FRUCTOSE-6-PHOSPHATE 3-EPIMERASE AND USE THEREOF

Abstract

The present disclosure relates to an epimerase protein of fructose-6-phosphate, nucleic acid molecule encoding the epimerase protein, a recombinant vector and a transgenic microorganism which comprise the nucleic acid molecule, and a composition for producing allulose by using them.

Claims

1. An epimerase protein of fructose-6-phosphate which has at least 70% of amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1 and converts fructose-6-phosphate to allulose-6-phosphate.

2. The epimerase protein according to claim 1, wherein the enzyme is encoded by a nucleotide sequence having at least 80% of nucleotide sequence identity to the nucleotide sequence of SEQ ID NO: 2.

3. The epimerase protein according to claim 1, wherein the enzyme is derived from Clostridium lundense, and has an enzyme reaction temperature of 40 to 70° C. and an enzyme reaction pH of pH 6 to 8.

4. The epimerase protein according to claim 1, wherein the enzyme is an incrased activity by a manganese ion, a cobalt ion, or a nickel ion.

5. A composition for producing allulose, comprising at least one selected from the group consisting of fructose-6-phosphate epimerase protein as set forth claim 1, a microorganism expressing the enzyme, a transgenic microorganism expressing the enzyme protein, a microbial cell of the microorganism, cell lysate of the micribial cell of the microorganism, a culture of the microorganism, a culture supernatant of the microorganism, a concentrate of the culture supernatant of the microorganisms and their powders.

6. The composition for producing allulose according to claim 5, wherein the composition further comprises a phosphatase enzyme of allulose 6-phosphate, a microorganism expressing the same, or a culture of the microorganism.

7. The composition for producing allulose according to claim 5, wherein the composition further comprises one or more metal ions selected from the group consisting of manganese ion, cobalt ion and nickel ion.

8. The composition for producing allulose according to claim 5, wherein the composition further comprises an isomerase converting glucose-6-phosphate to fructose-6-phosphate, a microorganism expressing the same, or a culture of the microorganism.

9. The composition for producing allulose according to claim 5, wherein the composition further comprises: (a) (i) starch, maltodextrin, sucrose, or a combination thereof; (ii) phosphate; (iii) allulose-6-phosphate phosphase; (iv) glucose-6-phosphate isomerase; (v) phosphoglucomutase or glucose phosphorylase; and (vi) α-glucanophosphorylase, starch phosphorylase, maltodextrin phosphorylase, sucrose phosphorylase, α-amylase, pullulanase, isoamylase, glucoamylase or sucrase; or (b) a microorganism expressing the enzyme of item (a) or a culture of the microorganism, and wherein the composition further comprises a phosphatase enzyme of allulose 6-phosphate.

10. A method for producing allulose, comprising a step of converting fructose-6-phosphate to allulose-6-phosphate using the epimerase of fructose-6-phosphate as set forth in claim 1.

11. The method according to claim 10, which further comprises a step of converting allulose-6-phosphate to allulose by contacting the allulose-6-phosphate with phytase, a microorganism expressing the same, or a culture of the microorganism.

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

13. The method according to claim 10, which further comprises a step of preparing fructose-6-phosphate from fructose or a fructose-containing material using hexokinase.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0063] FIG. 1 is an electrophoresis photograph confirming the expression and purification of fructose-6-phosphate 3-epimerase protein according to an embodiment of the present disclosure;

[0064] FIG. 2 shows the results of BIO-LC analysis of fructose-6-phosphate 3-epimerase protein according to an embodiment of the present disclosure;

[0065] FIG. 3 shows the results of LC analysis after dephosphorylating the phosphorylated saccharide in the reaction solution through the enzymatic reaction of allulose-6-phosphate phosphatase according to an example of the present disclosure;

[0066] FIG. 4 is a graph showing the results of analyzing the temperature characteristics of fructose-6-phosphate 3-epimerase protein according to an embodiment of the present disclosure;

[0067] FIG. 5 is a graph showing the results of analyzing the pH characteristics of fructose-6-phosphate 3-epimerase protein according to an embodiment of the present disclosure;

[0068] FIG. 6 is a graph showing the effect of metal ions of fructose-6-phosphate 3-epimerase protein according to an embodiment of the present disclosure; and

[0069] FIG. 7a and FIG. 7b is an HPLC analysis result of a reaction product obtained after performing allulose production using three types of conventionally known ribulose-phosphate 3-epimerase.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0070] The present disclosure will be described in more detail with reference to the following examples, but the scope of the lights is not limited to the following examples.

Example 1: Production of Fructose-6-Phosphate 3-Epimerase

[0071] Candidate enzymes expected to function as fructose-6-phosphate 3-epimerase were screened, and as the enzyme expected to show the best effect, polynucleotide (SEQ ID NO: 2) encoding the amino acid sequence (SEQ ID NO: 1) of the enzyme (ClFP3E) derived from Clostridium lundense DSM 17049 strain was obtained by requesting gene synthesis through IDT gene synthesis. A primer was designed based on the synthesized ClFP3E DNA sequence of SEQ ID NO: 2, and PCR was performed to amplify the nucleotide sequence of the gene. The forward and reverse primer sequences used for PCR amplification are as follows.

TABLE-US-00001 TABLE 1 SEQ ID Primer name Sequence (5′->3′) NO: pET21_CIRP3E_F tatacatatgAAATACGGCTTCGCACC 3 pET21_CIRP3E_R ggtgctcgagTTCTTTGTAGCTCAGGCTGTA 4

[0072] The ClFP3E gene obtained in large quantities was introduced into the pET21a vector using restriction enzymes NdeI and XhoI to prepare pET21_ClFP3E, which was transformed into Escherichia coli ER2566 strain. Recombinant. E. coli for enzyme protein expression was obtained as colonies on an agar plate prepared in LB medium containing 50 μg/ml ampicillin.

[0073] After seed culture in 4 ml LB medium, the main culture was performed in 100 ml LB medium. The culture conditions were incubated at 37° C. and 200 rpm until the absorbance value at 600 nm was 0.6, and then 0.1 mM IPTG was added thereto to induce expression of the target protein. After induction, the strain was cultured at 25° C. for about 16 hours, and then centrifuged to recover the cells. 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. The overexpression of the target protein ClFP3E from the cell disruption solution was confirmed by SDS-PAGE gel analysis. The results of overexpression analysis of the target protein ClFP3E are shown in FIG. 1. The molecular weight of ClFP3E confirmed by SDS-PAGE gel analysis was about 28 KDa.

[0074] Additionally, after removing the cell pellet, only the cell supernatant was obtained and bound to a Ni-NTA column (Ni-NTA superflow, Qiagen), and then proteins not bound to the column were removed with a washing buffer (50 mM sodium phosphate (pH 7.0) buffer, 300 mM NaCl, 20 mM imidazole). As a final step, the protein of interest was eluted with an elution buffer (50 mM sodium phosphate (pH 7.0) buffer, 300 mM NaCl, 200 mM imidazole). The finally secured protein was converted to 501 mM sodium phosphate buffer (pH 7.0) and stored for subsequent use.

Example 2: Evaluation of the Conversion Activity of Enzymes

[0075] 0.1 mg/ml of the purified ClFP3E enzyme obtained in Example 1 was added to a solution in which 20 g/L of fructose-6-phosphate was dissolved in 50 mM sodium phosphate (pH 7.0) buffer, and the enzymatic reaction was performed at 50° C.

[0076] The analysis of the enzymatic reaction solution confirmed a newly produced substance as compared with the substrate through Bio-LC analysis. However, since the allulose-6-phosphate standard did not exist, accurate confirmation was not possible, and thus, after further treatment with a phosphatase enzyme of allulose-6-phosphate (A6PP), the resulting allulose was finally confirmed. Bio-LC analysis confirmed that during the C1FP3E 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.

[0077] The result of LC analysis after dephosphorylating the phosphorylated saccharide in the reaction solution through the A6PP enzymatic reaction is as follows. Analysis was performed at a temperature of 80° C. and a flow rate of 0.6 ml/min using Aminex HPX-87C column, and the results are shown in FIG. 3. As a result of the analysis, fructose and allulose could be confirmed.

[0078] The final conversion rate of ClFP3E calculated by quantifying the amount of allulose produced, which is the final product of the reaction solution dephosphorylated by the Arlos-6-phosphate phosphatase (A6PP) enzymatic reaction, was calculated to be 34.3%. T The reaction product of this example is the final conversion of ClFP3E obtained by performing the enzymatic reaction for 16 hours, and the maximum conversion rate of allulose is calculated according to the following equation.

Equation 1

[0079] Allulose maximum conversion rate (%)=allulose production amount (g/L)/fructose-6-phosphate input amount (g/L)*(allulose molecular weight/fructose-6-phosphate molecular weight)*100

Example 3: Analysis of Temperature Characteristics of Enzymes

[0080] To determine the effect of temperature on ClFP3E enzyme activity, 10 g/L of fructose-6-phosphate was dissolved in 50 mM sodium phosphate (pH 7.0) buffer, and then 0.01 mg/ml of ClFP3E purified protein was added thereto, and the reaction was performed at various temperature conditions between 40 and 80° C. for 5 minutes.

[0081] Then, the A6PP enzyme was added to dephosphorylate all reaction compositions, and then, the amount of allulose produced was quantitatively analyzed through HPLC analysis. The relative activity of the enzyme according to the reaction temperature is shown in FIG. 4 based on the activity at 60° C. Where the highest activity was measured.

[0082] As a result of the experiment, it was confirmed that the optimum temperature condition for ClFP3E was confirmed to be 60° C., and the activity was 50% or more of the maximum enzyme activity in a wide temperature condition range of 40 to 70° C.

Example 4: Analysis of pH Characteristics of Enzymes

[0083] To confirm the effect of pH on ClFP3E enzyme activity, 10 of fructose-6-phosphate was dissolved in a buffer of pH 5.0˜8.5(pH 5.0˜6.5, sodium citrate/pH 6.5˜8.5, Tris -HCI), 0.01 mg/ml of purified ClFP3E protein was added thereto, and the enzymatic reaction was performed at a temperature of 60° C. for 5 minutes. Then, A6PP enzyme was added to dephosphorylate all reaction compositions, and then allulose production amount was quantitatively analyzed through HPLC analysis. The result of the enzyme. activity according to the reaction pH is shown in FIG. 5 as a relative activity based on pH 7.5, which had the best activity.

[0084] As a result of the experiment, it was possible to confirm the maximum activity at pH 7.0 to 7.5, and it was confirmed that it had 80% or more of the maximum enzyme activity in the range of pH 6.0 to 8.0.

Example 5: Analysis of the Effect of Metal Ions on Enzymes

[0085] In order to confirm the activity of ClFP3E according to the type of metal ion added during the reaction, 10 g/L of fructose-6-phosphate was dissolved in 50 mM sodium phosphate (pH 7.0) buffer, 5 mM of each metal ion (MgCl.sub.2, MnCl.sub.2, CaCl.sub.2, CoCl.sub.2, CuCl.sub.2, NiSO.sub.4, FeSO.sub.4, ZeSO.sub.4) was added. 0.01 mg/ml of ClFP3E purified protein was added to the reaction buffer containing each metal ion, and the reaction was carried out at a temperature of 60° C. for 5 minutes.

[0086] Then, the A6PP enzyme was added to dephosphorylate all reaction compositions, and then, the amount of allulose produced was quantitatively analyzed through HPLC analysis. The relative activity of the enzyme depending on the type of the metal ion is shown in FIG. 6 based on the experimental group in which no metal ion was added.

[0087] As a result of the experiment, when MnCl.sub.2 and CoCl.sub.2 were added, it was confirmed that the activity was three times higher than the condition in which no metal ions were added. it was found that the ClFP3E enzyme is an enzyme using manganese and cobalt as cofactors. Also, the activity was increased even by nickel. It was CaCl.sub.2, CuCl.sub.2, FeSO.sub.4, ZeSO.sub.4 that decreased the enzyme activity.

Comparative Example 1: Analysis of Allulose Production Using Ribulose-Phosphate 3-Epimerase

[0088] Polynucleotides of Ruminococcus sp. AF14-10-derived ribulose-phosphate 3-epimerase (RuFP3E: amino acid sequence of SEQ ID NO: 5 and nucleic acid sequence of SEQ ID NO: 6), Clostridium sp. DU-VDT-derived ribulose-phosphate 3-epimerase (CDFME: amino acid. sequence of SEQ ID NO: 7 and nucleic acid sequence of SEQ ID NO: 8), Paenibacillus kribbensis-derived ribulose-phosphate 3-epimerase (PkFP3E: amino acid sequence of SEQ ID NO: 9 and nucleic acid sequence of SEQ ID NO: 10) were obtained by requesting gene synthesis.

[0089] As a result of analyzing the amino acid sequence homology to the amino acid sequence (SEQ ID NO: 1) of fructose-6-phosphate 3-epimerase (ClFP3E) derived from the Clostridium lundense DSM 17049 strain, the amino acid sequence identity to RuFP3E having the amino acid sequence of SEQ ID NO: 5 was 59.05%, the amino acid sequence identity to CDFP3E having the amino acid sequence of SEQ ID NO: 7 was 63%, and the amino acid sequence identity to PkFP3E having the amino acid sequence of SEQ ID NO: 9 was 60.96%.

[0090] Based on the synthesized ClFP3E DNA sequence of SEQ ID NO: 2, a large amount of genes were obtained in substantially the same method as in Example 1. In substantially the same method as in Example 1, the obtained gene was introduced into and expressed in E. Coli and then the protein of interest was eluted. Finally, the obtained protein was convened to 50 mM sodium phosphate buffer (pH 7.0) and stored for subsequent use.

[0091] In order to analyze the conversion activity of fructose 6-phosphate to allulose 6-phosphate, an enzymatic reaction using fructose-6-phosphate as a substrate was performed using the obtained enzyme in the same method as in Example 2. After the enzymatic reaction, a dephosphorylation reaction using an allulose-6-phosphate phosphatase (A6PP) enzyme was performed, and then, the amount of produced allulose was measured for the reaction product. solution using High-Performance Liquid Chromatography (HPLC).

[0092] As for HPLC analysis conditions, RID (Refractive Index Detector Agilent 1260 RID) of HPLC (Agilent, USA) equipped with an Aminex HPX-87C column (BIO-RAD) was used. Water was used as the mobile phase solvent, and the temperature was 80° C. and the flow rate was 0.6 ml/min. The results of the HPLE analysis are shown in FIG. 7a and 7b. The reaction product of this example was obtained by performing the enzymatic reaction for 4 hours to obtain the allulose conversion rate of the enzyme. As an experiment for screening the FP3E candidate enzyme, the reaction solution in which the reaction was stopped at the intermediate stage of allulose production was analyzed. In FIG. 7a and FIG. 7b, the allulose conversion rate of ClFP3E was 16%, and the reaction proceeded to about 50% level of the maximum conversion rate of Example 2. In this Example, the allulose production ratio of total reaction products was 75%. The alullose production rate is calculated by the following Equation.

[0093] Allulose production ratio (% by weight)=production amount/(fructose production amount+allulose production amount)*100

[0094] As shown in FIG. 7a and FIG. 7b, no allulose peak was confirmed in other candidate enzymes except for the ClFP3E enzyme of Example 1. Therefore, because other candidate enzymes other than the ClFP3E enzyme had no activity against F6P, it was confirmed that when phosphatase was treated, only fructose of a dephosphorylated from produced from F6P as an initial substrate of the reaction step, was generated.