KR20190079351A - A novel thermostable fructose-6-phosphate 3-epimerase and methods for producing allulose using the same - Google Patents

Abstract

The present application relates to a fructose-6-phosphate-3-epimerase including the amino acid sequence of SEQ ID NO: 1, a nucleic acid encoding the fructose-6-phosphate-3-epimerase, and a transformant containing the nucleic acid. Also, the present application relates to a composition comprising the fructose-6-phosphate-3-epimerase of the present application for producing allulose, and a method for producing allulose by using the fructose-6-phosphate-3-epimerase of the present application.

Description

[0001] The present invention relates to a novel thermostable fructose-6-phosphate 3-epimerase and a method for producing the same,

The present application relates to fructose-6-phosphate 3-epimerase and a method for producing alulose using the same.

D-psicose 3-epimerase (EC 5.1.3.30) and D-tagatose 3-epimerase (EC 5.1.3.31) produced D-fructose in the presence of 3-epimerase (3-epimerization, 3-carbon epimerization), which is known as an enzyme capable of producing aluloses. When the enzyme is used to produce alulose in fructose by a single enzyme reaction, there is a certain level of reaction equilibrium between the substrate fructose and the product alulose (product / substrate = about 20% to 35% ). Therefore, in the case of producing high purity alululose using the single enzyme reaction, an additional purification step of separating and removing high concentration of fructose from the reaction product is required.

Chan et al. (2008. Biochemistry 47: 9608-9617) reported that 3-epimerization of D-fructose 6-phosphate and D-allulose 6-phosphate D-ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) derived from Streptococcus pyogenes and E. coli- derived alulose-6-phosphate 3 (D-allulose 6-phosphate 3-epimerase, EC 5.1.3.-) have been reported. However, these enzymes have no heat resistance and thus can not be used industrially.

Under these circumstances, the present inventors have made an effort to develop a method which can economically and industrially increase the conversion rate to aluloses. As a result, glucose or glucose-1-phosphate, glucose-6-phosphate and fructose-6-phosphate from glucose, starch or maltodextrin, which are economical raw materials, In the case of producing alulose-6-phosphate, it is possible to produce alululose using allulose-6-phosphate phosphatase, which performs an irreversible reaction pathway, Considering that alululose can be produced by one-pot enzymatic conversions in which a plurality of enzymes involved in the production pathway of aluloses can be used at the same time and the conversion rate to alulose can be remarkably increased, -Phosphoric acid into allylulose-6-phosphate, the present application has been completed.

One object of the present application is to provide a fructose-6-phosphate 3-epimerase comprising the amino acid sequence of SEQ ID NO: 1.

Another object of the present application is to provide a nucleic acid encoding the fructose-6-phosphate 3-epimerase of the present application.

Another object of the present invention is to provide a transformant comprising a nucleic acid encoding the fructose-6-phosphate 3-epimerase of the present application.

Another object of the present invention is to provide a composition for producing allulose comprising the fructose-6-phosphate 3-epimerase of the present application, a microorganism expressing the same, or a culture of the microorganism.

It is still another object of the present invention to provide a method for producing alulose using the fructose-6-phosphate 3-epimerase of the present application.

Hereinafter, the contents of the present application will be described in detail. On the other hand, the description and the embodiments of one aspect disclosed in this application can be applied to descriptions and embodiments of other aspects with respect to common items. Also, all combinations of the various elements disclosed in this application fall within the scope of the present application. In addition, the scope of the present application is not limited by the specific description described below.

To achieve the object of the present application, the present application provides, as one embodiment, a fructose-6-phosphate 3-epimerase consisting of the amino acid sequence of SEQ ID NO: 1.

In addition, the fructose-6-phosphate 3-epimerase of the present application may comprise a polypeptide having at least 80%, 90%, 95%, 97% or 99% homology with the amino acid sequence of SEQ ID NO: For example, an amino acid sequence having such homology and having an effect equivalent to that of the protein consisting of the amino acid sequence of SEQ ID NO: 1, even though some of the sequences have amino acid sequences that are deleted, modified, substituted or added, It is self-evident.

Further, in the case of a protein having an activity corresponding to the fructose-6-phosphate 3-epimerase consisting of the amino acid sequence of SEQ ID NO: 1 of the present application, it may be added to the amino acid sequence of SEQ ID NO: 1 or a naturally occurring mutation, Or a silent mutation thereof, and the protein comprising the amino acid sequence of SEQ ID NO: 1 is also within the scope of the present application.

Further, although not limited thereto, the fructose-6-phosphate 3-epimerase may be encoded by the nucleotide sequence of SEQ ID NO: 2 and may be at least 80%, 90%, 95% , 97%, or 99% homologous to the nucleotide sequence shown in SEQ ID NO. It is obvious that a polynucleotide capable of being translated into a protein consisting of the amino acid sequence of SEQ ID NO: 1 or a protein having homology thereto by codon degeneracy can also be included in the scope of the present application.

In the above, the term "homology" means the degree to which a given amino acid sequence or base sequence is consistent and can be expressed as a percentage. In the present specification, its homologous sequence having the same or similar activity as a given amino acid sequence or base sequence is referred to as "% homology ". For example, standard software for calculating parameters such as score, identity and similarity, specifically BLAST 2.0, or by sequential hybridization experiments under defined stringent conditions, And the appropriate hybridization conditions to be defined are within the skill of the art and can be determined by methods well known to those skilled in the art (for example, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York). The term " stringent conditions " as used herein refers to conditions that allow specific hybridization between polynucleotides. For example, these conditions are specifically described in the literature (e.g., J. Sambrook et al., Same as above).

In the present application, the stringent conditions can be adjusted to ascertain homology. To verify homology between polynucleotides, hybridization conditions of low stringency, corresponding to a Tm value of 55 ° C, may be used, for example, 5XSSC, 0.1% SDS, 0.25% milk, and formamide; Or 30% formamide, 5XSSC, 0.5% SDS conditions may be used. Hybridization conditions of mild stringency correspond to high Tm values, for example 40% formamide with 5X or 6X SSC can be used. High stringency hybridization conditions correspond to the highest Tm values, for example, 50% formamide, 5X or 6XSSC conditions may be used. However, the present invention is not limited to the above example.

Hybridization requires that two nucleic acids have a complementary sequence, although mismatches between bases are possible, depending on the severity of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases capable of hybridizing with each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Thus, the present application may also include substantially similar nucleic acid sequences as well as isolated nucleic acid fragments complementary to the entire sequence.

Specifically, polynucleotides having homology can be detected using hybridization conditions including the hybridization step at a Tm value of 55 ° C and using the conditions described above. In addition, the Tm value may be 60 ° C, 63 ° C, or 65 ° C, but is not limited thereto and may be suitably adjusted by those skilled in the art according to the purpose.

The appropriate stringency of hybridizing the polynucleotide depends on the length and complementarity of the polynucleotide, and the variables are well known in the art. The greater the similarity or degree of homology between two nucleotide sequences, the larger the Tm value for hybrids of polynucleotides with such sequences. The relative stability of the hybridization of the polynucleotide (corresponding to a higher Tm value) decreases in the following order: RNA: RNA, DNA: RNA, DNA: DNA. For hybrids greater than 100 nucleotides in length, the Tm calculation formula is known (see Sambrook et al., Supra, 9.50-9.51). For hybridization with shorter polynucleotides, e. G. Oligonucleotides, mismatch positions can be more important and the length of oligonucleotides can determine their specificity (Sambrook et al., Supra, 11.7-11.8 Reference).

Specifically, the polynucleotide can be detected using a hybridization condition that is lower than the 500 mM salt and includes at least a hybridization step at 37 ° C, and a washing step at 2X SSPE at least 63 ° C. The hybridization conditions may be lower than 200 mM salt and may include a hybridization step of at least 37 < 0 > C. Or the hybridization conditions may include 63 ° C and 2XSSPE in both the hybridization and washing steps.

The length of the hybridizing nucleic acid can be, for example, at least about 10 nucleotides, 15 nucleotides, 20 nucleotides, or at least 30 nucleotides. In addition, those skilled in the art will be able to adjust the temperature and wash solution salt concentration as needed depending on factors such as the length of the probe.

The fructose-6-phosphate 3-epimerase of the present application may be an enzyme derived from the genus Thermotoga , and specifically, the thermostat may be an enzyme derived from Thermotoga petrophila , no.

The fructose-6-phosphate 3-epimerase of the present application has a pH of 5.0 to 10.0, 5.0 to 9.0, 5.0 to 8.0, 5.0 to 7.5, 6.0 to 10.0, 6.0 to 9.0, 6.0 to 8.0, 6.0 to 7.5, 6.5 to 10.0 , 90-99.9% relative to the maximum activity in the range of 6.5 to 9.0, 6.5 to 8.0, 6.5 to 7.5, 7.0 to 10.0, 7.0 to 10.0, 7.0 to 9.0, 7.0 to 8.0, 7.0 to 7.5 or 7.5.

The fructose-6-phosphate 3-epimerase of the present application may be used in an amount of from 40 to 90, 40 to 80, 40 to 70, 40 to 60, 50 to 90, 50 to 80, 50 to 70, 50 to 60, 55 to 90, 60 to 99.9%, 70 to 99.9%, 80 to 99.9%, or 90 to 99.9% of the maximum activity at 55 to 80, 55 to 70, 55 to 65, 58 to 63, .

The fructose-6-phosphate 3-epimerase of the present application may be one whose activity increases in the presence of Mn, Mg or Ni ions.

The present application provides, in another aspect, a nucleic acid encoding the fructose-6-phosphate 3-epimerase of the present application.

The present application provides, as yet another embodiment, a transformant comprising a nucleic acid encoding the fructose-6-phosphate 3-epimerase of the present application.

The term "transformation" in the present application means introducing a vector comprising 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. The transformed nucleic acid may be either inserted into the chromosome of the host cell or located outside the chromosome, as long as it can be expressed in the host cell. 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 introduced into a host cell and expressed therefrom. For example, the nucleic acid may be introduced into the host cell in the form of an expression cassette, which is a gene construct containing all the elements necessary for its expression. The expression cassette can typically 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 may be in the form of an expression vector capable of self-replication. In addition, the nucleic acid may be introduced into the host cell in its own form and operably linked to the sequence necessary for expression in the host cell, but is not limited thereto.

Also, the term "operably linked" as used herein means that the gene sequence is functionally linked to a promoter sequence that initiates and mediates transcription of a nucleic acid encoding the protein of interest of the present application.

The method of transforming the vector of the present application includes any method of introducing a nucleic acid into a cell and may be carried out by selecting a suitable standard technique as known in the art depending on the host cell. For example, electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, A lithium acetate-DMSO method, and the like, but are not limited thereto.

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

In another aspect of the present invention, there is provided a method for producing a fructose-6-phosphate 3-epimerase of the present application, a microorganism expressing the fructose-6-phosphate 3-epimerase of the present application, 3-epimerase, and a culture of a microorganism expressing the 3-epimerase.

The composition for producing an alulose of the present application may be prepared by using an enzyme involved in an alulose production route (see FIG. 1) of the present application, a microorganism expressing an enzyme participating in the alulose production route of the present application, A culture of a microorganism expressing an enzyme participating in the pathway may be additionally included. However, if it is possible to produce alulose by using the fructose-6-phosphate-epimerizing enzyme of the present application as an example, it is possible that the enzymes contained in the composition for producing aluloses of the present application and the substrate used for the production of aluloses are limited It does not.

The composition for the production of aluloses of the present application is composed of alulose-6-phosphate dephosphorylase, a microorganism expressing the alulose-6-phosphate dephosphorylase or the alulose-6- A culture of a microorganism expressing the phosphorylase may be additionally included.

Also, the composition for producing aluloses of the present application comprises (a) (i) a starch, maltodextrin, sucrose or a combination thereof, glucose, glucose-1-phosphate, glucose-6-phosphate or fructose-6-phosphate; (ii) phosphate; (iii) alulose-6-phosphate dephosphorylase; (iv) Glucose-6-phosphate-isomerizing enzyme; (v) phosphoglucomutase or glucose phosphorylase; And / or (vi) a compound selected from the group consisting of? -Glucan phosphorylase, starch phosphorylase, maltodextrin phosphorylase, sucrose phosphorylase,? -Amylase, pullulanase, isoamylase, glucoamylase, ; Or (b) a microorganism expressing the enzyme of the above item (a) or a microorganism expressing the enzyme of the above item (a), but the present invention is not limited thereto.

Specifically, the starch / maltodextrin phosphorylase (EC 2.4.1.1) and? -Glucan phosphorylase of the present application phosphorylate phosphate to glucose and convert starch or maltodextrin to glucose -1-phosphate. ≪ / RTI > The sucrose phosphorylase (EC 2.4.1.7) of the present application may contain any protein as long as it is a protein having an activity of phosphorylating phosphate to glucose to produce glucose-1-phosphate from sucrose. Amylase (EC 3.2.1.1), pullulanse (EC 3.2.1.41), glucoamylase (EC 3.2.1.3), and isoamylase, which are starch hydrolyzate enzymes of the present application, May contain any protein as long as it is a protein having an activity of converting starch or maltodextrin to glucose. The sucrase (EC 3.2.1.26) of the present application may contain any protein as long as it is a protein having activity to convert sucrose to glucose. The phosphoglucomutase (EC 5.4.2.2) of the present application may contain any protein as long as it is a protein having an activity of converting glucose-1-phosphate into glucose-6-phosphate. Glucokinase may contain any protein that has the activity of converting phosphate into glucose and converting it to glucose-6-phosphate. Specifically, the glucose phosphorylase may be a polyphosphate-dependent glucose phosphorylase. More specifically, it may be a polyphosphate-dependent glucose phosphatase derived from Deinococcus geothermalis of amino acid sequence No. 5 and base sequence No. 7 or an amino acid sequence of SEQ ID NO: 8, Anaerolinea thermophila- derived polyphosphate-dependent glucose phosphatase. The glucose-6-phosphate-isomerizing enzyme of the present application may contain any protein as long as it is a protein having an activity of converting glucose-6-phosphate into fructose-6-phosphate. The allyl-6-phosphate dephosphorylase of the present application may contain any protein as long as the protein has an activity of converting alulos-6-phosphate to alulose. More specifically, the alulose-6-phosphate dephosphorylase may be a protein irreversibly active in converting alulose-6-phosphate into alulose.

In another aspect of the present application, a fructose-6-phosphate 3-epimerase comprising the amino acid sequence of SEQ ID NO: 1, fructose-6-phosphate 3- 6-phosphate is converted to allulose-6-phosphate by contacting a microorganism expressing the epimerase or a culture of the microorganism expressing the fructose-6-phosphate 3-epimerase Wherein the method comprises the steps of:

The production method of the present application is characterized in that after converting the fructose-6-phosphate of the present application to alululose-6-phosphate, alulose-6-phosphate dephosphorylase , Contacting the microorganism expressing the alulose-6-phosphate dephosphorylase or a culture of the microorganism expressing the alulos-6-phosphate dephosphorylase to convert the alulos-6-phosphate into alulose Step < / RTI >

The production method of the present application is also characterized in that glucose-6-phosphate isomerase is added to glucose-6-phosphate before the step of converting fructose-6-phosphate into alulose-6- 6-phosphate isomerizing enzyme or a culture of the microorganism expressing the glucose-6-phosphate isomerizing enzyme is brought into contact with the glucose-6-phosphate isomerizing enzyme to convert the glucose-6-phosphate isomer into fructose- .

In addition, the production method of the present application is characterized in that, prior to the step of converting the glucose-6-phosphate of the present application into fructose-6-phosphate, a phosphoglucomutase is added to glucose- The method may further comprise the step of bringing into contact a microorganism expressing glucomutase or a culture of a microorganism expressing the phosphoglucosimetase to convert the glucose-1-phosphate into glucose-6-phosphate.

In addition, the production method of the present application is characterized in that, prior to the step of converting glucose-6-phosphate of the present application into fructose-6-phosphate, glucose glucose oxidase, a microorganism expressing the glucose phosphorylase, , And converting the glucose to glucose-6-phosphate by contacting the culture with a phosphate and a culture of a microorganism expressing the glucose.

In addition, the production method of the present application can be carried out before the step of converting glucose-1-phosphate of the present application into glucose-6-phosphate, starch, maltodextrin, sucrose or a combination thereof with? -Glucan phosphorylase, Lyase, maltodextrin phosphorylase or sucrose phosphorylase; A microorganism expressing the phosphorylase; Or a culture of a microorganism expressing the phosphorylase, and a phosphate, and converting the starch, maltodextrin, sucrose or a combination thereof to glucose-1-phosphate.

In addition, the production method of the present application can also be carried out before the step of converting the glucose of the present application into glucose-6-phosphate, or by adding α-amylase, Isoamylase; A microorganism expressing the amylase, the pluranase or the sucrase; Or converting the starch, maltodextrin, sucrose, or a combination thereof into glucose by contacting the amylase, the plurasease or sucrase with a culture of the microorganism.

The production method of the present application is characterized in that 4-α-glucanotransferase, a microorganism expressing the 4-α-glucanotransferase, or a culture of the microorganism expressing the 4-α-glucanotransferase To convert the glucose to starch, maltodextrin or sucrose.

In the production process of the present application, the 'contact' of the present application can be carried out at a pH of 5.0 to 10.0, 50 to 90 ° C, and / or 1 minute to 24 hours. Specifically, the contact of the present application is carried out at a pH of 5.0 to 10.0, 5.0 to 9.0, pH 5.0 to 8.0, pH 5.0 to 7.0, pH 5.0 to 6.0, pH 6.0 to 10.0, pH 6.0 to 9.0, pH 6.0 to 8.0, 7.0, pH 7.0 to 10.0, pH 7.0 to 9.0, pH 7.0 to 8.0, pH 8.0 to 10.0, pH 8.0 to 9.0, or pH 9.0 to 10.0. The contact of the present application can be carried out at 50 캜 to 90 캜, 55 캜 to 90 캜, 60 캜 to 90 캜, 60 캜 to 75 캜, 65 캜 to 75 캜, or 60 캜 to 70 캜. In addition, the contact of the present application may be carried out for a period of 1 minute to 12 hours, 1 minute to 6 hours, 1 minute to 3 hours, 1 minute to 1 hour, 5 minutes to 24 hours, 5 minutes to 12 hours, 5 minutes to 6 hours, 5 Min to 3 hours, 5 minutes to 1 hour, 10 minutes to 24 hours, 10 minutes to 12 hours, 10 minutes to 6 hours, 10 minutes to 3 hours, or 10 minutes to 1 hour.

The manufacturing method of the present application may further include the step of adding Mn, Mg or Ni ions.

The present application provides, in yet another embodiment, a method of producing a starch, maltodextrin, sucrose, or a combination thereof, and (a) an allylose-6-phosphate dephosphorylase in a phosphate; A fructose-6-phosphate 3-epimerase consisting of the amino acid sequence of SEQ ID NO: 1; Glucose-6-phosphate-isomerizing enzyme; Phosphoglucomutase or glucose phosphorylase; And α-glucan phosphorylase, starch phosphorylase, maltodextrin phosphorylase, sucrose phosphorylase, α-amylase, pullulanase, isoamylase, glucoamylase or sucrase; Or (b) contacting a microorganism expressing the enzyme of the item (a) or a culture of the microorganism.

The heat-resistant fructose-6-phosphate 3-epimerase of the present application has heat resistance and can be industrially carried out to convert fructose-6-phosphate into allylulose-6-phosphate, and using an economical raw material, It is possible to proceed the synthesis pathway, and it is possible to produce aluloses by the allylulose-6-phosphate dephosphorylation reaction, which is an irreversible reaction pathway, so that the conversion to alulose can be markedly increased.

In addition, the method for producing aluloses using the fructose-6-phosphate 3-epimerase of the present application can increase the conversion rate to aluloses, so that the resultant reaction can include allylose at a high concentration to simplify or eliminate the separation and purification process The bar has a simple and economical manufacturing method.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the reaction pathways in which aluloses can be prepared from starch (e.g., maltodextrin), sucrose or glucose.
FIG. 2 shows the results of analysis of the molecular weight of the fructose-6-phosphate 3-epimerase (FP3E) of the present application by protein electrophoresis (SDS-PAGE).
FIG. 3 shows the conversion activity of fructose-6-phosphate 3-epimerase of the present application from fructose-6-phosphate to alulose-6-phosphate.
Fig. 4 shows the activity of the fructose-6-phosphate 3-epimerase of the present application according to pH.
FIG. 5 shows the activity of the fructose-6-phosphate 3-epimerase of the present application according to the temperature.
Fig. 6 shows the activity of the fructose-6-phosphate 3-epimerase of the present application upon addition of a metal ion.

Hereinafter, the present application will be described in more detail by way of examples. However, these embodiments are for illustrative purposes only, and the scope of the present application is not limited to these embodiments.

Example 1: Preparation of a recombinant expression vector containing a gene of fructose-6-phosphate 3-epimerase and a transforming microorganism

Thermotoga , a thermophilic microorganism, was isolated from Petotpila ( Thermotoga petrophila ) in order to discover a new heat-stable fructose-6-phosphate 3-epimerase, and a recombinant expression vector and a transformed microorganism were prepared.

Specifically, a genome registered in Genbank selects the gene fp3e expected to be a fructose-6-phosphate 3-epimerase based on the petrofilament gene sequences, and the amino acid sequence (SEQ ID NO: 1) and the nucleotide sequence 2 (SEQ ID NO: 3) and reverse primer (SEQ ID NO: 4) were designed and synthesized based on the information. Using the synthesized primers, Thermo was subjected to polymerase chain reaction (PCR) using a genomic DNA as a template, and plasmid vector pET24a (Novagen) for expression of E. coli was digested with restriction enzymes NdeI and XhoI. To prepare a recombinant expression vector named CJ_tp_fp3e. CJ_tp_fp3e was transformed into Escherichia coli BL21 (DE3) strain by a conventional transformation method (Sambrook et al. 1989) to prepare a microorganism transformed with a recombinant vector containing the nucleotide sequence of SEQ ID NO: 2, and E. coli BL21 (DE3) / CJ_tp_fp3e.

The strain E. coli BL21 (DE3) / CJ_tp_fp3e was deposited under the Budapest Treaty with the Korean Culture Center of Microorganisms (KCCM) on September 19, 2017, and received the deposit number KCCM12116P.

Example 2: Preparation of recombinant enzyme

To prepare recombinant enzyme (hereinafter FP3E), E. coli BL21 (DE3) / CJ_tp_fp3e was inoculated into a culture tube containing 5 ml of LB liquid medium and shaken at 37 ° C until the absorbance at 600 nm reached 2.0 The seed culture was performed in an incubator. Then, the cultured medium was cultured in a culture flask containing LB liquid medium. Then, when the absorbance at 600 nm was 2.0, 1 mM IPTG was added to induce the expression of FP3E. The seed culture and main culture were carried out at a stirring speed of 200 rpm and a temperature of 37. After completion of the culture, the culture was centrifuged at 8,000 xg for 4 to 20 minutes, and the cells were recovered. Cells recovered with 50 mM Tris-HCl (pH 7.0) buffer solution were washed twice, suspended in the same buffer solution, and then disrupted using an ultrasonic cell crusher. The cell lysate was centrifuged at 13,000 x g for 4 to 20 minutes and then only the supernatant was taken and FP3E was purified from the supernatant using His-tag affinity chromatography. The purified recombinant enzyme solution was dialyzed with 50 mM Tris-HCl (pH 7.0) buffer solution and used for characterization of the enzyme.

The molecular weight was confirmed by SDS-PAGE analysis, and it was confirmed that the molecular weight of the purified FP3E was about 25 kDa (FIG. 2).

Example 3: Identification of activity of FP3E

In order to analyze the conversion activity of FP3E from fructose-6-phosphate to alulose-6-phosphate, 50 mM fructose-6-phosphate was suspended in 50 mM Tris-HCl (pH 7.5) buffer and 0.15 mg / ml Purified FP3E was added and reacted at 50 ° C for 3 hours.

Since no alulose-6-phosphate standard substance is present at this time, it is impossible to determine whether or not alulose-6-phosphate is produced. As a result, alulose-6-phosphate phosphatase 6-phosphate was converted to alulose using phytase, and then the conversion activity was measured by whether or not alulose was produced. Specifically, after completion of the reaction, 10 units / ml of phytase was added and reacted at 37 ° C for 1 hour to dephosphorylate both the substrate fructose-6-phosphate and the product alululose-6-phosphate. The fructose and allylose were then analyzed by HPLC. HPLC analysis was performed using Aminex HPX-87C (Bio-rad) column at 80 ° C with water flowing at a flow rate of 0.5 ml / min to the mobile phase, and fructose and alulose were detected with a Refractive Index Detector.

As a result, fructose and alulose were detected in the reaction product of FP3E (FIG. 3), and it was confirmed that FP3E had 3-epimerization of fructose-6-phosphate to produce alulose-6-phosphate .

Example 4: Determination of FP3E activity by addition of pH, temperature and metal ion

4-1. Identification of activity by pH

To investigate the effect of pH on FP3E, 0.1 mg / ml of 20 mM fructose-6-phosphate suspended in a 20 mM buffer solution (6.5, potassium phosphate: pH 7.0-8.0, Tris-HCl) Of purified FP3E was added and reacted at 50 DEG C for 3 hours. Thereafter, the reaction was carried out with the phytase under the same conditions as in Example 3, and the allylus was quantitatively analyzed by HPLC.

As a result, it was confirmed that FP3E showed the maximum activity at pH 7.0, and maintained the activity over 90% of the maximum activity in the range of pH 6.5 to pH 7.5 (FIG. 4).

4-2. Identification of activity by temperature

For the analysis of the activity of FP3E according to the temperature, purified FP3E at 0.15 mg / ml was added to 20 mM fructose-6-phosphate suspended in 20 mM Tris-HCl (pH 7.5) Deg.] C and 70 [deg.] C for 10 minutes. Thereafter, the reaction was carried out with the phytase under the same conditions as in Example 3, and the allylus was quantitatively analyzed by HPLC.

As a result, it was confirmed that FP3E exhibited the maximum activity at 60 ° C and exhibited an activity of 50% or more of the maximum activity at 40 to 70 ° C (FIG. 5).

4-3. Identification of activity by metal ion addition

To investigate the effect of metal ion addition on the activity of FP3E, 20 mM fructose-6-phosphate suspended in 20 mM Tris-HCl (pH 7.0) buffer was added with each metal ion (NiSO ₄ , CuSO ₄ , MnSO ₄ , ZnSO ₄ , MgSO ₄ ) was added to a final concentration of 0.5 mM. For the removal of metal ions, 0.15 mg / ml of dialyzed FP3E was treated with 10 mM EDTA and reacted at 50 ° C for 3 hours. Thereafter, the reaction was carried out with the phytase under the same conditions as in Example 3, and the allylus was quantitatively analyzed by HPLC.

As a result, it was confirmed that the activity of FP3E was increased when Mn, Mg or Ni ion was added (FIG. 6).

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive. The scope of the present application is to be interpreted as being within the scope of the present application, all changes or modifications derived from the meaning and scope of the appended claims and from their equivalents rather than the detailed description.

Korea Microorganism Conservation Center KCCM12116P 20170919

<110> CJ CheilJedang Corporation <120> A novel thermostable fructose-6-phosphate 3-epimerase and methods          for producing all the same <130> KPA171036-KR <160> 8 <170> KoPatentin 3.0 <210> 1 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> fructose-6-phosphate 3-epimerase <400> 1 Met Ala Lys Ile Ala Ala Ser Ile Leu Ala Cys Asp Leu Ala Arg Leu   1 5 10 15 Ala Asp Glu Val Lys Arg Val Glu Glu His Val Asp Met Ile His Phe              20 25 30 Asp Val Met Asp Gly His Phe Val Pro Asn Ile Ser Phe Gly Leu Pro          35 40 45 Val Leu Lys Ala Leu Arg Lys Glu Thr Lys Leu Pro Ile Ser Val His      50 55 60 Leu Met Ile Thr Asn Pro Glu Asp Tyr Ile Asp Arg Phe Ile Glu Glu  65 70 75 80 Gly Ala Asp Met Val Ala Ile His Tyr Glu Ser Thr Phe His Leu His                  85 90 95 Arg Leu Val His Arg Val Lys Asp Leu Gly Ala Gln Ala Phe Val Ala             100 105 110 Ile Asn Pro His Thr Pro Ile Asn Leu Leu Ser Glu Ile Ile Thr Asp         115 120 125 Val Asp Gly Val Leu Val Met Ser Val Asn Pro Gly Phe Ser Gly Gln     130 135 140 Arg Phe Ile Ala Arg Ser Leu Glu Lys Ile Arg Asn Leu Arg Lys Met 145 150 155 160 Val Lys Glu Leu Gly Leu Glu Thr Glu Ile Met Val Asp Gly Gly Val                 165 170 175 Asn Glu Glu Asn Ala Ser Ile Leu Val Lys Asn Gly Ala Thr Ile Leu             180 185 190 Val Met Gly Tyr Gly Ile Phe Arg Asn Asp Asn Tyr Val Glu Leu Ile         195 200 205 Lys Ser Ile Lys Gln Glu Arg Glu Glu Ser Ala Asp     210 215 220 <210> 2 <211> 663 <212> DNA <213> Artificial Sequence <220> <223> fructose-6-phosphate 3-epimerase coding gene <400> 2 cgatgaagtg 60 aaaagagtgg aggagcacgt ggatatgatc catttcgatg tcatggatgg tcactttgtt 120 ccaaacattt cctttggact tcccgttctg aaagccctca ggaaagaaac aaaacttccc 180 ataagtgtac atctgatgat caccaatccc gaagattaca tcgaccgctt catagaagaa 240 ggcgcagata tggttgccat ccattacgaa tcgacctttc atctccacag gttggtccat 300 cgtgttaaag accttggagc acaggccttt gttgccataa atccgcatac ccccatcaat 360 cttctttcag agatcataac cgacgtcgat ggtgtactgg tgatgagcgt caatcccggt 420 ttctctggtc agaggttcat agcaaggagt cttgagaaaa taagaaacct gagaaagatg 480 gtgaaagaac tgggactgga aacggaaatc atggtcgacg gcggtgtaaa cgaagagaac 540 gcatcgatac tggttaaaaa cggtgcgacg atccttgtga tgggatacgg tatcttcaga 600 aacgacaatt acgttgaact gataaagagc atcaaacagg aaagagagga atctgctgac 660 tga 663 <210> 3 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 aaggagatat acatatggcg aaaatagcag cttca 35 <210> 4 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ggtggtggtg ctcgaggtca gcagattcct ctct 34 <210> 5 <211> 270 <212> PRT <213> Artificial Sequence <220> <223> Deinococcus geothermalis derived polyphosphate-dependent          glucokinase <400> 5 Met Leu Ala Ala Ser Asp Ser Ser Gln His Gly Gly Lys Ala Val Thr   1 5 10 15 Leu Ser Pro Met Ser Ile Leu Gly Ile Asp Ile Gly Gly Ser Gly              20 25 30 Ile Lys Gly Ala Pro Val Asp Thr Ala Thr Gly Lys Leu Val Ala Glu          35 40 45 Arg His Arg Ile Pro Thr Pro Glu Gly Ala His Pro Asp Ala Val Lys      50 55 60 Asp Val Val Val Glu Leu Val Arg His Phe Gly His Ala Gly Pro Val  65 70 75 80 Gly Ile Thr Phe Pro Gly Ile Val Gln His Gly His Thr Leu Ser Ala                  85 90 95 Ala Asn Val Asp Lys Ala Trp Ile Gly Leu Asp Ala Asp Thr Leu Phe             100 105 110 Thr Glu Ala Thr Gly Arg Asp Val Thr Val Ile Asn Asp Ala Asp Ala         115 120 125 Ala Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly     130 135 140 Glu Val Leu Leu Leu Thr Phe Gly Thr Gly Ile Gly Ser Ala Leu Ile 145 150 155 160 Tyr Asn Gly Val Leu Val Pro Asn Thr Glu Phe Gly His Leu Tyr Leu                 165 170 175 Lys Gly Asp Lys His Ala Glu Thr Trp Ala Ser Asp Arg Ala Arg Glu             180 185 190 Gln Gly Asp Leu Asn Trp Lys Gln Trp Ala Lys Arg Val Val Ser Arg Tyr         195 200 205 Leu Gln Tyr Leu Glu Gly Leu Phe Ser Pro Asp Leu Phe Ile Ile Gly     210 215 220 Gly Gly Val Ser Lys Lys Ala Asp Lys Trp Gln Pro His Val Ala Thr 225 230 235 240 Thr Arg Thr Arg Leu Val Pro Ala Ala Leu Gln Asn Glu Ala Gly Ile                 245 250 255 Val Gly Ala Ala Met Val Ala Ala Gln Arg Ser Gln Gly Asp             260 265 270 <210> 6 <211> 253 <212> PRT <213> Artificial Sequence <220> <223> Anaerolinea thermophila derived polyphosphate-dependent          glucokinase <400> 6 Met Gly Arg Gln Gly Met Glu Ile Leu Gly Ile Asp Ile Gly Gly Ser   1 5 10 15 Gly Ile Lys Gly Ala Pro Val Asp Val Glu Thr Gly Gln Leu Thr Ala              20 25 30 Glu Arg Tyr Arg Leu Pro Thr Pro Glu Asn Ala Leu Pro Glu Glu Val          35 40 45 Ala Leu Val Val Ala Gln Ile Val Glu His Phe Gln Trp Lys Gly Arg      50 55 60 Val Gly Ala Gly Phe Pro Ala Ala Ile Lys His Gly Val Ala Gln Thr  65 70 75 80 Ala Ala Asn Ile His Pro Thr Trp Ile Gly Leu His Ala Gly Asn Leu                  85 90 95 Phe Ser Glu Lys Cys Gly Cys Pro Val Ser Val Leu Asn Asp Ala Asp             100 105 110 Ala Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly Aly Gly Lly Gly Gln Lys         115 120 125 Gly Val Val Leu Met Ile Thr Ile Gly Thr Gly Ile Gly Thr Ala Leu     130 135 140 Phe Thr Asp Gly Ile Leu Val Pro Asn Thr Glu Leu Gly His Ile Glu 145 150 155 160 Ile Arg Gly Lys Asp Ala Glu Gln Arg Ser Ser Glu Ala Ala Arg Gln                 165 170 175 Arg Lys Asp Trp Thr Trp Gln Gln Trp Ala Lys Arg Leu Asn Glu His             180 185 190 Leu Glu Arg Leu Glu Ala Leu Phe Trp Pro Asp Leu Phe Ile Leu Gly         195 200 205 Gly Gly Ala Val Lys Asn His Glu Lys Phe Phe Pro Tyr Leu Lys Leu     210 215 220 Arg Thr Pro Phe Val Ala Lys Leu Gly Asn Leu Ala Gly Ile Val 225 230 235 240 Gly Ala Ala Trp Tyr Ala His Thr Gln Glu Thr Gln Ala                 245 250 <210> 7 <211> 813 <212> DNA <213> Artificial Sequence <220> <223> Deinococcus geothermalis derived polyphosphate-dependent          glucokinase coding gene <400> 7 atgctggcag ccagtgacag cagccagcat ggcgggaagg ctgttacgct atctcccatg 60 agcgtgatcc tcgggattga cataggtggg agcggcatca agggggcccc tgtggacacg 120 gcaaccggga agctggtggc cgagcgccac cgcatcccca cgcccgaggg cgcgcaccca 180 gggggtgga aggacgtggt ggttgagctg gtgcggcatt ttgggcatgc ggggccagtc 240 ggcatcactt tccctggcat cgtgcagcac ggccataccc tgagcgcagc caatgtggat 300 aaagcctgga ttggcctgga cgccgacacg ctttttactg aggcgaccgg tcgcgacgtg 360 accgtgatca acgacgcaga tgccgcgggg ctagcggagg cgaggttcgg ggccggggca 420 ggtgtgccgg gcgaggtgtt gctgttgacc tttgggacag gcatcggcag cgcgctgatc 480 tataacggcg tgctggtgcc caacaccgag tttgggcatc tgtatctcaa gggcgacaag 540 cacgccgaga catgggcgtc cgaccgggcc cgtgagcagg gcgacctgaa ctggaagcag 600 tgggccaaac gggtcagccg gtacctccag tatctggaag gtctcttcag tcccgatctc 660 tttatcatcg gtgggggcgt gagcaagaag gccgacaagt ggcagccgca cgtcgcaaca 720 acacgtaccc gcctggtgcc cgctgccctc cagaacgagg ccggaatcgt gggggccgcg 780 atggtggcgg cgcagcggtc acagggggac taa 813 <210> 8 <211> 762 <212> DNA <213> Artificial Sequence <220> <223> Anaerolinea thermophila derived polyphosphate-dependent          glucokinase coding gene <400> 8 atggggaggc agggcatgga aattttaggg attgatatcg gaggatccgg catcaaaggg 60 gctccggtgg atgtagaaac cggccagtta accgccgagc gataccgctt acccaccccc 120 gaaaatgcct tacctgaaga agtggctctg gtagttgccc aaattgtcga acactttcag 180 tggaaaggtc gtgtaggggc aggatttcct gctgccatca agcacggcgt ggcacagacg 240 gccgcaaaca tccaccctac atggattgga cttcatgctg gcaacctttt cagcgaaaaa 300 tgcggatgtc ctgtctcagt gttgaatgat gcggatgctg ccggactggc ggaaatgatc 360 tttggggcag gaaaaggcca gaaaggggtg gtgctgatga ttaccattgg cactggcatc 420 gggacagccc tgttcaccga tgggatattg gtccctaata ccgagttggg acatattgaa 480 attcggggca aagatgccga acagcgctct tcggaagccg cccgccagcg gaaggattgg 540 acctggcaac aatgggcaaa gcgtctgaat gagcatttgg agcgcctgga agccctgttc 600 tggcccgatt tattcatcct tggtggaggg gcagtaaaaa atcatgaaaa gttcttccct 660 tatctaaaac tgcgtactcc ctttgttgca gcaaaattgg ggaatctggc tgggattgta 720 ggcgcagcgt ggtatgctca cacccaggaa acgcaagcct ga 762

Claims (13)

Fructose-6-phosphate 3-epimerase consisting of the amino acid sequence of SEQ ID NO: 1.
The fructose-6-phosphate 3-epimerase according to claim 1, wherein the enzyme has an activity of 90 to 99.9% relative to the maximum activity at a pH of 5.0 to 10.0.
The fructose-6-phosphate 3-epimerase according to claim 1, wherein the enzyme has an activity of 50 to 99.9% relative to the maximum activity at 40 ° C to 90 ° C.
The fructose-6-phosphate 3-epimerase according to claim 1, wherein the enzyme increases in activity in the presence of Mn, Mg or Ni ions.
A nucleic acid encoding the fructose-6-phosphate 3-epimerase of claim 1.
A transformant comprising the nucleic acid of claim 5.
A composition for producing allulose comprising the fructose-6-phosphate 3-epimerase of claim 1, a microorganism expressing the same, or a culture of the microorganism.
6-phosphate 3-epimerase consisting of the amino acid sequence of SEQ ID NO: 1, a microorganism expressing the same, or a culture of the microorganism is contacted with fructose-6-phosphate To an allulose-6-phosphate. &Lt; RTI ID = 0.0 &gt; 18. &lt; / RTI &gt;
9. The method of claim 8, wherein the method is performed at a pH of from 5.0 to 10.0.
9. The process of claim 8, wherein the process is carried out at 40 to 90 &lt; 0 &gt; C.
9. The method of claim 8, further comprising adding Mn, Mg, or Ni ions.
12. The method according to any one of claims 8 to 11, wherein the contacting is carried out at a pH of 5.0 to 10.0, a temperature of 40 DEG C to 90 DEG C, and 1 minute to 24 hours.
Starch, maltodextrin, sucrose or a combination thereof, and phosphate; (a) alulose-6-phosphate dephosphorylase; A fructose-6-phosphate 3-epimerase consisting of the amino acid sequence of SEQ ID NO: 1; Glucose-6-phosphate isomerase; Phosphoglucomutase or glucose phosphorylase; And α-glucan phosphorylase, starch phosphorylase, maltodextrin phosphorylase, sucrose phosphorylase, α-amylase, pullulanase, isoamylase, glucoamylase or sucrase; Or (b) contacting a microorganism expressing the enzyme of item (a) or a culture of the microorganism.

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