phosphate to allulose-6-phosphate ) may be an epimerase. The enzyme may be encoded by a nucleotide sequence having 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more nucleotide sequence identity with the nucleotide sequence of SEQ ID NO: 18.

The enzyme is Clostridium lundense specifically derived from Clostridium lundense DSM 17049, has an enzyme reaction temperature of 40 to 70 ° C and an enzyme reaction pH of pH 6 to 8, and the activity is increased by manganese ions, cobalt ions or nickel ions. can

Specifically, the reaction temperature range of the Clostridium lundense -derived fructose-6-phosphate 3-epimerase may be 40 to 70 °C, 45 to 75 °C, 45 to 77 °C, 50 to 70 °C, or 50 to 75 °C. The optimum temperature may be, for example, a result of the reaction proceeding for 5 minutes at pH 7.0, but is not limited thereto. The optimum temperature condition for fructose-6-phosphate 3-epimerase is 60 °C, and it has an activity of 50% or more of the maximum enzyme activity in a wide temperature range from 40 to 70 °C.

The reaction pH range of the Clostridium lundense -derived fructose-6-phosphate 3-epimerase is pH 6 to 8, pH 6 to 7.5, pH 6.5 to 8, pH 6.5 to 7.5, pH 7 to 8, or pH 7 to 7.5 days. and has the maximum activity at pH 7.0 to 7.5, and has an activity of 80% or more of the maximum enzyme activity in the pH 6.0 to 8.0 range.

The maximum allulose production amount of the Clostridium lundense -derived fructose-6-phosphate 3-epimerase is 16 wt% or more, 18 wt% or more, 20 wt% or more, 25 wt% or more, 27 wt% or more, 30 wt% or more or 32% by weight or more. Specifically, the maximum allulose production amount of the enzyme may be a reaction performed by adding 0.1 mg/ml of the enzyme to a solution in which 20 g/L of fructose-6-phosphate is dissolved, and more specifically, 0.1 mg/ml of the enzyme It may be measured by adding 20 g/L fructose-6-phosphate to a dissolved solution and performing an enzymatic reaction at pH 7.0 and 50 °C.

The maximum conversion of allulose may be calculated by Equation 1 below. The time of the enzymatic reaction to obtain 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, and the upper limit of the reaction time may be 18 hours or less or 20 hours or less, , The specific reaction time may be a range combining the lower limit and the upper limit, for example, may be 16 hours to 20 hours.

The rate of allulose production (weight %) in the saccharides of the product of the enzymatic reaction can be calculated by the following equation. The enzyme reaction time for obtaining the allulose production rate may be 2 hours to 6 hours, 3 hours to 6 hours, or 4 hours to 6 hours, for example, 4 hours to 6 hours.

In the fructose-6-phosphate 3-epimerase protein according to the present invention, enzymatic activity may be increased or decreased by metal ions. Specifically, the enzyme activity of fructose-6-phosphate 3-epimerase protein is increased by Mn, Co and Ni ions, for example, 1.1 times or more, 1.2 times or more, 1.3 times or more, compared to the enzyme activity in the absence of metal ions. or more, or 1.5 times or more, in particular, Mn and Co ions are 2 times or more, or 3 times or more, specifically 2 to 5 times or less, 2 to 4 times or less, 3 times to less than the condition without metal ions 5-fold or less, or 3-fold to 5-fold or less activity. The fructose-6-phosphate 3-epimerase protein has a property that its activity is reduced by Ca, Cu, Fe, or Zn ions. Therefore, it is preferable that the conditions for producing allulose using fructose-6-phosphate 3-epimerase protein do not include at least one metal ion selected from the group consisting of Ca, Cu, Fe, and Zn ions. can

The fructose-6-phosphate 3-epimerase according to an embodiment of the present invention has 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 18. It contains the amino acid sequence above, and if it is an amino acid sequence exhibiting efficacy corresponding to the protein consisting of the amino acid sequence of SEQ ID NO: 18, it is included within the scope of the present invention even if some sequences have an amino acid sequence deleted, modified, substituted or added. self-evident

In another example, the fructose-6-phosphate 3-epimerase protein is Ruminococcus sp. AF14-10-derived ribulose-phosphate 3-epimerase (RuFP3E: amino acid sequence of SEQ ID NO: 19), Clostridium sp. DL-VIII-derived ribulose-phosphate 3-epimerase (CDFP3E: amino acid sequence of SEQ ID NO: 20), Paenibacillus kribbensis-derived ribulose-phosphate 3-epimerase (PkFP3E: amino acid sequence of SEQ ID NO: 21), or a combination thereof.

The composition for producing allulose according to the present invention comprises a hexokinase enzyme that converts fructose to fructose-6-phosphate, a transforming microorganism expressing the enzyme protein, a cell of the microorganism, a lysate of the microorganism, and a culture of the microorganism It may include one or more selected from the group consisting of water, a culture supernatant of the microorganism, a concentrate of the culture supernatant of the microorganism, and powders thereof.

The fructose-6-phosphate is preferably obtained by hexokinase treatment of fructose or a fructose-containing material, but it is also included in the protection scope of the present invention when provided by other chemical synthesis methods.

The fructose-6-phosphate may be prepared from glucose-6-phosphate, and the composition for producing allulose contains a glucose-6-phosphate isomerase that isomerizes glucose-6-phosphate and converts it to fructose-6-phosphate. may additionally include.

The glucose-6-phosphate may be prepared by direct phosphorylation of glucose, or may be converted from glucose-1-phosphate. Glucose may be glucose obtained by treating glucose-producing amylase on starch or a starch hydrolyzate, for example, dextrin, and glucose-1-phosphate may be obtained by treating the glucose with a phosphorylation enzyme. The composition for producing allulose may further include an enzyme system for producing glucose-6-phosphate.

Specifically, the enzyme included in the composition for producing allulose of the present invention and the substrate used for producing allulose are not limited. The composition for producing allulose of the present invention comprises (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 kinase; 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 enzyme of the above item (a) or a culture of a microorganism expressing the enzyme of the above item (a), but is not limited thereto.

Specifically, the starch/maltodextrin phosphorylase (EC 2.4.1.1) and α-glucan phosphorylase of the present invention transfer phosphate to glucose to obtain glucose from starch or maltodextrin. Any protein may be included as long as it has an activity to produce -1-phosphate.

The sucrose phosphorylase (EC 2.4.1.7) of the present invention may include any protein as long as it has an activity to produce glucose-1-phosphate from sucrose by transferring phosphate to glucose.

α-amylase (EC 3.2.1.1), pullulanse (EC 3.2.1.41), glucoamylase (EC 3.2.1.3) and isoamylase, which are starch liquid glycosylation enzymes of the present invention may include any protein as long as it has an activity to convert starch or maltodextrin into glucose.

The sucrase (EC 3.2.1.26) of the present invention may include any protein as long as it has an activity of converting sucrose into glucose. The phosphoglucomutase (EC 5.4.2.2) of the present invention may include any protein as long as it has an activity to convert glucose-1-phosphate to glucose-6-phosphate. Glucose kinase (glucokinase) may include any protein as long as it has an activity of converting phosphate to glucose to glucose-6-phosphate. Specifically, the glucose kinase may be a polyphosphate-dependent glucose kinase.

The glucose-6-phosphate isomerase of the present invention may include any protein as long as it has an activity to convert glucose-6-phosphate to fructose-6-phosphate.

In the allulose production method of the present invention, allulose derived from the Clostridiales genus strain according to the present invention as an enzyme protein having an activity of converting allulose-6-phosphate to allulose and having high substrate specificity for allulose-6-phosphate -6-phosphate dephosphorylation enzyme (CloA6PP), a microorganism expressing the allulose-6-phosphate dephosphorylation enzyme, or a culture of a microorganism expressing the allulose-6-phosphate dephosphorylation enzyme may be used.

Since the allulose-6-phosphate dephosphorylation enzyme according to the present invention has high activity properties in a wide pH range, a product can be made through the enzymatic reaction without restriction of conditions. In addition, since it has a high activity specifically for the A6P substrate, a final reaction product containing allulose in a high ratio can be obtained. By securing a reaction product with a high allulose content, there is an advantage in simplifying the process and reducing the production cost by omitting the separation and concentration process steps in the high-concentration allulose production process, which has been previously performed through several steps. Accordingly, the composition for producing allulose containing allulose-6-phosphatase is more sensitive to the pH condition of the enzymatic reaction than when phytase, which performs dephosphorylation reaction in allulose-6-phosphate, is used. The reaction can be carried out almost without restrictions, and by securing a reaction product with a high allulose content due to high substrate specificity, the separation and concentration process steps are omitted in the high-concentration allulose production process that has been previously performed several steps, thereby simplifying the process and This has the advantage of reducing the production cost.

The reaction temperature and reaction pH conditions using the enzyme for producing allulose or the microorganism producing the enzyme using the composition for producing allulose are the same as described above for the reaction temperature and reaction pH conditions of the enzyme.

In one embodiment of the present invention, the step of converting allulose-6-phosphate to allulose by contacting a dephosphorylation enzyme in allulose-6-phosphate, a microorganism expressing the enzyme, or a culture of a microorganism expressing the enzyme It provides a method for producing allulose comprising a.

In addition, in the production method of the present invention, before the step of converting allulose-6-phosphate to allulose, an epimerase for fructose-6-phosphate, a microorganism expressing the epimerase, or a method for expressing the epimerase The method may further include converting the fructose-6-phosphate to allulose-6-phosphate by contacting the culture of the microorganism. The description of the fructose-6-phosphate epimerase is the same as described above.

In the production method of the present invention, before the step of converting fructose-6-phosphate to allulose-6-phosphate, glucose-6-phosphate-isomerase and the glucose-6-phosphate-isomerase are expressed in glucose-6-phosphate. Contacting a microorganism or a culture of a microorganism expressing the glucose-6-phosphate-isomerase to convert the glucose-6-phosphate into fructose-6-phosphate may be additionally included.

In addition, in the production method of the present invention, before the step of converting glucose-6-phosphate to fructose-6-phosphate, phosphoglucomutase to glucose-1-phosphate, the phosphoglucomutase Contacting a culture of a microorganism expressing the phosphoglucomutase or a microorganism expressing the phosphoglucomutase, the step of converting the glucose-1-phosphate into glucose-6-phosphate may be additionally included.

In the production method of the present invention, α-glucan phosphorylase, starch phosphorylase is added to starch, maltodextrin, sucrose or a combination thereof before the step of converting glucose-1-phosphate of the present invention into glucose-6-phosphate. , maltodextrin phosphorylase or sucrose phosphorylase; a microorganism expressing the phosphorylase; Alternatively, the method may further include converting the starch, maltodextrin, sucrose, or a combination thereof into glucose-1-phosphate by contacting a culture of a microorganism expressing the phosphorylase and phosphate.

Before the step of converting the glucose of the present invention into glucose-1-phosphate, the production method of the present invention includes α-amylase, pullulanase, glucoamylase, sucrase or isoamylase in starch, maltodextrin, sucrose or a combination thereof. ; a microorganism expressing the amylase, fluranase or sucrase; Alternatively, the method may further include converting the starch, maltodextrin, sucrose or a combination thereof into glucose by contacting the amylase, fluranase or sucrase with a culture of a microorganism.

The production method of the present invention is a culture of 4-α-glucanotransferase, a microorganism expressing the 4-α-glucanotransferase in glucose, or a microorganism expressing the 4-α-glucanotransferase It may further include the step of converting the glucose into starch, maltodextrin or sucrose by contacting the

A specific example of the present invention is a method for preparing allulose from starch as a starting material, which may include the following steps:

(1) α-glucan phosphorylase, starch phosphorylase, maltodextrin phosphorylase or sucrose phosphorylase in starch, maltodextrin, sucrose or a combination thereof; a microorganism expressing the phosphorylase; or contacting a culture of a microorganism expressing the phosphorylase with phosphate to convert the starch, maltodextrin, sucrose or a combination thereof into glucose-1-phosphate;

(2) contacting glucose-1-phosphate with a culture of phosphoglucomutase, a microorganism expressing the phosphoglucomutase, or a microorganism expressing the phosphoglucomutase, converting the glucose-1-phosphate to glucose-6-phosphate;

(3) Contacting glucose-6-phosphate with a culture of a microorganism expressing the glucose-6-phosphate-isomerase, the glucose-6-phosphate-isomerase, or the glucose-6-phosphate-isomerase converting the glucose-6-phosphate to fructose-6-phosphate;

(4) contacting fructose-6-phosphate with an epimerase, a microorganism expressing the epimerase or a culture of a microorganism expressing the epimerase, and converting the fructose-6-phosphate to allulose-6-phosphate converting to , and

(5) converting allulose-6-phosphate to allulose by contacting a dephosphorylation enzyme in allulose-6-phosphate, a microorganism expressing the enzyme, or a culture of a microorganism expressing the enzyme.

In the method for producing allulose according to the present invention, the enzymatic reactions used in steps (1) to (5) are sequentially performed, or at least two or more steps are performed in one reactor in a complex reaction using at least two or more enzymes together. may be carried out, and preferably, a complex enzymatic reaction including all of the enzymes used in steps (1) to (5) may be performed in one reactor. Allulose can be produced at a higher conversion rate than allulose from fructose by proceeding with the above enzymatic reaction step as one-pot enzymatic conversions. Allulose produced in this way can be usefully added to functional foods and pharmaceuticals.

The dephosphorylation enzyme protein according to the present invention, more specifically, the allulose-6-phosphate dephosphorylation enzyme protein, which has high substrate specificity only for allulose-6-phosphate, has a high enzyme conversion rate, substrate specificity, acidic or neutral reaction pH conditions, and Since it satisfies the characteristics of thermal stability, it can be usefully used for the production of allulose using allulose-6-phosphate dephosphorylation enzyme on an industrial scale.

1 shows a candidate group of allulose-6-phosphate dephosphorylation enzymes according to an embodiment of the present invention comprising glucose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate, and allulose-6-phosphate. This is a graph showing the allulose production rate confirmed by analyzing the reaction product obtained by reacting the mixed substrate solution with HPLC.

2 shows the results of BIO-LC analysis of fructose-6-phosphate 3-epimerase protein according to an embodiment of the present invention.

3 is a result of LC analysis after dephosphorylating the phosphorylated saccharide in the reaction product solution through the enzymatic reaction of allulose-6-phosphate phosphatase according to an example of the present invention.

4 is a graph showing the effect of temperature on the activity of allulose-6-phosphate dephosphorylation enzyme (CloA6PP) according to an embodiment of the present invention.

5 is a graph showing the effect of pH conditions on the activity of allulose-6-phosphate dephosphorylation enzyme (CloA6PP) according to an embodiment of the present invention.

6 is a graph showing the effect of metal ions on the activity of allulose-6-phosphate dephosphorylation enzyme (CloA6PP) according to an embodiment of the present invention.

7 is a process for producing allulose as a starch substrate using a complex enzymatic reaction including allulose-6-phosphate dephosphorylation enzyme (CloA6PP) or TalA6PP (A6PP from Thermoleophilum album) according to an embodiment of the present invention. It is the HPLC analysis result showing the result.

The present invention will be described in more detail with reference to the following examples, but the scope of the rights is not limited to the following examples.

Example 1: Preparation of allulose-6-phosphate dephosphorylation enzyme

The amino acid information of the candidate enzymes expected to function as allulose-6-phosphate dephosphorase enzymes was obtained from NCBI, and polynucleotides encoding the amino acid sequences of each enzyme were obtained by requesting gene synthesis through IDT gene synthesis. PCR was performed using the synthetic gene fragment as a template to amplify the nucleotide sequence of the gene, and all were cloned into the pET21a vector with NdeI/XhoI restriction enzyme sites.

Candidate enzymes were obtained through the microbial culture and protein purification process, and the candidate enzymes are as follows, and the primer sequences used in each PCR are shown in Table 1 below.

(1) CloA6PP: It is known as an A6PP candidate enzyme (HAD family hydrolase) derived from Clostridiales sp. strain, and has the amino acid sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 2.

(2) CbaA6PP: Candidatus Bathyarchaeota archaeon-derived A6PP candidate enzyme known as (HAD family hydrolase, Genbank accession no. TEU11455.1), has the amino acid sequence of SEQ ID NO: 3

(3) TalA6PP: It is known as an A6PP candidate enzyme derived from Thermoleophilum album (HAD family hydrolase, Genbank accession no. NZ_FNWJ01000002.1) and has the amino acid sequence of SEQ ID NO: 4

(4) AbaA6PP: known as A6PP candidate enzyme derived from Acidobacteria bacterium (HAD family hydrolase, Genbank accession no. QHVS01000101.1), has the amino acid sequence of SEQ ID NO: 5

(5) ChbA6PP: Chloroflexi bacterium-derived A6PP candidate enzyme known as (HAD family hydrolase, Genbank accession no. VBIC01000113.1), has the amino acid sequence of SEQ ID NO: 6

Name	sequence (5'->3')	SEQ ID NO
Forward primer in CloA6PP		aaggagatatacatatgaacaactacaaagctgttttc	7
Reverse primer in CloA6PP		ggtggtggtgctcgagctcttcgaagatgtccagcag	8
Forward primer in CbaA6PP		aaggagatatacatatgcgtttcccggtcgttatc	9
Reverse primer in CbaA6PP		ggtggtggtgctcgagagactgatcaatcagttcacc	10
Forward primer in TalA6PP	aaggagatatacatatgcgtgcactggtattcgacc	11
Reverse primer in TalA6PP	ggtggtggtgctcgagttcggctgcagtttccagg	12
Forward primer in AbaA6PP	aaggagatatacatatgccagcgttaatctttgatctg	13
Reverse primer in AbaA6PP	ggtggtggtgctcgagtggcagcacgcccagctc	14
Forward primer in ChbA6PP	aaggagatatacatatgacaccggtacttttattc	15
Reverse primer in ChbA6PP	ggtggtggtgctcgagagatgaaggttcacgaacac	16

Each of the five types of recombinant vectors was transformed into Escherichia coli ER2566 strain. Recombinant E. coli for enzyme protein expression was obtained as colonies on an agar plate prepared with LB medium containing 100 μg/ml ampicillin.

After incubation in 4ml LB medium, the main culture was performed in 100ml LB medium, and the culture condition was 37℃ 200rpm until the absorbance value was 0.6 at 600nm, and then 0.1mM of IPTG was added to induce expression of the target protein. After induction, the strain was cultured at 25 °C for about 16 hours, and the cells were recovered by centrifugation. The recovered cells were suspended in a lysis buffer (50 mM sodium phosphate (pH 7.0) buffer, 300 mM NaCl, 10 mM imidazole), and the cells were disrupted using a beadbeater to obtain a cell disruption solution.

After removing the cell pellet from the cell disruption solution, only the cell supernatant was obtained and bound to a Ni-NTA column (Ni-NTA superflow, Qiagen), followed by washing buffer (50 mM sodium phosphate (pH 7.0) buffer, 300 mM NaCl, 20 mM imidazole) to remove the protein not bound to the column, and as a final step, the target protein was eluted with an elution buffer (50 mM sodium phosphate (pH 7.0) buffer, 300 mM NaCl, 200 mM imidazole). Finally, the obtained protein was converted to 50mM sodium phosphate buffer (pH 7.0) and stored for later use.

Example 2: Preparation of substrate solution using fructose-6-phosphate epimerization (FP3E) enzyme

2-1. Evaluation of Fructose-6-Phosphate Epimerization (FP3E) Enzyme for Preparation of Substrate Solution

In this experiment, to prepare a substrate solution for allulose-6-phosphate dephosphorylation enzyme, 50 g/L of fructose-6-phosphate was dissolved in 50 mM sodium phosphate (pH 7.0) buffer, followed by fructose-6-phosphate epimerization. (FP3E) enzyme (enzyme (ClFP3E) derived from Clostridium lundense DSM 17049 strain, having the amino acid sequence of SEQ ID NO: 17 and the nucleotide sequence of SEQ ID NO: 18, and the amino acid sequence of the enzyme (SEQ ID NO: 17)) using allulose A -6-phosphate conversion solution was obtained.

To evaluate whether ClFP3E enzyme produces a substrate for allulose-6-phosphate dephosphorylation enzyme from fructose-6-phosphate, 0.1 mg/ml ClFP3E enzyme was added to 50 mM sodium phosphate (pH 7.0) buffer with 20 g/L fructose-6 - The conversion activity of ClFP3E enzyme was evaluated by adding phosphoric acid to the dissolved solution and performing the enzymatic reaction at 50 °C.

In the analysis of the enzyme reaction product, the newly produced material was confirmed by comparing it with the substrate through Bio-LC analysis. (A6PP) was finally confirmed allulose produced after treatment with the dephosphorylation enzyme. Bio-LC analysis confirmed that during the ClFP3E enzyme reaction, a decrease in F6P and a new peak thought to be A6P were generated. The results of the Bio-LC analysis are shown in FIG. 2 below.

The results of LC analysis after dephosphorylating the phosphorylated sugar in the reaction product solution through the A6PP enzymatic reaction are as follows. It was analyzed using an Aminex HPX-87C column at a temperature of 80° C. and a flow rate of 0.6 ml/min, and the results are shown in FIG. 3 . As a result of the analysis, fructose and allulose were confirmed.

2-2: Preparation of mixed substrate solution

As an enzyme substrate solution, 10 g/L of fructose-6-phosphate was dissolved in 50 mM sodium phosphate (pH 7.0) buffer, and 0.01 mg/ml of purified ClFP3E protein was added to the conversion reaction at 50 ° C., pH 7.0 for 2 hours. to obtain a reaction product containing fructose 6-phosphate and allulose-6-phosphate. A reaction product containing fructose 6-phosphate and allulose-6-phosphate in a weight ratio of 3:2 was used to prepare a mixed substrate solution for allulose-6-phosphate dephosphorylation enzyme activity analysis.

To prepare a mixed substrate solution for enzyme activity analysis, a predetermined amount of glucose-1-phosphate, glucose A substrate mixture was prepared by adding -6-phosphoric acid and used in the experiment, and the mixing weight ratio of each substrate was G-1-P: G-6-P: F-6-P: A-6-P = 2: 2 : 3 : 2 was used, and the total content of 4 phosphorylated sugars included in the mixed substrate was 10 g/L.

Example 3: Enzyme screening using substrate reaction

When allulose-6-phosphate dephosphorylation enzyme having low substrate specificity acts on the mixed substrate, glucose-1-phosphate and glucose-6-phosphate are dephosphorylated to produce glucose, and fructose-6-phosphate is fructose and allulose. -6-phosphate is converted to allulose.

The purified enzyme protein prepared in Example 1-1 was added in an amount of 0.1 mg/ml to the prepared mixed substrate solution, and the enzyme reaction was performed at 50° C. and pH 7.0 for 2 hours, and the resulting enzyme reaction product was analyzed by HPLC. to analyze the sugar contained in the enzymatic reaction product solution, and the experimental results are shown in FIG. 1 . The HPLC analysis was performed using an Aminex HPX-87C column at a temperature of 80° C. and a flow rate of 0.6 ml/min.

1 shows the candidate group of allulose-6-phosphate dephosphorylation enzymes reacted with a mixed substrate solution containing glucose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate, and allulose-6-phosphate. It is a graph showing the allulose production rate confirmed by analyzing the obtained reaction product solution by HPLC. The production ratio of each product shown in the HPLC graph of FIG. 1 is digitized and shown in Table 1 below.

The substrate specificity was expressed as the proportion of allulose in the total product (allulose production rate, weight %), and was specifically 89.1%. The allulose production ratio (weight %) was calculated by the formula of allulose production/(glucose production + fructose production + allulose production) * 100. The unit of production amount of the following product is (g/L). The conversion rate in Table 2 below shows the content ratio of the generated dephosphorylation product based on the content of the corresponding substrate contained in the mixed substrate. For example, the content of allulose-6-phosphate substrate contained in the mixed substrate (g/L ) means the weight ratio of the allulose content (g/L) produced by dephosphorylation.

division	enzyme	CloA6PP	TalA6PP	AbaA6PP
glucose	Production (g/L)	-	1.4	1.4
	Conversion rate (%)	-	63.0	63.0
fruit sugar	Production (g/L)	0.2	0.6	0.8
	Conversion rate (%)	6.1	18.0	24.0
allulose	Production (g/L)	2.1	2.2	2.2
	Conversion rate (%)	95.5	99.0	99.0
Allulose ratio of reaction product (%)		89.1	51.5	51.3

As shown in FIG. 1 , it was confirmed that CbaA6PP and ChbA6PP did not have an allulose product confirmed by HPLC and thus had no dephosphorylation enzyme activity of allulose-6-phosphate. In addition, CloA6PP, TalA6PP and AbaA6PP represent allulose-6-phosphate dephosphorylation enzymes, and the quantitative results are shown in Table 2 in more detail. In addition, CloA6PP had an allulose-6-phosphate dephosphorylation activity, but exhibited an irreversible enzymatic activity that did not perform a reverse reaction.

As shown in Figure 1 and Table 2, in the enzymatic reaction product to which TalA6PP and AbaA6PP were added, there was activity of the additive enzyme, but the substrate specificity acting specifically for A6P was very poor, so glucose, fructose, and allulose were all produced, and fructose, glucose and The production rate showed a tendency to increase in the order of allulose. In addition, the allulose ratio (%) of the reaction product is 51.5% and 51.3% for TalA6PP and AbaA6PP, respectively, and the allulose production ratio is low, so the industrial use value is low.

1 and Table 2, it can be seen that CloA6PP is excellent in both enzyme activity and substrate specificity, so that only allulose is produced in excess. As a result of the enzymatic reaction using CloA6PP, it was confirmed that the ratio of allulose in the total product was about 89.1%, and at the same time, a very small amount of fructose was converted, and glucose was not confirmed and thus was not produced. Specifically, the CloA6PP enzyme produces allulose with a very high ratio of allulose conversion to fructose conversion of 15.7 times, but TalA6PP shows a low conversion ratio of 5.5 times and AbaA6PP 4.3 times. Also, looking at the ratio of the allulose conversion rate to the total conversion rate of fructose and glucose, the CloA6PP enzyme does not produce glucose, so it is 15.7 times as high as the allulose conversion rate for fructose, but TalA6PP and AbaA6PP with high glucose production in the product shows a low conversion ratio of 1.2 times.

Example 4: Temperature and pH Characterization of Enzymes

4-1: Confirmation of enzyme activity according to temperature

In order to confirm the effect of temperature on CloA6PP enzyme activity, an enzyme reaction experiment was performed under various temperature conditions.

After dissolving 50 g/L of fructose-6-phosphate in the 50 mM sodium phosphate (pH 7.0) buffer prepared in Example 2, a fructose-6-phosphate epimerization (FP3E) enzyme was used to convert allulose-6-phosphate. was obtained and used as a substrate. The enzyme reaction was carried out with the CloA6PP enzyme prepared in Example 1-1 0.1 mg/ml at each temperature condition for 30 min. saved The experimental results are shown in FIG. 4 .

As shown in Figure 4, the optimum temperature condition of CloA6PP was confirmed to be 55 ℃, it can be seen that although high activity at 40 ~ 55 ℃ condition, the activity rapidly decreases at 60 ℃.

4-2: Confirmation of enzyme activity according to pH conditions

To confirm the pH effect of CloA6PP, enzyme reaction experiments were performed under various pH conditions.

After dissolving 50 g/L of fructose-6-phosphate in the sterile water prepared in Example 2, a fructose-6-phosphate epimerization (FP3E) enzyme was used to obtain an allulose-6-phosphate conversion solution and used as a substrate. did After diluting the FP3E conversion reaction product to a concentration of 10 g/L in a buffer solution between pH 5.0 and 8.5 (pH 5.5 to 6.5, sodium citrate / pH 6.5 to 9, Tris-HCl), 0.1 mg/ml of CloA6PP purification The protein was added and the enzymatic reaction was carried out under conditions of 50° C. and 60 min. The obtained reaction product was analyzed by HPLC to quantitatively analyze the amount of allulose produced to determine the relative enzyme activity. The experimental results are shown in FIG. 5 .

As shown in FIG. 5, it was confirmed that it had 80% or more of activity in a wide range of pH 5.5 to 9.0, and it was confirmed that there was no reaction inhibition by pH.

Example 5: Analysis of metal ion properties of enzymes

An experiment was performed to confirm the activity of CloA6PP according to the type of metal ion added to the enzymatic reaction.

After dissolving 50 g/L of fructose-6-phosphate in the 50 mM sodium phosphate (pH 7.0) buffer prepared in Example 2, a fructose-6-phosphate epimerization (FP3E) enzyme was used to convert allulose-6-phosphate. was obtained and used as a substrate.

After diluting the substrate 1/2-fold, 5 mM of each metal ion (MgSO ₄ , MnCl ₂ , CaCl ₂ , CoCl ₂ , CuCl ₂ , NiSO ₄ , FeSO ₄ , ZnSO ₄ ) was added.

0.1mg/ml of CloA6PP purified protein was added to the reaction buffer containing each metal ion, and the enzymatic reaction was performed under conditions of 50°C and 60min. The obtained reaction product was analyzed by HPLC to quantitatively analyze the amount of allulose produced, activity was obtained. The experimental results are shown in FIG. 6 .

As shown in Figure 6, MgSO ₄ , MnCl ₂ , CoCl ₂ , CaCl ₂ , When NiSO ₄ was added, higher activity was confirmed than the control group not containing metal ions, and in particular, when MnCl ₂ was added, 11-fold or more activity could be confirmed.

Example 6: Production of allulose through a complex enzyme reaction

In order to produce allulose from the maltodextrin substrate, five enzymes required for the entire reaction step were reacted simultaneously. The enzymes used were GP, PGM, PGI, FP3E, and CloA6PP, each used at a concentration of 0.1 mg/ml. An experiment in which A6PP having low substrate specificity was added as a control was also conducted under the same conditions.

Alpha-glucan kinase (αGP) derived from Corynebacterium glutamicum (CAF20007.1), Glucose phosphate mutase (PGM) derived from Geobacillus thermocatenulatus (AST00503.1), Glucose phosphatase (PGI) (ASS98370.1), Examples 2 according to Clostridium sp. Maltodextrin → glucose-6-phosphate → fructose-6-phosphate → allulose-6- using fructose-6-phosphate-3-epimerization agent (FP3E) derived from and CloA6PP derived from Clostridiales according to Example 1 A sequential enzymatic conversion reaction was carried out from phosphoric acid to allulose.

Genes for each enzyme were cloned into pET21a vector or pET28a vector, and then transformed into Escherichia coli ER2566 strain. Recombinant E. coli for enzyme protein expression was obtained as colonies on an agar plate prepared with LB medium containing 100 μg/ml ampicillin or 30 μg/ml kanamycine.

After culturing one colony in 4ml LB medium, the main culture was performed in 100ml LB medium, and cultured under the condition of 37°C and 200rpm until the absorbance value was 0.6 at 600nm, and then 0.1mM of IPTG was added to express the target protein. was induced. After induction, the strain was cultured at 25 °C for about 16 hours, and the cells were recovered by centrifugation. The recovered cells were suspended in a lysis buffer (50 mM sodium phosphate (pH 7.0) buffer, 300 mM NaCl, 10 mM imidazole), and the cells were disrupted using a beadbeater to obtain a cell disruption solution.

After removing the cell pellet from the cell disruption solution, only the cell supernatant was obtained and bound to a Ni-NTA column (Ni-NTA superflow, Qiagen), followed by washing buffer (50 mM sodium phosphate (pH 7.0) buffer, 300 mM NaCl, 20 mM imidazole) to remove the protein not bound to the column, and as a final step, the target protein was eluted with an elution buffer (50 mM sodium phosphate (pH 7.0) buffer, 300 mM NaCl, 200 mM imidazole). Finally, the obtained protein was converted to 50mM sodium phosphate buffer (pH 7.0) and then used for the enzymatic conversion reaction.

Specifically, 20g/L maltodextrin substrate was prepared by dissolving in 50mM sodium phosphate buffer (pH 7.0), and 5 enzymes quantified at 0.1mg/ml each and 5mM MnCl2 were added for enzymatic reaction at 50℃ for 5 hours. The enzymatic reaction was carried out, and the reaction product was analyzed by analyzing the obtained reaction product by HPLC. The quantitative analysis results are shown in FIG. 8 .

For HPLC analysis, Aminex HPX-87C (Bio-rad) column was used, and the mobile phase was flowed at a flow rate of 0.6ml/min at 80°C, and the product was detected with a Reactive Index Detector (RID).

Comparative Example 1: Allulose production through complex enzyme reaction

It was carried out in substantially the same manner as in Example 6, but in a complex enzymatic reaction, four enzymes (alpha-glucan kinase (αGP) (CAF20007.1) from Corynebacterium glutamicum (CAF20007.1) and glucose phosphate mutase (PGM) from Geobacillus thermocatenulatus ) ( AST00503.1), glucose phosphate isomerase (PGI) (ASS98370.1), and a fructose-6-phosphate-3-epimerase (FP3E) derived from Clostridium sp. according to Example 2) were the same, and CloA6PP was Instead, the reaction was performed using the TalA6PP enzyme used in Example 1-1, and the resulting reaction product was analyzed by HPLC. The quantitative analysis results are shown in FIG. 7 .

As shown in the HPLC analysis result of FIG. 7 , it was confirmed that five conversion reactions of starch, G1P, G6P, F6P, A6P, and allulose were sequentially performed in one reactor through the five complex enzyme reactions.

In addition, by using CloA6PP as a dephosphoryase, a substrate-specific rapid reaction was performed only for A6P, and it was confirmed that an excess of allulose was produced while minimizing the production of glucose or fructose. All phosphorylated sugars are dephosphorylated without substrate specificity, and glucose-1-phosphate (G-1-P) or glucose-6-phosphate (G-6-P), which are the initial substances in the reaction step, is dephosphorylated to generate excess glucose confirmed that it has been These results could be seen through the analysis results of the reaction product solution using TalA6PP and the reaction product solution using CloA6PP in the graph of FIG. 8 .

enzyme	glucose Production (g/L)	Fructose production (g/L)	allulose Production (g/L)	allulose Substrate specificity (%)	allulose Conversion rate (%)
TalA6PP	6.51	1.6	0.5	5.8	2.5
CloA6PP	0.6	1.8	12.9	84.3	64.5

Claims (13)

1. Allulose-6-phosphate dephosphorylation enzyme having 30% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1 and converting allulose-6-phosphate to allulose by dephosphorylating it protein.
2. The enzyme protein according to claim 1, wherein the enzyme is encoded by a nucleotide sequence having 50% or more nucleotide sequence identity with the nucleotide sequence of SEQ ID NO: 2.
3. The enzyme protein according to claim 1, wherein the ratio of the conversion rate of allulose-6-phosphate to allulose to the conversion rate of glucose-6-phosphate or glucose-1-phosphate to glucose is 20 times or more.
4. The method according to claim 1, wherein when the enzyme is applied to a mixed substrate containing fructose-6-phosphate and allulose-6-phosphate, 60% by weight or more of allulose based on 100% by weight of total dephosphorylated sugar of the reaction product An enzyme protein having a production amount.
5. The enzyme protein according to claim 1, wherein the ratio of the conversion rate of allulose-6-phosphate to allulose to the conversion rate of fructose-6-phosphate to fructose is 6 to 20 times for the enzyme.
6. According to claim 1, wherein the enzyme is allulose-6-phosphate for the sum of the conversion rate of conversion of fructose-6-phosphate to fructose and the conversion rate of glucose-1-phosphate and glucose-6-phosphate to glucose. Enzyme protein in which the ratio of conversion rate to allulose is at least 2 to 20 times.
7. According to claim 1, wherein the enzyme is derived from Clostridium lundense , the enzyme reaction temperature of 40 to 55 ℃ and pH 5.5. An enzyme protein having an enzyme reaction pH of to 9.0.
8. According to claim 1, wherein the enzyme is Mg, Mn, Co, Ca, or An enzyme protein whose activity is increased by Ni ions.
9. The enzyme protein according to claim 1, wherein the enzyme activity is reduced by Cu, Fe or Zn ions.
10. The allulose-6-phosphate dephosphorylation enzyme according to any one of claims 1 to 9, a microorganism expressing the enzyme protein, a transforming microorganism expressing the enzyme protein, a cell body of the microorganism, a cell body of the microorganism A composition for producing allulose, comprising at least one selected from the group consisting of a lysate, a culture of the microorganism, and an extract thereof.
11. The composition for producing allulose according to claim 10, wherein the composition further comprises a fructose 6-phosphate epimerase, a microorganism expressing the same, or a culture of the microorganism.
12. 10. A method for producing allulose, comprising the step of converting allulose-6-phosphate to allulose using an allulose-6-phosphate dephosphorylation enzyme according to any one of claims 1 to 9.
13. 13. The method of claim 12, wherein the step of converting fructose 6-phosphate to allulose-6-phosphate by contacting the fructose 6-phosphate epimerase, a microorganism expressing the same, or a culture of the microorganism with fructose 6-phosphate. How to include additionally.

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