RU2792229C2 - Ribulose phosphate-3-epimerase motif with low side reactivity and an enzyme containing this motif - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention represents the use of ribulose phosphate-3-epimerase for the production of psicose-6-phosphate or psicose. The invention also relates to the use of a ribulose phosphate-3-epimerase producing microorganism for the production of psicose-6-phosphate or psicose.

EFFECT: invention makes it possible to effectively obtain psicose-6-phosphate or psicose.

17 cl, 2 dwg, 3 tbl, 4 ex

Description

PRIOR ART

1. Scope of invention

The present invention relates to ribulose phosphate-3-epimerase, in particular to ribulose phosphate-3-epimerase with low 3-epimerization activity against psicose, to a composition for producing psicose-6-phosphate or psicose containing said ribulose phosphate-3-epimerase, and to a process for producing psicose-6-phosphate or psicose using said ribulose phosphate-3-epimerase or said composition.

2. Description of the prior art

Psicose (allulose), which is the epimer of fructose at the C3 position, is a monosaccharide known as a rare sugar found in nature in extremely small amounts. Psicose has approximately 70% of the sweetness of sucrose with almost zero energy value and has attracted considerable attention as a new food raw material used in functional foods due to its functions such as inhibition of blood glucose, fat synthesis and so on.

In view of these properties, psicose is being considered for use in various foods as a sugar replacement sweetener. However, since it exists in nature in very small amounts, there is a continuing need for a method that provides efficient production of psicose.

One known method for producing psicose is a method for producing psicose-6-phosphate by converting into glucose or glucose-1-phosphate, glucose-6-phosphate, and fructose-6-phosphate (Korean Patent Publication No. 10-2018-0004023), but There is a growing need to develop technology to produce psicose more efficiently and economically.

Psicose-3-epimerase (D-psicose-3-epimerase, EC 5.1.3.30) is known as an enzyme that allows the production of allulose by 3-epimerization (C3-epimerization) of fructose (D-fructose). In the production of allulose from fructose by a single enzymatic reaction using the above enzyme, there is a certain level of reaction equilibrium between the substrate fructose and the product allulose (product/substrate approximately 20% to 35%). Thus, in the case of obtaining allulose with a high degree of purity using a single enzymatic reaction, an additional purification process is necessary to separate and remove fructose in high concentration from the resulting reaction mixture.

In addition, since previously known psicose-6-phosphate-3-epimerases have psicose 3-epimerization activity, they cannot be called psicose-6-phosphate 3-epimerization-specific enzymes and are not suitable for the practical production of psicose. (WO2018/129275, WO2018/112139).

By examining the sequences of specific motifs that can influence psicose 3-epimerization activity, the present inventors have found that certain motifs are particularly critical for psicose-6-phosphate 3-epimerization.

SUMMARY OF THE INVENTION

One object of the present invention is to provide ribulose phosphate-3-epimerase.

Another object of the present invention is to provide a nucleic acid encoding said ribulose phosphate-3-epimerase.

Another object of the present invention is to provide a transformant containing said nucleic acid encoding said ribulose phosphate-3-epimerase.

Another object of the present invention is to provide a psicose-6-phosphate composition comprising said ribulose phosphate-3-epimerase, a microorganism expressing said ribulose phosphate-3-epimerase, or a culture of said microorganism.

Another object of the present invention is to provide a process for the production of psicose 6-phosphate comprising the step of contacting fructose 6-phosphate with said ribulose phosphate 3-epimerase, a microorganism expressing said ribulose phosphate 3-epimerase, or a culture of said microorganism.

Yet another object of the present invention is to provide a composition for producing psicose comprising said ribulose phosphate-3-epimerase, a microorganism expressing said ribulose phosphate-3-epimerase, or a culture of said microorganism.

Another object of the present invention is to provide a method for producing psicose, comprising the step of contacting fructose-6-phosphate with ribulose phosphate-3-epimerase, a microorganism expressing said ribulose phosphate-3-epimerase, or a culture of said microorganism.

BRIEF DESCRIPTION OF GRAPHICS

FIG. 1 shows the protein structure predicted from the amino acid sequence of ribulose phosphate-3-epimerase (SEQ ID NO:9: KPL22606); And

in FIG. 2 shows an HPLC (High Performance Liquid Chromatography) histogram showing the previously known psicose-6-phosphate-3-epimerase (ADL69228; solid line) and the enzyme of the present invention (SEQ ID NO:20; broken line).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Hereinafter, the present invention will be described in detail as follows. At the same time, each of the descriptions and embodiments disclosed in this invention can also be applied to other descriptions and embodiments. That is, the scope of the present invention includes all combinations of the various elements disclosed in this invention. In addition, the scope of the present invention is not limited to the specific description below.

To solve the above problems, according to one aspect of the present invention proposed ribulose phosphate-3-epimerase.

Specifically, the ribulose phosphate-3-epimerase of the present invention may be a ribulose phosphate-3-epimerase containing motif I consisting of the amino acid sequence of SEQ ID NO:1 and motif III consisting of the amino acid sequence of SEQ ID NO:3, and having a high activity and resistance to heat, and more specifically not containing motif II, consisting of the amino acid sequence of SEQ ID NO:2, but not limited to this.

As used herein, the term "ribulose phosphate-3-epimerase" refers to an enzyme whose ribulose phosphate-3-epimerase activity is known, or which has ribulose phosphate-3-epimerase activity, and in particular to an enzyme that can act as fructose-6- phosphate-3-epimerase; or psicose-6-phosphate-3-epimerase. When ribulose phosphate-3-epimerase has fructose-6-phosphate-3-epimerase activity or psicose-6-phosphate-3-epimerase activity, it may contain an amino acid sequence having a deletion, modification, substitution, conservative substitution, or addition of certain sequences.

Specifically, the enzyme of the present invention is an enzyme having a reversible conversion activity of reversibly converting psicose-6-phosphate to fructose-6-phosphate or fructose-6-phosphate to psicose-6-phosphate, and in the present invention, the term "ribulose phosphate -3-epimerase" can be used interchangeably with the terms "psicose-6-phosphate-3-epimerase" or "enzyme".

The enzyme of the present invention may be an enzyme that converts glucose-1-phosphate (D-glucose-1-phosphate), glucose-6-phosphate (D-glucose-6-phosphate) or fructose-6-phosphate (D-fructose- 6-phosphate) to psicose-6-phosphate when mixed together. For example, the enzyme of the present invention may exhibit a conversion rate to psicose-6-phosphate when mixed in equal amounts of psicose-6-phosphate, glucose-1-phosphate, glucose-6-phosphate, and fructose-6-phosphate of 1% or more. , 10% or more or 30% or more. As described, due to the selective activity of the enzyme of the present invention, it can show a high rate of conversion to psicosis when enzymatically converted in a single vessel, where multiple enzymes and substrates are used simultaneously.

As used herein, "motif" refers to a portion (region) of an enzyme's sequence having a particular sequence, and may refer to a sequence having a particular protein function or activity, and may be, but is not limited to, a sequence that is conserved across microbial species. The ribulose phosphate-3-epimerase of the present invention may comprise a motif I consisting of the amino acid sequence of SEQ ID NO:1 and a motif III consisting of the amino acid sequence of SEQ ID NO:3. In addition, ribulose phosphate-3-epimerase may be characterized in that it does not contain motif II, consisting of the amino acid sequence of SEQ ID NO:2. Since said enzyme does not contain motif II, it may have low psicose-3-epimerase activity, but is not limited to this.

Specifically, said enzyme may have 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less psicose to fructose conversion activity or no activity compared to an enzyme without motif I and motif III, but not limited to this.

In addition, the enzyme of the present invention may further comprise a motif consisting of the amino acid sequence of SEQ ID NO:4 or 5.

The enzyme of the present invention is characterized in that it contains or substantially contains a specific motif, and also does not contain a specific motif. Specifically, motifs I and III of the present invention can be included in the site (binding site) of the enzyme, which partially or completely binds to and interacts with a substrate and/or metal ion (for example, Mg, Mn, Zn, etc.), and can retain their own enzymatic activity while reducing side reactivity. More specifically, motifs I and III can be incorporated into the TIM barrel structure at the binding site. An enzyme "containing" a specific motif may or may not additionally contain another motif, domain, amino acid sequence, fragment, and so on, in addition to the corresponding motif, and an enzyme "substantially containing" a specific motif may essentially contain the corresponding motif to obtain the desired property or characteristic, and may or may not also contain another motif, domain, amino acid sequence, fragment, and so on, in addition to, but not limited to, the corresponding motif. An enzyme "not containing" a specific motif cannot contain a sequence corresponding to the corresponding motif, and may contain an insertion, substitution or deletion with a different amino acid sequence at the site of the corresponding motif, or a combination of these, but without limitation.

In addition, each of the motifs included or not included in the enzyme of the present invention may or may not be included independently, and they are not limited to a particular order or position.

For example, the enzyme of the present invention may be an enzyme containing only motif I of SEQ ID NO:1, an enzyme containing motif I of SEQ ID NO:1 and motif III of SEQ ID NO:3 without motif II of SEQ ID NO: 2, or an enzyme containing but not limited to motif I of SEQ ID NO:1, motif III of SEQ ID NO:3, and motifs of SEQ ID NO:4 and 5 without motif II of SEQ ID NO:2.

When used here, "motif I" may consist of the amino acid sequence of the following SEQ ID NO:1. Obviously, a sequence containing insertion, substitution, deletion, and so on of insignificant amino acid residues can also be included in motif I of the present invention, provided that this does not affect the activity of the amino acid sequence of SEQ ID NO:1.

Motif I (SEQ ID NO:1): V-D-G.

Motif I can be included in the binding site, interacting with the substrate ribulose phosphate-3-epimerase and metal ion, but not limited to this. Specifically, said motif can be located at amino acid positions 173-184 from the first amino acid of the N-terminus of ribulose phosphate-3-epimerase, but is not limited to this. In other words, valine (V), which is the first amino acid residue of motif I, can be located between positions 173-182, and glycine (G), which is the last residue of motif I, can be located between positions 175-184 from the first amino acid of the N-terminus. ribulose phosphate-3-epimerase, but not limited to this.

In the domain where the first beta-fold structure - helical structure - alpha-helical structure - second beta-fold structure are linked, the first amino acid residue of motif I can begin at the C-terminus relative to the second beta-fold, and the domain can be included in the binding site or may have a structure in which some areas overlap (FIG. 1).

When used here, "motif II" may consist of the amino acid sequence of the following SEQ ID NO:2. Obviously, a sequence containing insertion, substitution, deletion, and so on of insignificant amino acid residues can also be included in motif II of the present invention, provided that this does not affect the activity of the amino acid sequence of SEQ ID NO:2.

Motif II (SEQ ID NO:2): M-X-X-D-P-G (X is any amino acid).

Motif II can be included in the N-terminal region of ribulose phosphate-3-epimerase, but is not limited to this. Specifically, said motif can be located at amino acid positions 136-150 from the first amino acid of the N-terminus of ribulose phosphate-3-epimerase, but is not limited to this. In other words, methionine (M), which is the first amino acid residue of motif II, can be located between positions 136-145, and glycine (G), which is the last residue of motif II, can be located between positions 141-150 from the first amino acid of the N-terminus. ribulose phosphate-3-epimerase, but not limited to this. In the domain where the first beta helix structure is bound alpha helix structure the second beta helix structure, the first amino acid residue of motif II may begin at the C-terminus of the first beta helix and the domain may be included in the binding site or may have the structure , in which some areas overlap, and the domain may be identical to the domain containing motif I (FIG. 1).

Specifically, X may include, but is not limited to, any amino acid, and specifically threonine (T) or valine (V). More specifically, motif II may contain the sequence M-(T/A/M/L)-(V/N/I)-D-P-G, but is not limited to this.

When used here, "motif III" may consist of the amino acid sequence of the following SEQ ID NO:3. Obviously, a sequence containing insertion, substitution, deletion, and so on of insignificant amino acid residues can also be included in motif III of the present invention, provided that this does not affect the activity of the amino acid sequence of SEQ ID NO:3.

Motif III (SEQ ID NO:3): M-X-X-X'-P-G (X is any amino acid).

Motif III can be included in the N-terminal region of ribulose phosphate-3-epimerase, but is not limited to this. Specifically, said motif can be located at amino acid positions 136-150 from the first amino acid of the N-terminus of ribulose phosphate-3-epimerase, but is not limited to this. In other words, methionine (M), which is the first amino acid residue of motif III, can be located between positions 136-145, and glycine (G), which is the last residue of motif III, can be located between positions 141-150 from the first amino acid of the N-terminus. ribulose phosphate-3-epimerase, but not limited to this. In the domain where the first beta-fold structure is bound alpha-helical structure the second beta-fold structure, the first amino acid residue of motif III may begin at the C-terminus of the first beta-fold, and the domain may be included in the binding site or may have the structure , in which some areas overlap, and the domain may be identical to the domain containing motif I (FIG. 1).

More specifically, motifs II and III may be motifs included in the same region when aligned in the enzyme of the present invention, without being limited to this.

Specifically, X may include any amino acid without limitation, and specifically threonine (T), alanine (A), methionine (M), leucine (L), valine (V), asparagine (N) or isoleucine (I), but without restrictions on this.

In addition, X' may include any amino acid except aspartic acid (D), without limitation, and specifically, an uncharged amino acid or a positively charged amino acid. Specifically, the uncharged amino acid may include all polar amino acids and non-polar amino acids, and may be any of serine, threonine, cysteine, asparagine, glutamine, glycine, alanine, proline, valine, leucine, isoleucine, and methionine. In addition, the positively charged amino acid may be any of lysine, arginine, and histidine. For example, a positively charged amino acid may include asparagine (N) and lysine (K). More specifically, motif III may contain the sequence M-(T/A/M/L)-(V/N/I)-N-P-G, but is not limited to this.

The enzyme of the present invention may further comprise a motif consisting of the amino acid sequence of the following SEQ ID NO:4. Obviously, a sequence containing the insertion, substitution, deletion, and so on of insignificant amino acid residues can also be included in the enzyme of the present invention, provided that this does not affect the activity of the amino acid sequence of SEQ ID NO:4.

SEQ ID NO:4: S-X-M/I-C (X is any amino acid).

A motif having the amino acid sequence of SEQ ID NO:4 may be included in the N-terminal region of ribulose phosphate 3-epimerase, but is not limited to this. Specifically, said motif can be located at amino acid positions 5-20, and more particularly at amino acid positions 7-19 from the first amino acid of the N-terminus of ribulose phosphate-3-epimerase, but is not limited to this. In other words, series (S) being the first amino acid residue of the motif having the amino acid sequence of SEQ ID NO:4 can be located between positions 7-16, and cysteine (C) being the last residue of the motif having the amino acid sequence of SEQ ID NO: 4 may be located between positions 10-19 from the first amino acid of the N-terminus of ribulose phosphate 3-epimerase, but is not limited to this. In addition, the motif of SEQ ID NO:4 may be formed after the beta sheet structure, and specifically, a portion of the sequence of the motif of SEQ ID NO:4 may be included in the alpha helical structure formed after the beta sheet structure.

Specifically, X may include any amino acid without limitation, specifically, but not limited to methionine, isoleucine, leucine, or valine. More specifically, the motif consisting of the amino acid sequence SEQ ID NO:4 may contain the sequence SIMC (SEQ ID NO:27), SMMC (SEQ ID NO:28), SLMC (SEQ ID NO:29), or SVMC (SEQ ID NO :30), but not limited to this.

The enzyme of the present invention may further comprise a motif consisting of the amino acid sequence of the following SEQ ID NO:5. Obviously, a sequence containing insertion, substitution, deletion, and so on of insignificant amino acid residues can also be included in the enzyme of the present invention, provided that this does not affect the activity of the amino acid sequence of SEQ ID NO:5.

SEQ ID NO:5: G-X-X-X-X-F/L (X is any amino acid).

A motif consisting of the amino acid sequence of SEQ ID NO:5 may be included in the C-terminal region of ribulose phosphate-3-epimerase, but is not limited to this. Specifically, said motif can be located at amino acid positions 190-210, and more particularly at amino acid positions 196-210 from the first amino acid of the N-terminus of ribulose phosphate 3-epimerase, but is not limited to this. In other words, glycine (G), which is the first amino acid residue of the motif consisting of the amino acid sequence of SEQ ID NO:5, can be located between positions 196-205, and phenylalanine (F), which is the last residue of the motif, consisting of the amino acid sequence of SEQ ID NO:5 may be located between positions 201-210 from the first amino acid of the N-terminus of ribulose phosphate 3-epimerase, but is not limited to this. In addition, the motif of SEQ ID NO:5 may be formed after the beta sheet structure, and specifically, a portion of the sequence of the motif of SEQ ID NO:5 may be included in the alpha helical structure formed after the beta sheet structure.

Specifically, each X motif consisting of the amino acid sequence of SEQ ID NO:5 can independently be threonine, serine, glycine, leucine, cysteine, isoleucine, asparagine, lysine, alanine, valine, or glutamine, but is not limited thereto. More specifically, the motif consisting of the amino acid sequence SEQ ID NO:5 may contain the amino acid sequence GNSGLF (SEQ ID NO:31), GSSGLFGSSSLF (SEQ ID NO:32), GSTSLF (SEQ ID NO:33), GTAGLF (SEQ ID NO:33). NO:34), GTKGLF (SEQ ID NO:35), GTQSLF (SEQ IDNO:36), GTSCLF (SEQ ID NO:37), GTSGLF (SEQ ID NO:38), GTSSIF (SEQ ID NO:39), GTSGIF (SEQ ID NO:40), GTSSLF (SEQ ID NO:41), or GTSSVF (SEQ ID NO:42), but not limited to this. The enzyme of the present invention is characterized in that it has a high activity of converting fructose-6-phosphate to psicose-6-phosphate due to the fact that it contains the motif(s) of SEQ ID NO:4 and/or 5.

Appropriate amino acid residues included in the motif of the present invention can be independently combined with each other to form a motif.

The ribulose phosphate 3-epimerase of the present invention may not contain a specific motif, i.e. motif II, whereby it exhibits very low psicose 3-epimerization activity, indicating that a high yield psicose end product can be obtained by reducing side interactions during the time of the process of obtaining psicose-6-phosphate from fructose-6-phosphate. In addition, ribulose phosphate-3-epimerase can be used in combination with other enzymes (eg, psicose-6-phosphate phosphatase) for effective use in producing psicose.

The alpha-helix (α-helix) structure, the beta-sheet (β-sheet) structure, and the helical structure of the present invention can be understood in accordance with the generally accepted definition given in Kwangsoo Kim et al. (Crystal Structure of d-Psicose 3-epimerase from Agrobacterium tumefaciens and its Complex with True Substrate d-Fructose, Volume 361, Issue 5, 1 September 2006, Pages 920-931) and so on, and the alpha helix may be a right-handed alpha helix. The structure can be obtained by direct prediction using a well-known method such as NMR (Nuclear Magnetic Resonance), X-ray crystallography and so on, or using Rosetta algorithms or Internet server (I-TASSER, ROBETTA and so on) based on amino acid sequences.

In addition, in the present invention, the domain in which structures are assembled can be expressed as structure 1 structure 2.

In addition, the ribulose phosphate-3-epimerase of the present invention may be derived from any one selected from the group consisting of Chthonomonas, Geobacillus, Mahella, Thermoanaerobacterium, Tepidanaerobacter, Ardenticatenia, Firmicutes, Aeribacillus, Epulopiscium and Thermoflavimicrobium, and more particularly may be derived from any selected from the group consisting of Chthonomonas calidirosea T49, Geobacillus sp. 8, Geobacillus thermocatenulatus, Mahella australiensis 50-1 BON, Thermoanaerobacterium sp. PSU-2, Thermoanaerobacterium thermosaccharolyticum, Tepidanaerobacter syntrophicus, Ardenticatenia bacteria, Firmicutes HGW-Firmicutes-5 bacteria, Aeribacillus pallidus, Epulopiscium sp. SCG-B05WGA-EpuloA1 and but not limited to Thermoflavimicrobium dichotomicum.

In addition, the ribulose phosphate-3-epimerase of the present invention may comprise any sequence selected from the group consisting of the amino acid sequences of SEQ ID NO:15-26, or may consist of any sequence selected from the group consisting of the amino acid sequences of SEQ ID NO. :15-26, but not limited to this. More specifically, the enzyme may consist of the amino acid sequence of SEQ ID NO:19, 20 or 22, but is not limited to this.

The presence or absence of a specific motif in the ribulose phosphate-3-epimerase of the present invention has an important effect on enzymatic activity, and therefore enzyme sequences that exclude the motif region may have low identity. Specifically, the enzyme may be composed of any sequence selected from the group consisting of amino acid sequences having 26%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85 %, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with a region excluding motifs I and III in the amino acid sequence and amino acid sequences having 24%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with the exclusion region motifs I and III, and but not limited to SEQ ID NOs: 4 and 5.

In addition, the enzyme may contain any of the amino acid sequences of SEQ ID NO:15-26, or an amino acid sequence having 70% or more homology or identity with such a sequence, but not limited to this. Specifically, the amino acid sequence may comprise sequences of SEQ ID NOs: 15-26 and amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with the indicated sequences. In addition, it is clear that a protein having an amino acid sequence with a deletion, modification, substitution or addition of part of the sequence can also be included in the scope of the present invention, provided that its amino acid sequence has such homology or identity and demonstrates the efficiency corresponding to the protein in question.

In other words, although the present invention discloses "a protein having an amino acid sequence indicated by a certain sequence number", provided that the protein has an activity identical or equivalent to that of a protein consisting of an amino acid sequence with the corresponding sequence number, it is obvious that that the scope of the present invention also includes proteins having an amino acid sequence with a partial deletion, modification, substitution, conservative substitution or addition. For example, when a protein has an activity identical or equivalent to that of an enzyme, it is clear that the addition of a sequence that does not change the function of the protein before and after the amino acid sequence, its natural mutation, silent mutation, or conservative substitution is not excluded, and the scope of the present invention includes the addition or mutation sequences.

As used herein, the term "homology" or "identity" refers to the degree of correspondence between two given amino acid sequences or nucleotide sequences, and may be expressed as a percentage.

The terms "homology" and "identity" can often be used interchangeably with each other.

Homology or sequence identity of conserved polynucleotides or polypeptides can be determined using standard alignment algorithms and can be used in conjunction with the gap penalty set in the default program being used. Substantially homologous or identical sequences will be expected to hybridize over the entire length or over about 50%, about 60%, about 70%, about 80%, or about 90% or more of the total length of the sequences under conditions of moderate or high stringency. Also contemplated are polynucleotides containing degenerate codons instead of the codons of hybridizing polynucleotides.

The presence of homology, similarity, or identity of any two polynucleotide or polypeptide sequences can be determined using a known computer algorithm, such as the "FASTA" program, as described in Pearson et al. (1988) Proc. Natl. Acad. sci. USA 85:2444 using the default settings. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-153) using the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al ., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later), GCG software package (Devereux, J. et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J MOLEC BIOL 215:403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and CARILLO et al. (1988) SIAM J Applied Math 48:1073). For example, homology, similarity or identity can be determined using BLAST or Clusta1W of the National Center for Biotechnology Information.

Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using, for example, a GAP computer program such as Needleman et al. (1970), J Mol Biol. 48:443 as disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the GAP program defines homology, similarity, or identity as the value obtained by dividing the number of similarly aligned characters (ie, nucleotides or amino acids) by the total number of characters in the shorter of the two sequences. The default parameters for the GAP program may include: (1) a single comparison matrix (including a value of 1 for identical positions and 0 for non-identical ones) and a weighted comparison matrix according to Gribskov et al. (1986) Nucl. Acids Res. 14:6745 as described in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979), (alternatively, EDNAFULL substitution matrix (EMBOSS version of NCBI NUC4.4)); (2) a penalty of 3.0 for each break and a penalty of 0.10 for each symbol of each break (or a gap start penalty of 10 and a gap continuation penalty of 0.5); and (3) no penalties for ending breaks. Accordingly, as used herein, the term "homology" or "identity" refers to sequence similarity.

At the same time, the ribulose phosphate-3-epimerase of the present invention may be heat-resistant, but not limited thereto.

As used herein, the term "heat resistance" refers to the ability to display the original activity of the enzyme without losing its activity even under conditions of high temperature, and the resistance of the enzyme to heat has many advantages in the process of obtaining the desired product. Specifically, the ribulose phosphate-3-epimerase of the present invention may have psicose-6-phosphate 3-epimerization activity at 40°C or more, more specifically at 50°C or more, and much more specifically at 60°C or more, but not limited to this. Even more specifically, the enzyme of the present invention may have psicose-6-phosphate 3-epimerization activity under conditions of pH 5.0 to 10.0 and 50°C to 90°C for 1 minute to 24 hours. , but not limited to this.

The ribulose phosphate-3-epimerase of the present invention can be obtained by transforming a strain with the enzyme itself or DNA expressing the enzyme, culturing the strain to obtain a culture, breaking the culture, and then purifying the product using a column, and so on. The transformation strain may be, but is not limited to, Escherichia coli, Corynebacterium glutamicum, Aspergillus oryzae, Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, or Bacillus subtilis, and could potentially be transformed into a GRAS (Generally Recognized as Safe) strain in the future. safe).

There are no particular restrictions on the method for purifying ribulose phosphate-3-epimerase of the present invention, and a method commonly used in the field to which the present invention pertains can be applied. Their non-limiting examples may include chromatography, heat treatment, adsorption, filtration, ion purification, and so on. Only one of the cleaning methods may be applied, and two or more methods may be applied in combination.

According to another aspect of the present invention, a nucleic acid encoding ribulose phosphate-3-epimerase or a vector containing said nucleic acid is provided.

As used herein, "nucleic acid" has a meaning that includes both DNA molecules and RNA molecules. Nucleotides, which are the basic structural units of nucleic acids, may include not only natural nucleotides, but also their modified counterparts, in which sugar or basic groups are modified.

The nucleic acid of the present invention may be a DNA or RNA sequence, where the nucleotide units are linked to each other by covalent bonds, and, specifically, it may be any of all nucleotide sequences possible when converting the amino acid sequences of SEQ ID NO:15-26 to DNA (with a 61 codon amino acid substitution), and more specifically, it may include a nucleic acid having 90% or more, 95% or more, 97% or more, 99% or more, or 100% or more homology, similarity or identity with each nucleotide that can be translated into any amino acid sequence of the amino acid sequences of SEQ ID NO:15-26 of the present invention, while still being able to show the desired enzymatic activity after translation. It is also clear that a polynucleotide which, due to the degeneracy of the genetic code, can be translated to produce a protein having the same activity or having the same amino acid sequence after translation, specifically consisting of any of the amino acid sequences of SEQ ID NOs: 15-26, or a protein having homology, similarity or identity thereto may also be included within the scope of the present invention. More specifically, the nucleic acid sequence of the present invention is not specifically specified, and the nucleic acid may, without limitation, consist of any number of DNA codons that can be translated to give the amino acid sequences of SEQ ID NO:15-26.

Alternatively, a probe may be included, without limitation, that may be derived from a known gene sequence, such as any sequence that hybridizes under stringent conditions to a sequence that is complementary to all or part of the nucleotide sequence encoding the enzyme of the present invention.

"Stringent conditions" refers to conditions that allow specific hybridization of polynucleotides. Such conditions are detailed in the literature (eg, J. Sambrook et al., cited below). For example, stringent conditions may include conditions under which genes having a high degree of homology or identity, genes having a homology or identity of 80% or more or 85% or more, specifically 90% or more, more specifically 95% or more, even more specifically 97% or more, and especially specifically 99% or more, hybridize with each other, while genes having lower homology or identity than those indicated above do not hybridize with each other, or may include conventional Southern washing conditions. -hybridization, that is, one wash, specifically two or three times washing, at a salt concentration and temperature corresponding to 60°C, 1xSSC (saline - sodium citrate), 0.1% SDS (sodium dodecyl sulfate), specifically 60°C, 0 .1xSSC, 0.1% SDS, and more specifically 68°C, 0.1xSSC, 0.1% SDS.

Hybridization requires two polynucleotides to have complementary sequences, however, depending on the stringency of the hybridization, base mismatches are possible. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in relation to DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Thus, the present invention may also include an isolated nucleic acid fragment that is complementary to the complete sequence, as well as a nucleic acid sequence substantially similar to it.

Specifically, a polynucleotide having homology or identity can be determined using hybridization conditions including a hybridization step at a T _m value of 55°C under the conditions described above. In addition, the value of T _m may be 60°C, 63°C or 65°C, but without limitation, and experts in this field can properly control it depending on the task.

The appropriate stringency for polynucleotide hybridization depends on the length and degree of complementarity of the polynucleotides, and these parameters are well known in the art.

As used herein, the term "vector" refers to a DNA construct containing a nucleic acid nucleotide sequence encoding an enzyme of the present invention, operably linked to a suitable expression regulation sequence for expression of a desired protein variant in a suitable host. A regulatory sequence includes a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable ribosome binding site on an mRNA (messenger RNA), and a sequence for regulating transcription termination and translation. The vector can be transformed into a suitable host cell and then can replicate or function independently of the host genome, or can be integrated into the genome itself.

There are no particular restrictions on the vector used in the present invention, provided that the vector is capable of replication in the host cell, and any vector known in the art can be used. Examples of commonly used vectors may include native or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A and so on can be used as a phage vector or cosmid vector, and pBR-based plasmids, pUC based, pBluescriptII based, pGEM based, pTZ based, pCL based, pET based and so on. Specifically, the vectors pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC and so on can be used.

According to another aspect of the present invention, there is provided a transformant containing a nucleic acid encoding an enzyme of the present invention, or a vector containing a nucleic acid encoding an enzyme of the present invention.

When used herein, “transformant containing a nucleic acid encoding an enzyme” or “transformant containing a vector containing a nucleic acid encoding an enzyme” may refer to a microorganism recombined to express the ribulose phosphate-3-epimerase of the present invention, and, for example, refers to a host cell or microorganism that may contain a nucleic acid encoding ribulose phosphate-3-epimerase, or may be transformed with a vector containing a nucleic acid encoding ribulose phosphate-3-epimerase to express ribulose phosphate-3-epimerase. For purposes of the present invention, the ribulose phosphate-3-epimerase expressed by the transformant may be any of the amino acid sequences of SEQ ID NO:15-26, but is not limited thereto.

As used herein, "transformation" refers to introducing a vector containing a nucleic acid encoding a ribulose phosphate-3-epimerase of the present invention into a host cell such that expression of the protein encoded by the nucleic acid occurs in the host cell. Provided that the nucleic acid used for transformation can be expressed in the host cell, it may include any nucleic acid used for transformation, whether the nucleic acid used for transformation is introduced and located on the chromosome of the host cell or located outside the chromosome. In addition, the nucleic acid includes DNA and RNA encoding a nucleic acid encoding a ribulose phosphate-3-epimerase of the present invention. The nucleic acid can be introduced in any form, as long as the nucleic acid can be introduced into a host cell for expression. For example, the nucleic acid can be introduced into a host cell in the form of an expression cassette, which is a genomic structure containing all the elements necessary for autonomous expression. Typically, the expression cassette may contain 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 a self-replicating expression vector. In addition, the nucleic acid may also be introduced into the host cell itself and may be operably linked to a sequence required for expression in the host cell, but is not limited to this.

In addition, the term "operably linked" means that the gene sequence is operably linked to a promoter sequence that initiates and mediates the transcription of a nucleic acid encoding a psicose-6-phosphate phosphatase of the present invention.

Introduction of a nucleic acid or vector into a chromosome can be accomplished by any method known in the art, such as, but not limited to, homologous recombination. A selection marker may optionally be included to confirm insertion into the chromosome. The selection marker is intended to select cells transformed with the vector, ie to confirm the introduction of the target nucleic acid molecule. Markers conferring selective phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic drugs, or expression of modified surface proteins can be used. The selection of transformed cells can be carried out in a medium treated with a selective agent, since only cells expressing the selection marker survive or show different phenotypes.

The vector transformation method of the present invention includes any method of introducing a nucleic acid into a host cell and can be carried out by selecting an appropriate standard technique known in the art, depending on the host cell. Method examples may include electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, retroviral infection, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, acetate method. lithium and DMSO (dimethyl sulfoxide) and so on, but not limited to this.

As the host cell, it is preferable to use a host with a high efficiency of introducing DNA into the host cell and a high efficiency of expression of the introduced DNA. For example, it may be a Corynebacterium sp. microorganism, an Escherichia sp. microorganism, a Serratia sp. microorganism, a Bacillus sp. microorganism, a Saccharomyces cerevisiae sp. All GRAS strains can be used.

More specifically, the transformant of the present invention may be E. coli BL21(DE3)/pET-CJ-ef7, E. coli BL21(DE3)/pET-CJ-ef12, or E. coli BL21(DE3)/pET-CJ- ef15, but not limited to this.

According to another aspect of the present invention, a composition for producing psicose-6-phosphate is provided, comprising ribulose phosphate-3-epimerase, a microorganism expressing said ribulose phosphate-3-epimerase, or a culture of said microorganism.

The composition for producing psicose-6-phosphate of the present invention may contain a ribulose phosphate-3-epimerase showing activity to convert fructose-6-phosphate to psicose-6-phosphate, a microorganism expressing said ribulose 3-epimerase, or a culture of said microorganism, and thus, by bringing said composition into contact (reacting) with fructose-6-phosphate, psicose-6-phosphate can be obtained from fructose-6-phosphate.

Specifically, the composition may further contain fructose-6-phosphate as a substrate, but is not limited to this.

The psicose-6-phosphate composition of the present invention may further comprise any suitable excipient commonly used in the psicose-6-phosphate composition. The excipient may include, for example, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizing agent, an isotonic agent, and so on, but is not limited thereto.

The psicose-6-phosphate composition of the present invention may further contain a metal ion or metal salt. In one embodiment, the metal ion may be a divalent cation, and specifically one or more metal ions selected from the group consisting of Ni, Mg, Ni, Co, Mn, Fe, and Zn. More specifically, the composition for producing psicose-6-phosphate of the present invention may further contain a metal salt. More specifically, the metal salt may be one or more selected from the group consisting of NiSO ₄ , MgSO ₄ , MgCl ₂ , NiCl ₂ , COSO ₄ , COCl ₂ , MnCl ₂ , MnSO ₄ , FeSO ₄ and ZnSO ₄ .

According to another aspect of the present invention, there is provided a method for producing psicose-6-phosphate, comprising the step of contacting fructose-6-phosphate with a ribulose phosphate-3-epimerase, a microorganism expressing said ribulose phosphate-3-epimerase, or a culture of said microorganism.

The ribulose phosphate-3-epimerase of the present invention can exhibit the activity of converting fructose-6-phosphate to psicose-6-phosphate, and thus when ribulose phosphate-3-epimerase, a microorganism expressing said ribulose 3-epimerase, or a culture of said microorganism in contact with fructose-6-phosphate, psicose-6-phosphate can be obtained from fructose-6-phosphate.

Conditions for contacting and reacting with fructose-6-phosphate and psicose-6-phosphate can be appropriately selected by one skilled in the art, taking into account the substrate, the enzyme, and so on.

Specifically, the step of obtaining psicose-6-phosphate by bringing fructose-6-phosphate into contact with ribulose phosphate-3-epimerase can be carried out at a pH of 5.0 to 9.0 and a temperature of 40°C to 80°C and/or within 2-24 hours, more specifically at a pH of 6.0 to 8.0 and a temperature of 40°C to 60°C and/or within 20-24 hours, and more specifically at a pH of 7.0 and a temperature of 50° With within 24 hours, but not limited to this.

The method for producing psicose-6-phosphate according to the present invention may further include the step of isolating and/or purifying the resulting psicose-6-phosphate, but without being limited to this.

The step of isolating and/or purifying the psicose-6-phosphate can be carried out using a method known in the art, but not limited to any particular method.

According to another aspect of the present invention, a composition for producing psicose is provided, comprising ribulose phosphate-3-epimerase, a microorganism expressing said ribulose phosphate-3-epimerase, or a culture of said microorganism. Specifically, the composition may further contain fructose-6-phosphate as a substrate, but is not limited to this.

The composition for producing psicose of the present invention can provide psicose production, since it additionally contains an enzyme necessary for the production of psicose, for example, psicose-6-phosphate phosphatase, providing production of psicose by dephosphorylation of psicose-6-phosphate, in addition to ribulose phosphate-3-epimerase, providing obtaining psicose-6-phosphate from fructose-6-phosphate.

Specifically, the composition for producing psicose of the present invention may further comprise one or more enzymes selected from the group consisting of glucose-6-phosphate isomerase, phosphoglucomutase, polyphosphate glucokinase, α-glucan phosphorylase, starch phosphorylase, maltodextrin phosphorylase or sucrose phosphorylase, α-amylase, pullulanase , isoamylase, α-glucan transferase, glucoamylase, sucrase and psicose-6-phosphate phosphatase, a microorganism expressing said enzyme, or a culture of said microorganism, and specifically, may further contain psicose-6-phosphate phosphatase, but is not limited thereto.

More specifically, the composition for producing psicose of the present invention may further comprise: (a) (1) starch, maltodextrin, sucrose, or a combination thereof, glucose, glucose-1-phosphate, glucose-6-phosphate, or fructose-6-phosphate; (2) phosphate; (3) psicose-6-phosphate phosphatase; (4) glucose-6-phosphate isomerase; (5) phosphoglucomutase or glucokinase; and/or (6) α-glucan phosphorylase, starch phosphorylase, maltodextrin phosphorylase, sucrose phosphorylase, α-amylase, pullulanase, isoamylase, glucoamylase, or sucrase; or

(b) a microorganism expressing the enzyme of (a), or a culture of a microorganism expressing the enzyme of (a), but not limited to this.

However, this is for illustrative purposes only and, provided that psicose can be produced using the ribulose phosphate-3-epimerase of the present invention, limitations regarding the enzymes included in the composition for producing psicose of the present invention and the substrate used to get psicosis, no.

Specifically, the starch/maltodextrin phosphorylase (EC 2.4.1.1) and α-glucan phosphorylase of the present invention may include any protein, as long as it is a protein having the activity of generating glucose-1-phosphate from starch or maltodextrin via phosphoryl transfer of phosphate to glucose. Starch/maltodextrin phosphorylase (EC 2.4.1.1) and α-glucan phosphorylase can include any protein, as long as it is a protein that has the activity of generating glucose-1-phosphate from starch or maltodextrin via phosphoryl transfer of phosphate to glucose. Sucrose phosphorylase (EC 2.4.1.7) may include any protein, provided that it is a protein having the activity of generating glucose-1-phosphate from sucrose via phosphoryl transfer of phosphate to glucose. α-amylase (EC 3.2.1.1), pullulanase (EC 3.2.1.41), isoamylase (EC 3.2.1.68), 4-α-glucan transferase (EC 2.4.1.25) and glucoamylase (EC 3.2.1.3), which are enzymes that degrade starch may include any protein, provided that it is a protein having the activity of converting starch or maltodextrin to straight-chain maltooligosaccharide or glucose. Sucrase (EC 3.2.1.26) may include any protein, provided that it is a protein having the activity of converting sucrose to glucose. Phosphoglucomutase (EC 5.4.2.2) of the present invention may include any protein, provided that it is a protein having the activity of converting glucose-1-phosphate to glucose-6-phosphate. Polyphosphate glucokinase (EC 2.7.1.63) may include any protein, provided that it is a protein having the activity of converting glucose to glucose-6-phosphate by transferring phosphate from polyphosphate to glucose. The glucose-6-phosphate isomerase of the present invention may include any protein, as long as it is a protein having the activity of converting glucose-6-phosphate to fructose-6-phosphate. The psicose-6-phosphate phosphatase of the present invention may include any protein, as long as it is a protein having the activity of converting psicose-6-phosphate to psicosis. More specifically, psicose-6-phosphate phosphatase may be a protein having the activity of irreversibly converting psicose-6-phosphate to psicosis.

The enzymes included in the composition for producing psicose of the present invention can exhibit a high rate of conversion to psicose when enzymatically converted in a single vessel that uses multiple enzymes and substrates simultaneously.

The psicose composition of the present invention may further comprise any suitable excipient commonly used in the psicose composition. The excipient may include, for example, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizing agent, an isotonic agent, and so on, but without limitation.

The psicose composition of the present invention may further comprise a metal ion or metal salt. In one embodiment, the metal ion may be a divalent cation, and specifically one or more metal ions selected from the group consisting of Ni, Mg, Ni, Co, Mn, Fe, and Zn. More specifically, the composition for producing psicose of the present invention may further comprise a metal salt, and more specifically, the metal salt may be one or more selected from the group consisting of NiSO ₄ , MgSO ₄ , MgCl ₂ , NiCl ₂ , CoSO ₄ , CoCl ₂ , MnCl ₂ , MnSO ₄ , FeSO ₄ and ZnSO ₄ .

According to another aspect of the present invention, there is provided a method for producing psicose, comprising the step of contacting fructose-6-phosphate with ribulose phosphate-3-epimerase, a microorganism expressing said ribulose phosphate-3-epimerase, or a culture of said microorganism.

Specifically, the process for producing psicose according to the present invention may sequentially include the steps of:

contacting fructose-6-phosphate with ribulose phosphate-3-epimerase, a microorganism expressing said ribulose phosphate-3-epimerase, or a culture of a microorganism expressing said ribulose phosphate-3-epimerase to convert fructose-6-phosphate to psicose-6-phosphate ;

contacting psicose-6-phosphate with psicose-6-phosphate phosphatase, a microorganism expressing said psicose-6-phosphate phosphatase, or a culture of said microorganism to convert psicose-6-phosphate to psicose; but not limited to this.

In addition, the method for producing psicose of the present invention may further include the step of contacting glucose-6-phosphate with glucose-6-phosphate isomerase, a microorganism expressing said glucose-6-phosphate isomerase, or a culture of a microorganism expressing said glucose-6-phosphate isomerase, with the conversion of glucose-6-phosphate to fructose-6-phosphate before the step of converting fructose-6-phosphate to psicose-6-phosphate.

The method for producing psicose of the present invention may further include the step of contacting glucose-1-phosphate with phosphoglucomutase, a microorganism expressing said phosphoglucomutase, or a culture of a microorganism expressing said phosphoglucomutase, to convert glucose-1-phosphate to glucose-6-phosphate before the step conversion of glucose-6-phosphate to fructose-6-phosphate.

The method for producing psicose of the present invention may further include the step of contacting glucose with a polyphosphate glucokinase, a microorganism expressing said polyphosphate glucokinase, or a culture of a microorganism expressing said polyphosphate glucokinase, and a polyphosphate to convert glucose to glucose-6-phosphate before the step of converting glucose-6-phosphate to fructose-6-phosphate.

The method for producing psicose according to the present invention may further include the step of bringing starch, maltodextrin, sucrose, or a combination thereof into contact with α-glucan phosphorylase, starch phosphorylase, maltodextrin phosphorylase or sucrose phosphorylase, a microorganism expressing said phosphorylase, or a culture of a microorganism expressing said phosphorylase, and phosphate to convert starch, maltodextrin, sucrose, or combinations thereof to glucose-1-phosphate before the step of converting glucose-1-phosphate to glucose-6-phosphate.

The method for producing psicose according to the present invention may further include the step of bringing starch, maltodextrin, sucrose, or a combination thereof into contact with α-amylase, pullulanase, isoamylase, glucoamylase or sucrase, a microorganism expressing said α-amylase, pullulanase, glucoamylase, sucrase or isoamylase, or a culture of a microorganism expressing said α-amylase, pullulanase, glucoamylase, sucrase, or isoamylase, converting starch, maltodextrin, sucrose, or combinations thereof into glucose before the step of converting starch, maltodextrin, sucrose, or combinations thereof into glucose-1-phosphate.

Ribulose phosphate-3-epimerase, psicose-6-phosphate phosphatase, α-glucan phosphorylase, phosphoglucomutase (or phosphomannomutase), glucose-6-phosphate isomerase, psicose-6-phosphate-3-epimerase (or ribulose-5-phosphate-3-epimerase), pullulanase (or isoamylase), 4-α-glucan transferase, polyphosphate glucokinase, and so on, used in the method for producing psicose of the present invention may have no side reactivity or have less side reactivity with respect to the final product of psicose.

The method for producing psicose according to the present invention decomposes a high concentration of starch to obtain the optimal/maximum amount of psicose in complex combination with glucose phosphate converting enzymes. Up to eight types of enzymes can be used in combination to ensure maximum productivity against psicosis.

First, glucan phosphorylase (glycogen phosphorylase, EC 2.4.1.1), which is an enzyme that breaks down starch to produce glucose-1-phosphate, is specific for α-1,4-linked starch to produce glucose-1-phosphate. Secondly, in the intermediate complex enzymatic reaction, phosphoglucomutase (EC 2.7.5.1) or phosphomannomuthase (EC 5.4.2.8) is used, which converts the thus obtained glucose-1-phosphate into glucose-6-phosphate. Thirdly, glucose-6-phosphate isomerase (EC 5.3.1.9) is used, which is an enzyme that converts glucose-6-phosphate to fructose-6-phosphate. Fourth, fructose-6-phosphate-3-epimerase, which is an enzyme that converts fructose-6-phosphate to psicose-6-phosphate, is used to produce psicose-6-phosphate in a reversible reaction.

In addition, to increase the rate of starch utilization, the enzyme pullulanase (EC 3.2.1.41) or isoamylase (EC 3.2.1.68) is also used to cleave the branched α-1,6 bonds in addition to the α-1,4 bonds of amylopectin. In addition, glucan transferase (4-alpha-glucan transferase, EC 2.4.1.25) can be used to increase starch utilization by glucan phosphorylase. The rate of utilization of segmented starch substrates can be increased by binding oligosaccharides in α-1,4-linked form with maltose or other oligosaccharides, which are substrates with relatively low activity. In addition, it is possible to obtain additional amounts of psicose using polyphosphate glucokinase (polyphosphate glucose phosphotransferase, EC 2.7.1.63) through a complex enzymatic reaction with cleaved glucose obtained from the utilization of starch.

In addition, in the process for producing psicose of the present invention, the contacting of the present invention can be carried out at a pH of 5.0 to 9.0, specifically a pH of 6.0 to 8.0.

In the process for producing psicose of the present invention, the contacting of the present invention can be carried out at a temperature of 40°C to 80°C, specifically at a temperature of 40°C to 60°C or 50°C to 60°C.

In the process for producing psicose of the present invention, the contacting of the present invention can be carried out for 2 to 24 hours, specifically 6 hours to 24 hours.

In the method for producing psicose according to the present invention, the contacting according to the present invention can be carried out at a pH of 5.0 to 9.0 and a temperature of 40°C to 80°C and/or within 2-24 hours. Specifically, bringing into contact can be carried out at a pH of 6.0 to 8.0 and a temperature of 40°C to 60°C or 50°C to 60°C and/or within 6-24 hours.

The method for producing psicose according to the present invention may further include the step of purifying psicose. There are no particular restrictions on the purification of the present invention, and it can be carried out using a method commonly used in the field to which the present invention pertains. Its non-limiting examples may include chromatography, fractional crystallization, ion purification, and so on. These purification methods may be performed singly, or a combination of two or more of these methods may be performed. For example, the resulting psicose can be purified by chromatography, and isolation of sugars by chromatography can be done using the difference in weak binding strengths between the recovered sugars and the metal ions associated with the ionic resin.

In addition, the present invention may further include performing bleaching, desalting, or both bleaching and desalting before or after the purification step of the present invention. The implementation of bleaching and/or desalting allows you to get a more purified product psicose without impurities.

Hereinafter, the present invention will be described in more detail with reference to typical embodiments. However, these exemplary embodiments are provided only for a better understanding of the present invention, and the scope of the present invention should not be limited to them.

In the present invention, amino acids may be represented by the following abbreviations or amino acid names.

Example 1: Obtaining a Recombinant Expression Vector for Each Enzyme and Transformed Microorganism

Heat resistant enzyme genes were selected to provide the appropriate enzymes required for the psicose production routes of the present invention. In addition, enzyme genes having the amino acid sequence S-X-M-C (SEQ ID NO:4) or G-X-X-X-X-F (SEQ ID NO:5) were selected for high epimerization activity (Table 2).

Selected amino acid genes were amplified by gene synthesis or polymerase chain reaction (PCR) against each gene from the genomic DNA of each strain, and each amplified DNA was introduced into the plasmid vector pET21a (Novagen) for expression in E. coli using DNA assembly methods, obtaining each case a recombinant expression vector. This expression vector was transformed into E. coli strain BL21(DE3) according to the conventional transformation method (Sambrook et al. 1989), obtaining in each case the transformed microorganism.

Specifically, E. coli strain BL21(DE3) was transformed with psicose-6-phosphate-3-epimerases of SEQ ID NOS:6-26, respectively. Of these, the microorganisms transformed by the above production method using SEQ ID NOs: 19, 20 and 22 were deposited with the Korean Culture Center of Microorganisms (KCCM), an international depository body, on April 16, 2019 under accession numbers KCCM12494P (E. coli BL21(DE3)/pET-CJ-fep19), KCCM12495P (E. coli BL21(DE3)/pET-CJ-fep20), and KCCM12496P (E. coli BL21(DE3)/pET-CJ- fep22), respectively.

Example 2 Preparation of Recombinant Enzymes

To obtain recombinant enzymes, each transformed microorganism obtained in Example 1 was introduced into a culture tube containing 5 ml of liquid LB medium, and the seed culture was cultivated in a shaking incubator at 37°C until an optical density at 600 im of 2.0 was reached. The culture medium of the seed culture was introduced into the culture flask containing liquid LB medium to perform the main culture. When the optical density at 600 im reached 2.0, 1 mM IPTG (Isopropyl-β-D-thiogalactoside) was added to induce expression and obtain a recombinant enzyme. During cultivation, the stirring speed was 180 rpm, and the culture temperature was maintained at 37°C. The culture medium was centrifuged at 8000 g and 4°C for 20 minutes to obtain a cell pellet. The resulting cell pellet was washed with a buffer containing 50 mM Tris-HCl (pH 8.0), suspended in the same buffer, and the cells were sonicated. The cell lysate was centrifuged at 13000 g and 4°C for 20 minutes, taking only the supernatant. The recombinant enzyme from the supernatant was purified using His-tag affinity chromatography, dialyzed against 50 mM Tris-HCl buffer (pH 8.0) and then used in the reactions.

Example 3 Modeling of psicose-6-phosphate-3-epimerase, analysis of 3-epimerization activity against psicosis and comparison of their sequences

The amino acid sequence of SEQ ID NO:9 disclosed as ribulose phosphate-3-epimerase was introduced into the I-TASSER server, Phyre2, Galaxyweb for protein structure analysis.

As a result, it was confirmed that the enzyme had a TIM-barrel structure, where the β-folding is located in the center, which is surrounded by an α-helix, and in the structure (TIM-barrel structure), motifs I (V-D-G) and III (M- X-X-X'-P-G), presumed to be the binding site of psicose or psicose-6-phosphate (FIG. 1, blue dotted line (motif I), red dotted line (motif III), an example of a structural model (SEQ ID NO: 9)).

At the same time, since the known psicose-6-phosphate-3-epimerases (ADL69228, WP_034772999, WP_029098887; WO2018/129275, WO2018/112139) are active in producing fructose through the epimerization of psicose, the production yield of psicose is reduced.

Accordingly, the inventors of the present invention suggested that aspartic acid (D) in the structure (M-X-X-D-P-G) having a negative charge could affect the 3-epimerization activity against psicosis, and based on this, enzymes were selected that were presumably not affecting the 3- epimerization activity against psicosis (WP_085113038, PKM55438, WP 117016900), where X' motif III (M-X-X-X'-P-G) is N/K, the charge of which is different from aspartic acid.

As a result, it was confirmed that psicose 3-epimerization activity may vary depending on the type of particular motif in the enzyme.

Example 4 Assay of Ribulose Phosphate-3-Epimerase Conversion Activity to Psicose-6-Phosphate

To analyze the activity of ribulose phosphate-3-epimerase of the present invention, which is an enzyme that converts to psicose-6-phosphate, 50 mM fructose-6-phosphate or 20 mM glucose-1-phosphate was suspended in buffer with 50 mM Tris-HCl (pH 7.0), or 50 mM sodium phosphate (pH 6-7), or 50 mM potassium phosphate (pH 6-7), phosphoglucomutase or phosphomannomutase, and glucose-6-phosphate isomerase, and psicose-6-phosphate phosphatase, and recombinant ribulose phosphate-3-epimerase obtained in Example 2, each at 0.1 units/ml, and allowed to interact at 45-70°C for 1-24 hours.

To analyze the activity of psicose-3-epimerase, psicose was suspended at a concentration of 1% (w/v) in buffer with 50 mM Tris-HCl (pH 7.0), or 50 mM sodium phosphate (pH 6-7), or 50 mM potassium phosphate (pH 6-7), ribulose phosphate-3-epimerase, 0.1 units/ml, was added and allowed to react at 45-70° C. for 1-24 hours. The formation of glucose, fructose or psicose was analyzed by HPLC. HPLC analysis was performed using a SP_0810 column (Shodex) and an Aminex HPX-87C column (Bio-RAD) at 80°C using a mobile phase with a flow rate of 0.6 ml/min and detection was performed using a refractive index detector (RID) . Carried out qualitative and quantitative analysis of glucose, fructose and psicose, which are the main sugars, each of which is obtained by mixing with the above enzymes. For quantification, the tolerance in the rate of conversion to fructose relative to the initial concentration of psicose was set to 5%, taking into account spontaneous experimental error according to LC (liquid chromatography) sensitivity, high substrate conversion rate and substrate concentration.

In Table 2, among several ribulose phosphate-3-epimerases demonstrating psicose-6-phosphate 3-epimerization activity, enzymes lacking psicose 3-epimerization activity were divided based on the presence or absence of the M-X-X-N/K-P-G and V-D-G motifs. . Among these enzymes, the enzyme containing the specific motif, specifically the motif of SEQ ID NO:1 without the motif of SEQ ID NO:2, and containing the motif of SEQ ID NO:3 at the same location instead of the motif of SEQ ID NO: was confirmed to be: 2 had high epimerization activity specific for psicose-6-phosphate. In addition, as confirmed in the case of sequences A and B, when the enzyme contains motifs I and III, it has a high epimerization activity specific for psicose-6-phosphate.

In addition, it was confirmed that the enzyme having the amino acid sequence of SEQ ID NO:20 of the present invention showed significantly less fructose production compared to the known psicose-6-phosphate-3-epimerase (FIG. 2).

This shows that the presence or absence of a motif has a significant effect on the yield of allulose production.

In addition, it was confirmed that the sequence containing SEQ ID NO:4 and/or SEQ ID NO:5 showed high 3-epimerization activity against psicose-6-phosphate.

Example 4 Psicose Formation Activity Assay in a Complex Enzymatic Reaction (Multiple Enzymes)

To obtain psicose from maltodextrin, glucan phosphorylase, pullulanase, 4-alpha-glucan transferase, phosphoglucomutase, glucose-6-phosphate isomerase, psicose-6-phosphate phosphatase, and fructose-6-phosphate-3-epimerase of the present invention were reacted in one container.

Specifically, each of the seven types of enzymes, 0.1 units/ml, was added to a solution where 5% (w/v) maltodextrin was added to 1-5 mM MgCl ₂ , 10-50 mM sodium phosphate (pH 7.0) , and allowed them to interact at a temperature of 50°C for 12 hours.

After completion of the reaction, the psicose present in the reaction products was analyzed by HPLC. HPLC analysis was performed using an Aminex HPX-87C column (Bio-Rad) at 80°C using a mobile phase with a flow rate of 0.6 ml/min and detection was performed using a refractive index detector. As a result, it was confirmed that psicose was obtained from maltodextrin through a complex enzymatic reaction.

Based on the above description, it will be clear to those skilled in the art that the present invention may be embodied in various specific forms without changing its technical idea or essential features. In this regard, it should be understood that, in all aspects, the embodiments described above are for illustrative purposes and are not restrictive. The scope of the invention is defined in the appended claims and not in the foregoing description, and therefore the claims are intended to include all changes and modifications not exceeding the bounds and limits of the claims or the equivalents of such bounds and limits.

Invention Effect

Since the ribulose phosphate-3-epimerase of the present invention does not contain a specific motif, it has a low psicose 3-epimerization activity and is heat resistant, and thus is advantageous in its industrial applications such as the production of psicose and so on.

Claims (17)

1. The use of ribulose phosphate-3-epimerase, for the production of psicose-6-phosphate, containing motif I, consisting of the amino acid sequence of SEQ ID NO: 1, and motif III, consisting of the amino acid sequence of SEQ ID NO: 3, while ribulose phosphate-3 -epimerase does not contain motif II, consisting of the amino acid sequence of SEQ ID NO: 2. 2. The use according to claim 1, wherein the ribulose phosphate-3-epimerase has no psicose to fructose conversion activity or has 5% or less of said activity. 3. Use according to claim 1, wherein the ribulose phosphate-3-epimerase further comprises a motif consisting of the amino acid sequence of SEQ ID NO: 4 or 5. 4. Use according to claim 1, wherein the ribulose phosphate-3-epimerase contains motif I at positions 173-184 from the N-terminal amino acid. 5. Use according to claim 1, wherein the ribulose phosphate-3-epimerase contains motif III at positions 136-150 from the N-terminal amino acid. 6. Use according to claim 1, wherein the ribulose phosphate-3-epimerase has 3-epimerization activity against psicose-6-phosphate at a temperature of 50 to 90°C. 7. Use according to claim 1, wherein the ribulose phosphate-3-epimerase is of any origin selected from the group consisting of Chthonomonas, Geobacillus, Mahella, Thermoanaerobacterium, Tepidanaerobacter, Ardenticatenia, Firmicutes, Aeribacillus, Epulopiscium and Thermoflavimicrobium. 8. Use according to claim 1, wherein the ribulose phosphate-3-epimerase is of any origin selected from the group consisting of Chthonomonas calidirosea T49, Geobacillus sp. 8, Geobacillus thermocatenulatus, Mahella australiensis 50-1 BON, Thermoanaerobacterium sp. PSU-2, Thermoanaerobacterium thermosaccharolyticum, Tepidanaerobacter syntrophicus, Ardenticatenia bacteria, Firmicutes HGW-Firmicutes-5 bacteria, Aeribacillus pallidus, Epulopiscium sp. SCG-B05WGA-EpuloA1 and Thermoflavimicrobium dichotomicum. 9. Use according to claim 1, wherein the ribulose phosphate-3-epimerase consists of any sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 15-26 and amino acid sequences having at least 26% identity with the motif exclusion region I and III in amino acid sequences. 10. The use of nucleic acid encoding ribulose phosphate-3-epimerase, as defined in paragraphs. 1-9 for the production of psicose-6-phosphate. 11. The use of a microorganism containing a nucleic acid as defined in claim 10 for the production of psicose-6-phosphate. 12. A composition for producing psicose-6-phosphate containing ribulose phosphate-3-epimerase, a microorganism expressing said ribulose phosphate-3-epimerase, or a culture of said microorganism, wherein the ribulose phosphate-3-epimerase contains motif I consisting of the amino acid sequence of SEQ ID NO : 1, and motif III, consisting of the amino acid sequence of SEQ ID NO: 3, where the composition additionally contains fructose-6-phosphate, while ribulose phosphate-3-epimerase does not contain motif II of the amino acid sequence of SEQ ID NO: 2. 13. A method for producing psicose-6-phosphate, comprising the step of bringing fructose-6-phosphate into contact with ribulose phosphate-3-epimerase, a microorganism expressing said ribulose phosphate-3-epimerase, or a culture of said microorganism, wherein the ribulose phosphate-3-epimerase contains the motif I, consisting of the amino acid sequence of SEQ ID NO: 1, and motif III, consisting of the amino acid sequence of SEQ ID NO: 3, while ribulose phosphate-3-epimerase does not contain motif II of the amino acid sequence of SEQ ID NO: 2. 14. Composition for producing psicose, containing ribulose phosphate-3-epimerase, a microorganism expressing the specified ribulose phosphate-3-epimerase, or a culture of the specified microorganism, and psicose-6-phosphate phosphatase, a microorganism expressing the specified psicose-6-phosphate phosphatase, or a culture of the specified microorganism , where ribulose phosphate-3-epimerase contains motif I, consisting of the amino acid sequence of SEQ ID NO: 1, and motif III, consisting of the amino acid sequence of SEQ ID NO: 3, while ribulose phosphate-3-epimerase does not contain motif II of the amino acid sequence of SEQ ID NO: 2. 15. The composition according to claim 14, additionally containing one or more enzymes selected from the group consisting of glucose-6-phosphate isomerase, phosphoglucomutase, polyphosphate glucokinase, α-glucan phosphorylase, starch phosphorylase, maltodextrin phosphorylase or sucrose phosphorylase, α-amylase, pullulanase, isoamylase , α-glucan transferase, glucoamylase, sucrase and psicose-6-phosphate phosphatase, a microorganism expressing said enzyme, or a culture of said microorganism. 16. A method for producing psicose, comprising the steps of bringing fructose-6-phosphate into contact with ribulose phosphate-3-epimerase, a microorganism expressing said ribulose phosphate-3-epimerase, or a culture of said microorganism and bringing psicose-6-phosphate derived from fructose-6 -phosphate, in contact with a psicose-6-phosphate phosphatase, a microorganism expressing said psicose-6-phosphate phosphatase, or a culture of said microorganism, wherein the ribulose phosphate-3-epimerase contains motif I, consisting of the amino acid sequence of SEQ ID NO: 1, and motif III , consisting of the amino acid sequence of SEQ ID NO: 3, while ribulose phosphate-3-epimerase does not contain motif II of the amino acid sequence of SEQ ID NO: 2. 17. The method of claim 16 further comprising the step of isolating psicose derived from psicose-6-phosphate.

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