[One]The present application relates to a ribulose-phosphate 3-epimerase, particularly a ribulose-phosphate 3-epimerase having low psychoses 3-epimerization activity, and a composition for producing psicose-6-phosphate or psicose comprising the same; And it relates to a method for producing psicose-6-phosphate or psicose using the same.
[2]
background
[3]
Psychose (allulose) is an epimer of carbon 3 of fructose, and is a monosaccharide known as a rare sugar that exists in a very small amount in nature. It has about 70% sweetness of sugar, but it is close to zero calories, and it is attracting a lot of attention as a new food ingredient that can be used in functional foods because of its functions such as suppressing blood sugar rise and fat synthesis.
[4]
Due to these characteristics, Psychos is being considered for use in various foods as a sugar substitute sweetener, but since it exists in very small amounts in nature, the need for a method for efficiently manufacturing Psychos is increasing.
[5]
[6]
As one of the methods for producing psicose, it undergoes conversion to glucose or glucose-1-phosphate, glucose-6-phosphate and fructose-6-phosphate. Although the process for producing -6-phosphate is known (Korean Publication 10-2018-0004023), the demand for technology development for more efficient and economical psychic production is increasing.
[7]
[8]
Psychose 3-epimerase (D-psicose 3-epimerase, EC 5.1.3.30) is a 3-epimerization (3-epimerization, 3-carbon epimerization) of fructose (D-fructose) that can produce allulose known as enzymes. When allulose is produced from fructose in a single enzymatic reaction using the enzyme, a certain level of reaction equilibrium exists between fructose as a substrate and allulose as a product (product/substrate = about 20% to 35%) ). Therefore, when high-purity allulose is prepared using the single enzymatic reaction, an additional purification process for separating and removing high-concentration fructose from the reaction product is required.
[9]
[10]
In addition, because the previously known psicose-6-phosphate 3-epimerization enzymes have psicose 3-epimerization activity, they cannot be said to be specific enzymes for psicose-6-phosphate 3-epimerization and actually produce psicose. also not suitable (WO2018/129275, WO2018/112139).
[11]
DETAILED DESCRIPTION OF THE INVENTION
technical challenge
[12]
The Applicant confirmed that certain motifs are specifically important for psycho 3-epimerization through investigation of specific motif sequences that can affect the 3-epimerization activity of psychoses.
[13]
means of solving the problem
[14]
One object of the present application is to provide a ribulose-phosphate 3-epimerase.
[15]
Another object of the present application is to provide a nucleic acid encoding a ribulose-phosphate 3-epimerase.
[16]
Another object of the present application is to provide a transformant comprising a nucleic acid encoding a ribulose-phosphate 3-epimerase.
[17]
Another object of the present application is to provide a ribulose-phosphate 3-epimerase, a microorganism expressing the same, or a composition for producing psycho-6-phosphate comprising a culture of the microorganism.
[18]
Another object of the present application comprises the step of contacting a ribulose-phosphate 3-epimerase, a microorganism expressing the same, or a culture of the microorganism to fructose-6-phosphate To provide a method for producing psicose-6-phosphate.
[19]
Another object of the present application is to provide a ribulose-phosphate 3-epimerase, a microorganism expressing the same, or a composition for producing psychosis comprising a culture of the microorganism.
[20]
Another object of the present application comprises the step of contacting a ribulose-phosphate 3-epimerase, a microorganism expressing the same, or a culture of the microorganism to fructose-6-phosphate It is to provide a method for manufacturing psychosis.
[21]
Effects of the Invention
[22]
The ribulose-phosphate 3-epimerase of the present application does not include a specific motif and thus has a low psicose 3-epimerization activity, and has heat resistance, so it has an advantage in industrial applications such as producing psicose. .
[23]
Brief description of the drawing
[24]
1 is a diagram showing the protein structure predicted from the amino acid sequence of ribulose-phosphate 3-epimerase (SEQ ID NO: 9: KPL22606).
[25]
Figure 2 is a diagram showing the HPLC chromatogram of the conventionally known psicose-6-phosphate 3-epimerase (ADL69228; solid line) and the enzyme of the present application (SEQ ID NO: 20; dotted line).
[26]
Best mode for carrying out the invention
[27]
Hereinafter, the content of the present application will be described in detail as follows. On the other hand, descriptions and embodiments of one aspect disclosed in the present application may be applied to descriptions and embodiments of other aspects with respect to common matters. Also, all combinations of the various elements disclosed in this application are within the scope of this application. In addition, it cannot be seen that the scope of the present application is limited by the detailed description described below.
[28]
[29]
One aspect of the present application for achieving the above object is to provide a ribulose-phosphate 3-epimerase.
[30]
[31]
Specifically, the ribulose-phosphate 3-epimerase of the present application includes 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 high activity and heat resistance ribulose-phosphate 3 - May be an epimerase, and more specifically, may not include Motif II consisting of the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.
[32]
In the present application, "ribulose-phosphate 3-epimerase" means an enzyme for which ribulose-phosphate 3-epimerase activity is known or has ribulose-phosphate 3-epimerase activity, in particular, fructose- It means that it can act as a 6-phosphate 3-epimerase or psicose-6-phosphate 3-epimerase. The ribulose-phosphate 3-epimerase has some sequence when it has the activity of fructose-6-phosphate 3-epimerase or psicose-6-phosphate 3-epimerase. These deletions, modifications, substitutions, conservative substitutions or added amino acid sequences may also be included.
[33]
Specifically, the enzyme of the present application is an enzyme having a reversible conversion activity having an activity of reversibly converting psicose-6-phosphate to fructose-6-phosphate or fructose-6-phosphate to psicose-6-phosphate, In the present application, "ribulose-phosphate 3-epimerase" may be used interchangeably with "psychose-6-phosphate 3-epimerase" or "enzyme".
[34]
The enzyme of the present application is glucose-1-phosphate (D-glucose-1-phosphate), glucose-6-phosphate (D-glucose-6-phosphate) or fructose-6-phosphate (D-fructose-6-phosphate) When mixed, it may be an enzyme that converts to psicose-6-phosphate. For example, the enzyme of the present application has a conversion rate of 1% or more to psicose-6-phosphate when the same amounts of psicose-6-phosphate, glucose-1-phosphate, glucose-6-phosphate, and fructose-6-phosphate are mixed. , 10% or more, or 30% or more. Due to the selective activity of the enzyme of the present application as described above, it may exhibit a high psycho-conversion rate in one-pot enzymatic conversion using a plurality of enzymes and substrates at the same time.
[35]
[36]
In the present application, "motif" refers to a site (region) having a specific sequence in an enzyme sequence, and may mean a sequence having a specific function or activity of a protein, and may be a sequence conserved between microbial species, but It is not limited. The ribulose-phosphate 3-epimerase of the present application may include Motif I composed of the amino acid sequence of SEQ ID NO: 1 and Motif III composed of the amino acid sequence of SEQ ID NO: 3. Additionally, it may be characterized in that it does not include the motif II consisting of the amino acid sequence of SEQ ID NO: 2, and by not including the motif II, the enzyme is characterized in that it has low psychoses-3-epimerization activity. However, the present invention is not limited thereto.
[37]
Specifically, the enzyme has an activity of 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or activity for converting psychoses into fructose compared to enzymes that do not include motifs I and III It may be absent, but is not limited thereto.
[38]
[39]
In addition, the enzyme of the present application may further include a motif consisting of the amino acid sequence of SEQ ID NO: 4 or 5.
[40]
[41]
The enzyme of the present application includes, or may necessarily include, a specific motif, and is characterized in that it does not include a specific motif. Specifically, motifs I and III of the present application are included in the site (binding site, binding site) where the enzyme is partially or fully bound to a substrate and/or metal ion (eg, Mg, Mn, Zn, etc.) It may be to lower the side-reactivity while maintaining the activity of the enzyme itself. More specifically, the motifs I and III may be included in the TIM-barrel fold within the binding site. An enzyme "comprising" a specific motif may or may not further include other motifs, domains, amino acid sequences, fragments, etc. in addition to the motif, and an enzyme "essentially comprising" a specific motif is Including, it is possible to obtain the desired properties or characteristics, and also may or may not further include other motifs, domains, amino acid sequences, fragments, etc. in addition to the corresponding motif, but is not limited thereto. An enzyme "not including" a specific motif does not include a sequence corresponding to the motif in the enzyme, and other amino acid sequences are inserted, substituted, deleted, or a combination thereof at the position of the motif, but is not limited thereto .
[42]
[43]
In addition, motifs included or not included in the enzyme of the present application may or may not be included independently of each other, and are not limitedly arranged in a specific order or position.
[44]
[45]
For example, the enzyme of the present application comprises only motif I of SEQ ID NO: 1; comprising motif I of SEQ ID NO: 1, and motif III of SEQ ID NO: 3, and no motif II of SEQ ID NO: 2; It may be an enzyme that includes the motif I of SEQ ID NO: 1, the motif III of SEQ ID NO: 3, and the motif of SEQ ID NO: 4 and 5, but does not include the motif II of SEQ ID NO: 2, but is not limited thereto.
[46]
[47]
In the present application, "motif I" may be composed of the amino acid sequence of SEQ ID NO: 1, but includes insertions, substitutions, deletions, etc. of meaningless amino acid residues that do not affect the activity of the amino acid sequence of SEQ ID NO: 1 It is apparent that even the sequence corresponds to motif I of the present application.
[48]
[49]
Motif I (SEQ ID NO: 1): VDG
[50]
[51]
The motif I may be included in a binding site that reacts with a substrate and a metal ion of a ribulose-phosphate 3-epimerase, but is not limited thereto. Specifically, the motif may be located at amino acids 173 to 184 from the first amino acid of the N-terminus of ribulose-phosphate 3-epimerase, but is not limited thereto. That is, valine (V), which is the first amino acid residue of the motif I, may be located between positions 173 and 182 from the first amino acid of the N-terminus of the ribulose-phosphate 3-epimerase, and the last residue of the motif I Glycine (G) may be positioned between positions 175 and 184, but is not limited thereto.
[52]
In addition, the motif I may be one in which the first amino acid residue starts at the C-terminus from the domain to which the first beta sheet structure-coil structure-alpha helix structure-second beta sheet structure is connected to the second beta sheet, and the domain is a binding It may be included in the site or may have a structure in which some regions overlap (FIG. 1).
[53]
[54]
In the present application, "Motif II" may be composed of the amino acid sequence of SEQ ID NO: 2, but includes insertions, substitutions, deletions, etc. of meaningless amino acid residues that do not affect the activity of the amino acid sequence of SEQ ID NO: 2 It is apparent that even the sequence corresponds to motif II of the present application.
[55]
[56]
Motif II (SEQ ID NO: 2): MXXDPG (X is any amino acid residue)
[57]
[58]
The motif II may be included in the N-terminal region of the ribulose-phosphate 3-epimerase, but is not limited thereto. Specifically, the motif may be located at amino acids 136 to 150 from the first amino acid of the N-terminus of the ribulose-phosphate 3-epimerase, but is not limited thereto. That is, methionine (M), which is the first amino acid residue of motif II, may be located between positions 136 and 145 from the first amino acid of the N-terminal of ribulose-phosphate 3-epimerase, and the last residue of motif II. Glycine (G) may be located between positions 141 to 150, but is not limited thereto. In addition, the motif II may be a domain in which the first beta sheet structure-coil structure-alpha helix structure-second beta sheet structure is connected, wherein the first amino acid residue starts at the C-terminus of the first beta sheet, and the domain is a binding site It may have a structure included in or overlapping some regions, and the domain may be the same as the domain including the motif I ( FIG. 1 ).
[59]
[60]
Specifically, X may include any amino acid without limitation, and specifically may be threonine (T) or valine (V), but is not limited thereto. More specifically, the motif II may include a sequence of M-(T/A/M/L)-(V/N/I)-DPG, but is not limited thereto.
[61]
[62]
In the present application, "Motif III" may be composed of the amino acid sequence of SEQ ID NO: 3, but includes insertions, substitutions, deletions, etc. of meaningless amino acid residues that do not affect the activity of the amino acid sequence of SEQ ID NO: 3 It is apparent that even the sequence corresponds to motif III of the present application.
[63]
[64]
Motif III (SEQ ID NO: 3): MXX-X'-PG (X is any amino acid residue)
[65]
[66]
The motif III may be included in the N-terminal region of the ribulose-phosphate 3-epimerase, but is not limited thereto. Specifically, the motif may be located at amino acids 136 to 150 from the first amino acid of the N-terminus of the ribulose-phosphate 3-epimerase, but is not limited thereto. That is, methionine (M), the first amino acid residue of motif III, may be located between positions 136 and 145 from the first amino acid of the N-terminus of ribulose-phosphate 3-epimerase, and the last residue of motif III Glycine (G) may be located between positions 141 to 150, but is not limited thereto. In addition, the motif III may be a domain in which the first beta sheet structure-coil structure-alpha helix structure-second beta sheet structure is connected, wherein the first amino acid residue starts at the C-terminus of the first beta sheet, and the domain is a binding site It may have a structure included in or overlapping some regions, and the domain may be the same as the domain including the motif I ( FIG. 1 ).
[67]
More specifically, the motifs II and III may be motifs included in the same position when the enzymes of the present application are aligned, but is not limited thereto.
[68]
[69]
Specifically, X may include any amino acid without limitation, specifically threonine (T), alanine (A), methionine (M), leucine (L), valine (V), asparagine (N), isoleucine ( I) may be, but is not limited thereto.
[70]
In addition, the X' may include any amino acid except for aspartic acid (D) without limitation, and specifically, an amino acid residue having no charge or a positive charge may be included in the amino acid residue. Specifically, the uncharged amino acid includes both polar amino acids and non-polar amino acids, and may be any one of serine, threonine, cysteine, asparagine, glutamine, glycine, alanine, proline, valine, leucine, isoleucine, and methionine. In addition, the amino acid having a positive charge may be any one of lysine, arginine, and histidine. For example, asparagine (N) and lysine (K) may be included. More specifically, the motif III may include a sequence of M-(T/A/M/L)-(V/N/I)-NPG, but is not limited thereto.
[71]
[72]
The enzyme of the present application may be one that additionally includes a motif consisting of the amino acid sequence of SEQ ID NO: 4, but includes insertions, substitutions, deletions, etc. of meaningless amino acid residues that do not affect the activity of the amino acid sequence of SEQ ID NO: 4 It is apparent that even the sequence is included in the enzyme of the present application.
[73]
[74]
SEQ ID NO: 4: SXM/IC (X is any amino acid residue)
[75]
[76]
The 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 thereto. Specifically, the motif may be located at amino acids 5 to 20, more specifically, amino acids 7 to 19 from the first amino acid of the N-terminus of ribulose-phosphate 3-epimerase, but is not limited thereto. . That is, serine (S), which is the first amino acid residue of the motif having the amino acid sequence of SEQ ID NO: 4, may be located between 7 and 16 from the first amino acid of the N-terminal of ribulose-phosphate 3-epimerase, and , cysteine ​​(C), which is the last residue of the motif having the amino acid sequence of SEQ ID NO: 4, may be located between positions 10 to 19, but is not limited thereto. In addition, the motif of SEQ ID NO: 4 may be formed after the beta sheet structure, and specifically, a portion of the motif sequence of SEQ ID NO: 4 may be included in the alpha helix structure formed after the beta sheet structure.
[77]
[78]
Specifically, X may include any amino acid without limitation, and specifically may be methionine, isoleucine, leucine, or valine, but is not limited thereto. More specifically, the motif consisting of the amino acid sequence of SEQ ID NO: 4 may include a sequence of SIMC (SEQ ID NO: 27), SMMC (SEQ ID NO: 28), SLMC (SEQ ID NO: 29) or SVMC (SEQ ID NO: 30), However, the present invention is not limited thereto.
[79]
[80]
The enzyme of the present application may additionally include a motif consisting of the amino acid sequence of SEQ ID NO: 5, but including insertions, substitutions, deletions, etc. of meaningless amino acid residues that do not affect the activity of the amino acid sequence of SEQ ID NO: 5 It is apparent that even the sequence is included in the enzyme of the present application.
[81]
[82]
SEQ ID NO: 5: GXXXXF/L (X is any amino acid residue)
[83]
[84]
The motif composed 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 thereto. Specifically, it may be located at amino acids 190 to 210, more specifically, amino acids 196 to 210 from the N-terminal amino acid of the ribulose-phosphate 3-epimerase, but is not limited thereto. That is, glycine (G), the first amino acid residue of the motif consisting of the amino acid sequence of SEQ ID NO: 5, may be located between positions 196 and 205 from the first amino acid of the N-terminus of ribulose-phosphate 3-epimerase, 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 to 210, but is not limited thereto. In addition, the motif of SEQ ID NO: 5 may be formed after the beta sheet structure, and specifically, a portion of the motif sequence of SEQ ID NO: 5 may be included in the alpha helix structure formed after the beta sheet structure.
[85]
[86]
Specifically, each X of the motif consisting of the amino acid sequence of SEQ ID NO: 5 may be, independently of each other, threonine, serine, glycine, leucine, cysteine, isoleucine, asparagine, lysine, alanine, valine, or glutamine, It is not limited thereto. More specifically, the motif consisting of the amino acid sequence of SEQ ID NO: 5 is GNSGLF (SEQ ID NO: 31), GSSGLFGSSSLF (SEQ ID NO: 32), GSTSLF (SEQ ID NO: 33), GTAGLF (SEQ ID NO: 34), GTKGLF (SEQ ID NO: 35), Amino acids of GTQSLF (SEQ ID NO: 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) sequences may include, but are not limited to. The enzyme of the present application is characterized in that it has a high activity of converting fructose-6-phosphate to psicose-6-phosphate by including the motif of SEQ ID NOs: 4 and/or 5.
[87]
Each amino acid residue included in the motif of the present application may be independently combined with each other to constitute the motif.
[88]
[89]
The ribulose-phosphate 3-epimerase of the present application may exhibit very low psicose 3-epimerization activity by not including a specific motif, ie, II, which is from fructose-6-phosphate to psicose-6- By lowering the side reaction in the process of producing phosphoric acid, it suggests that the final product, Psychos, can be obtained in high yield. In addition, it can be effectively used to produce psicose through combination with other enzymes (eg, psicose-6-phosphate dephosphorylation enzyme).
[90]
[91]
The alpha-helix structure, the beta-sheet structure, and the coil structure of the present application are described in KwangsooKim et. al (Crystal Structure of d-Psicose 3-epimerase from Agrobacterium tumefaciens and its Complex with True Substrate d-FructoseVolume 361, Issue 5, 1 September 2006, Pages 920-931), etc. The alpha helix may be a right handed alpha helix. The structure can be obtained by performing directly by a commonly known method such as NMR or X-ray crystallography, or by predicting using Rosetta or a web server (I-TASSER, ROBETTA, etc.) based on the amino acid sequence.
[92]
In addition, in the present application, the domains in which the structures are aggregated may be expressed in the same form as Structure 1 - Structure 2.
[93]
[94]
In addition, the present application ribul Ross-phosphate 3-epimerase is keusso eggplant grandma ( Chthonomonas ), geo Bacillus ( Geobacillus ), e HeLa ( Mahella ), Thermo unloading area tumefaciens ( Thermoanaerobacterium ), te the blood Nero bakteo ( Tepidanaerobacter ) , Arden urticae'll ah ( Ardenticatenia ), permeation kyucheu ( Firmicutes ), Arianna Bacillus ( Aeribacillus ), fish Titanium (as yipul Epulopiscium ), and the thermopile away flaviviruses micro ( Thermoflavimicrobium of any one of the origin is selected from the group consisting of genus) It may be, and more specifically, Chthonomonas calidirosea ( Chthonomonas calidirosea ) T49, Geobacillus genus 8 ( Geobacillus sp. 8) Geo Bacillus Thermo category Cronulla tooth ( Geobacillus thermocatenulatus ), HeLa Au village host Raleigh N-Sys ( Mahella australiensis ) 50-1 BON, Thermo Aero tumefaciens frozen in 2-PSU ( Thermoanaerobacterium sp . PSU-2), Thermo frozen Aero tumefaciens Thermo saccharide with utility glutamicum ( Thermoanaerobacterium thermosaccharolyticum), te the blood Nero bakteo syntropy kusu ( Tepidanaerobacter syntrophicus ), Arden urticae'll ah tumefaciens ( Ardenticatenia bacterium ), permeation kyucheu tumefaciens HGW-Firmicutes-5 ( Firmicutes bacterium HGW-Firmicutes -5), Aeribacillus pallidus , SCG-B05WGA-EpuloA1 ( Epulopiscium sp. SCG-B05WGA-EpuloA1), And Thermoflavimicrobium dichotomicum ( Thermoflavimicrobium dichotomicum ) It may be derived from any one selected from the group consisting of, but is not limited thereto.
[95]
[96]
In addition, the ribulose-phosphate 3-epimerase of the present application may include any one sequence selected from the group consisting of the amino acid sequence of SEQ ID NOs: 15 to 26, or the amino acid sequence of SEQ ID NOs: 15 to 26 It may be composed of any one sequence selected from the group consisting of, but is not limited thereto. More specifically, the enzyme may be composed of the amino acid sequence of SEQ ID NO: 19, 20, or 22, but is not limited thereto.
[97]
Alternatively, in the ribulose-phosphate 3-epimerase of the present application, since the presence or absence of a specific motif has an important effect on enzyme activity, the enzyme sequence excluding the motif region may have low sequence identity. Specifically, 26%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90 of the amino acid sequence except for the motifs I and III. %, 95%, 96%, 97%, 98%, or 99% or more, motifs I, III, a region excluding the region of SEQ ID NOs: 4 and 5 and 24%, 40%, 45%, 50%, 55%, Any one selected from the group consisting of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity. It may be composed of a sequence of, but is not limited thereto.
[98]
In addition, it may include an amino acid sequence of any one of SEQ ID NOs: 15 to 26 or an amino acid sequence having at least 70% homology or identity therewith, but is not limited thereto. Specifically, the amino acid has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology with the sequence of SEQ ID NOs: 15 to 26 and the sequence. Or it may include amino acids having the same identity. In addition, it is apparent that proteins having amino acid sequences in which some sequences are deleted, modified, substituted or added are included within the scope of the present application as long as the amino acid sequence has such homology or identity and exhibits efficacy corresponding to the protein.
[99]
That is, even if it is described as 'a protein having an amino acid sequence described in a specific SEQ ID NO:' in the present application, if it has the same or corresponding activity as a protein consisting of the amino acid sequence of the corresponding SEQ ID NO: some sequences are deleted, modified, It is apparent that proteins having substituted, conservatively substituted or added amino acid sequences may also be used in the present application. For example, if it has the same or corresponding activity as the enzyme, adding a sequence that does not change the function of the protein before or after the amino acid sequence, a naturally occurring mutation, a silent mutation, or a conservative substitution It is not excluded, and it is evident that such sequence addition or mutation falls within the scope of the present application.
[100]
[101]
As used herein, the term 'homology' or 'identity' refers to the degree to which two given amino acid sequences or base sequences are related to each other and may be expressed as a percentage.
[102]
The terms homology and identity are often used interchangeably.
[103]
[104]
Sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, with default gap penalties established by the program used may be used. Substantially homologous or identical sequences under moderate or high stringent conditions generally contain at least about 50%, 60%, 70%, 80% of the total or full-length of the sequence. or more than 90% hybrid. Hybridization is also contemplated for polynucleotides containing degenerate codons instead of codons in the polynucleotides.
[105]
Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined, for example, by Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444, using a known computer algorithm such as the “FASTA” program. or, as performed in the Needleman Program (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) of the EMBOSS package, The Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) can be used to determine. (GCG program 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 ETA/.] (1988) including SIAM J Applied Math 48: 1073) For example, BLAST of the National Center for Biotechnology Information Database, or ClustalW, can be used to determine homology, similarity or identity.
[106]
Homology, similarity or identity of polynucleotides or polypeptides is described, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482, see, for example, Needleman et al. (1970), J Mol Biol. 48: 443 by comparing sequence information using a GAP computer program. In summary, the GAP program is defined as the total number of symbols in the shorter of two sequences divided by the number of similarly aligned symbols (ie, nucleotides or amino acids). Default parameters for the GAP program are: (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps. Thus, as used herein, the term "homology" or "identity" refers to a relevance between sequences.
[107]
[108]
On the other hand, the ribulose-phosphate 3-epimerase of the present application may be heat-resistant, but is not limited thereto.
[109]
In the present application, "heat resistance" means a property capable of exhibiting the original activity without losing the activity of the enzyme even in a high-temperature environment, and the heat resistance of the enzyme has various advantages in the process of producing the target product. Specifically, the ribulose-phosphate 3-epimerization enzyme of the present application may be one having psicose-6-phosphate 3-epimerization activity at 40°C or higher, more specifically 50°C or higher, and more specifically 60°C or higher. , but is not limited thereto. More specifically, the enzyme of the present application may have a 3-epimerization activity of psicose-6-phosphate for 1 minute to 24 hours at a pH of 5.0 to 10.0, 50° C. to 90° C., but is not limited thereto.
[110]
[111]
The ribulose-phosphate 3-epimerase of the present application transforms the enzyme itself or the DNA expressing the same into a strain, culturing it to obtain a culture, crushing the culture, and purified through a column, etc. it could be As the transformation strain, E. coli ( Escherichia coli ), Corynebacterium glutamicum ( Corynebacterum glutamicum ), Aspergillus oryzae ), Saccharomyces cerevisiae ), Yarrowia Lee Polytica ( Yarrowia lipolytica ), Pichia pastoris ( Pichia pastoris ) or Bacillus subtilis ( Bacillus subtilis ), but is not limited thereto, there may be a potential for transformation in a GRAS (Generally Recognized as Safe) strain in the future .
[112]
The purification method of the ribulose-phosphate 3-epimerase of the present application is not particularly limited, and a method commonly used in the technical field of the present application may be used. Non-limiting examples include chromatography, heat treatment, adsorption, filtration and ion purification. The purification method may be performed only one, or two or more methods may be performed together.
[113]
[114]
Another aspect of the present application is to provide a nucleic acid encoding the ribulose-phosphate 3-epimerase, or a vector including the nucleic acid.
[115]
In the present application, "nucleic acid" has a meaning to encompass DNA or RNA molecules, and nucleotides, which are basic structural units in nucleic acids, may include not only natural nucleotides, but also analogs in which sugars or base sites are modified.
[116]
The nucleic acid of the present application may be a DNA or RNA sequence in which nucleotides, which are units, are covalently linked, and specifically, when converting the amino acid sequence of SEQ ID NOs: 15 to 26 into DNA conversion (amino acids are transformed into 61 codons), all possible It may be any one of the number of nucleotide sequences, and more specifically, 90% or more, 95% or more, 97 of each nucleotide that can be translated into any one of the amino acid sequences of SEQ ID NOs: 15 to 26 of the present application. % or more, 99% or more, or 100% homology, similarity or identity, and may include a nucleic acid capable of being translated and exhibiting a desired enzymatic activity. A protein having the same activity by codon degeneracy, a protein having the same amino acid sequence after translation, specifically, a protein consisting of any one of SEQ ID NOs: 15 to 26, or homology, similarity, or identity thereto It is apparent that polynucleotides that can be translated into a protein may also be included in the scope of the present application. More specifically, the sequence of the nucleic acid of the present application is not separately indicated, and may consist of all the number of DNA codons that can be translated into the amino acid sequence of SEQ ID NOs: 15 to 26, but is not limited thereto.
[117]
[118]
Also, a probe that can be prepared from a known gene sequence, for example, a sequence encoding the enzyme of the present application by hydridation under stringent conditions with a sequence complementary to all or part of the nucleotide sequence may be included without limitation. .
[119]
The "stringent condition" means a condition that enables specific hybridization between polynucleotides. These conditions are specifically described in the literature (eg, J. Sambrook et al., supra). For example, genes having high homology or identity, 80% or more, 85% or more, specifically 90% or more, more specifically 95% or more, more specifically 97% or more, In particular, the conditions in which genes having 99% or more homology or identity hybridize with each other and genes with lower homology or identity do not hybridize, or at 60° C., which is a washing condition for normal Southern hybridization , 1 X SSC, 0.1% SDS, specifically at a salt concentration and temperature equivalent to 60° C., 0.1 X SSC, 0.1% SDS, more specifically 68° C., 0.1 X SSC, 0.1% SDS, once, specifically Conditions for washing 2 to 3 times can be exemplified.
[120]
Hybridization requires that two nucleic acids have complementary sequences, although mismatch between bases is possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases capable of hybridizing to each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the present application may also include isolated nucleic acid fragments that are complementary to substantially similar nucleic acid sequences as well as the entire sequence.
[121]
Specifically, polynucleotides having homology or identity can be detected using hybridization conditions including a hybridization step at a Tm value of 55° C. and using the above-described conditions. In addition, the Tm value may be 60 °C, 63 °C, or 65 °C, but is not limited thereto and may be appropriately adjusted by those skilled in the art according to the purpose.
[122]
The appropriate stringency for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides, and the parameters are well known in the art.
[123]
In the present application, "vector" refers to a DNA preparation containing a nucleotide sequence of a nucleic acid encoding an enzyme of the present application operably linked to a suitable regulatory sequence so as to express a desired mutant protein in a suitable host. The regulatory sequences may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. After transformation into an appropriate host cell, the vector can replicate or function independently of the host genome, and can be integrated into the genome itself.
[124]
The vector used in the present application is not particularly limited as long as it is capable of replication in a host cell, and any vector known in the art may be used. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage vectors or cosmid vectors, and pBR-based, pUC-based, and pBluescriptII-based plasmid vectors may be used. , pGEM-based, pTZ-based, pCL-based and pET-based and the like can be used. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors and the like can be used.
[125]
[126]
Another aspect of the present application provides a transformant comprising a vector comprising a nucleic acid encoding the enzyme of the present application or a nucleic acid encoding the enzyme of the present application.
[127]
In the present application, "transformants comprising a nucleic acid encoding an enzyme" or "transformants comprising a vector comprising a nucleic acid encoding an enzyme" means that the ribulose-phosphate 3-epimerase of the present application is It may refer to a microorganism recombinant to be expressed. For example, the ribulose-phosphate 3-epimerase-encoding nucleic acid is contained, or the ribulose-phosphate 3-epimerase is transformed with a vector containing the nucleic acid encoding the ribulose-phosphate 3-epimerase enzyme. It refers to a host cell or microorganism capable of expressing a merging enzyme. For the purpose of the present application, the ribulose-phosphate 3-epimerase expressed by the transformant may be composed of the amino acid sequence of any one of SEQ ID NOs: 15 to 26, but is not limited thereto.
[128]
[129]
In the present application, the term “transformation” refers to introducing a vector containing a nucleic acid encoding a ribulose-phosphate 3-epimerase of the present application into a host cell so that the protein encoded by the nucleic acid can be expressed in the host cell. means to do As long as the transformed nucleic acid can be expressed in the host cell, it may include all of them regardless of whether they are inserted into the chromosome of the host cell or located outside the chromosome. In addition, the nucleic acid includes DNA and RNA encoding the nucleic acid encoding the ribulose-phosphate 3-epimerase of the present application. The nucleic acid may be introduced in any form as long as it can be expressed and introduced into a host cell. For example, the nucleic acid may be introduced into a host cell in the form of an expression cassette, which is a gene construct including all elements necessary for self-expression. The expression cassette may include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, which are usually operably linked to the nucleic acid. The expression cassette may be in the form of an expression vector capable of self-replication. In addition, the nucleic acid may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.
[130]
In addition, the term "operably linked" as used herein refers to a promoter sequence that initiates and mediates transcription of a nucleic acid encoding a dephosphorylation enzyme of psycho-6-phosphate of the present application and the gene sequence is functionally linked. means that
[131]
Insertion of the nucleic acid or vector into the chromosome may be accomplished by any method known in the art, for example, homologous recombination, but is not limited thereto. It may further include a selection marker (selection marker) for confirming whether the chromosome is inserted. The selection marker is used to select cells transformed with a vector, that is, to determine whether a target nucleic acid molecule is inserted, and a selectable phenotype such as drug resistance, auxotrophic tolerance, resistance to cytotoxic agents, or expression of a surface variant protein. Markers that give , can be used. In an environment treated with a selective agent, only cells expressing a selectable marker survive or exhibit other expression traits, and thus transformed cells can be selected.
[132]
The method for transforming the vector of the present application includes any method of introducing a nucleic acid into a cell, and may be performed by selecting a suitable standard technique as known in the art depending on the host cell. For example, electroporation, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, retroviral infection, microinjection, polyethylene glycol (PEG) method, DEAE-dex Trans method, cationic liposome method, lithium acetate-DMSO method, etc., but are not limited thereto.
[133]
As the host cell, it is preferable to use a host with high DNA introduction efficiency and high expression efficiency of the introduced DNA, for example, a microorganism of the genus Coryne, a microorganism of the genus Escherichia, a microorganism of the genus Serratia, a microorganism of the genus Bacillus, Saccharin It may be a microorganism of the genus Romansis cerevisiae or a microorganism of the genus Pichia, specifically, E. coli , but is not limited thereto, and is applicable to all GRAS strains.
[134]
More specifically, the transformant of the present application is E.coil BL21(DE3)/pET-CJ-ef7 , E.coil BL21(DE3)/pET-CJ-ef12 , or E.coil BL21(DE3)/ pET- It may be CJ-ef15 , but is not limited thereto.
[135]
[136]
Another aspect of the present application provides a ribulose-phosphate 3-epimerase, a microorganism expressing the same, or a composition for producing psycho-6-phosphate comprising a culture of the microorganism.
[137]
The composition for producing psicose-6-phosphate of the present application is a ribulose-phosphate 3-epimerase that exhibits an activity of converting fructose-6-phosphate to psicose-6-phosphate, a microorganism expressing the same, or the microorganism Since it includes a culture, when the composition is brought into contact (reacted) with fructose-6-phosphate, psicose-6-phosphate can be produced from the fructose-6-phosphate.
[138]
Specifically, the composition may further include fructose-6-phosphate as a substrate, but is not limited thereto.
[139]
[140]
The composition for producing psycho-6-phosphate of the present application may further include any suitable excipients commonly used in the composition for producing psycho-6-phosphate. Such excipients may include, but are not limited to, for example, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizing agents, and isotonic agents.
[141]
The composition for producing psicose-6-phosphate of the present application may further include a metal ion or a metal salt. In one embodiment, the metal ion may be a divalent cation, and specifically, may be one or more metal ions selected from the group consisting of Ni, Mg, Ni, Co, Mn, Fe, and Zn. More specifically, the composition for production of psicose-6-phosphate of the present application may further include a metal salt, and more specifically, the metal salt is NiSO 4 , MgSO 4 , MgCl 2 , NiCl 2 , CoSO 4 , CoCl 2 , MnCl 2 , MnSO 4 , FeSO 4 and ZnSO 4 It may be at least one selected from the group consisting of.
[142]
[143]
Another aspect of the present application comprises the step of contacting fructose-6-phosphate with ribulose-phosphate 3-epimerase, a microorganism expressing the same, or a culture of the microorganism Provided is a method for producing psicose-6-phosphate.
[144]
Since the ribulose-phosphate 3-epimerase of the present application exhibits an activity of converting fructose-6-phosphate to psicose-6-phosphate, the ribulose-phosphate 3-epimerase, a microorganism expressing the same, or When the culture of the microorganism is brought into contact with fructose-6-phosphate, psicose-6-phosphate can be produced from the fructose-6-phosphate.
[145]
Conditions for contacting and reacting the fructose-6-phosphate and psicose-6-phosphate may be appropriately selected by those skilled in the art in consideration of substrates and enzymes.
[146]
Specifically, the step of producing psicose-6-phosphate by contacting the fructose-6-phosphate and ribulose-phosphate 3-epimerase is at a pH of 5.0 to 9.0, a temperature of 40°C to 80°C, and/or 2 hours. to 24 hours, more specifically pH 6.0 to pH 8.0, a temperature of 40 °C to 60 °C, and/or may be carried out for 20 hours to 24 hours, more specifically pH 7.0, 50 It may be carried out at ℃ temperature, 24 hours, but is not limited thereto.
[147]
Psychos-6-phosphate manufacturing method of the present application may additionally include a step of obtaining and/or purifying the prepared psicose-6-phosphate, but is not limited thereto.
[148]
The step of obtaining and / or purifying the psicose-6-phosphoric acid may be performed by a method known in the art, and is not limited to a specific method.
[149]
[150]
Another aspect of the present application is to provide a ribulose-phosphate 3-epimerase, a microorganism expressing the same, or a composition for producing psychosis comprising a culture of the microorganism. Specifically, the composition may further include fructose-6-phosphate as a substrate, but is not limited thereto.
[151]
[152]
In addition to the ribulose-phosphate 3-epimerization enzyme for producing psicose-6-phosphate from fructose-6-phosphate, the composition for psicose production of the present application, an enzyme necessary to produce psicose, for example, psicose Psychos-6-phosphate dephosphorylation enzyme that dephosphorylates -6-phosphate to dephosphorylate phosphate may be further included to produce psicose.
[153]
[154]
Specifically, the composition for production of psicose of the present application is glucose-6-phosphate-isomerase, phosphoglucomutase, polyphosphate glucose kinase, α-glucan phosphorylase, starch phosphorylase, maltodextrin phosphate group consisting of phosphorylase or sucrose phosphorylase, α-amylase, pullulanase, isoamylase, α-glucanotransferase, glucoamylase, sucrase, and psicose-6-phosphate dephosphorylation enzyme It may further include one or more enzymes selected from, a microorganism expressing the same, or a culture of the microorganism, specifically, may be to further include a psicose-6-phosphate dephosphorylation enzyme, but is limited thereto no.
[155]
[156]
More specifically, the composition for producing psycho of the present application is
[157]
(a) (i) starch, maltodextrin, sucrose or a combination thereof, glucose, glucose-1-phosphate, glucose-6-phosphate, or fructose-6-phosphate; (ii) phosphate; (iii) psicose-6-phosphate dephosphorylation enzyme; (iv) glucose-6-phosphate-isomerase; (v) phosphoglucomutase or glucose kinase; and/or (vi) α-glucan phosphorylase, starch phosphorylase, maltodextrin phosphorylase, sucrose phosphorylase, α-amylase, pullulanase, isoamylase, glucoamylase or sucrase ; or
[158]
(b) may additionally include a culture of a microorganism expressing the enzyme of the item (a) or a microorganism expressing the enzyme of the item (a), but is not limited thereto.
[159]
However, this is exemplary and if it is possible to produce psicose using the ribulose-phosphate 3-epimerase of the present application, the enzyme included in the composition for psicose production of the present application and the substrate used for psicose production are limited. doesn't happen
[160]
[161]
Specifically, starch/maltodextrin phosphorylase (EC 2.4.1.1) and α-glucan phosphorylase of the present application convert phosphate to glucose by phosphorylation transfer to glucose from starch or maltodextrin. Any protein may be included as long as it has an activity to produce -1-phosphate. The starch/maltodextrin phosphorylase (starch/maltodextrin phosphorylase, EC 2.4.1.1) and α-glucan phosphorylase phosphorylate and transfer phosphate to glucose to convert glucose-1-phosphate from starch or maltodextrin. Any protein may be included as long as it has an activity to produce it. The sucrose phosphorylase (EC 2.4.1.7) may include any protein as long as it has an activity to produce glucose-1-phosphate from sucrose by phosphorylation transfer of phosphate to glucose. The starch liquid glycosylation enzyme α-amylase (α-amylase, EC 3.2.1.1), pullulanase (pullulanse, EC 3.2.1.41), isoamylase (EC 3.2.1.68), 4-α-glucanotrense Perease (4-α-glucanotransferase, EC 2.4.1.25) and glucoamylase (EC 3.2.1.3) are any protein having an activity to convert starch or maltodextrin into debranched maltooligosaccharide or glucose. may include The sucrase (EC 3.2.1. 26) may include any protein as long as it has an activity of converting sucrose into glucose. The phosphoglucomutase (EC 5.4.2.2) of the present application may include any protein as long as it has an activity to convert glucose-1-phosphate to glucose-6-phosphate. Polyphosphate glucokinase (EC 2.7.1.63) may include any protein as long as it has an activity of converting phosphate of polyphosphate to glucose and converting it into glucose-6-phosphate. The glucose-6-phosphate-isomerase of the present application may include any protein as long as it has an activity to convert glucose-6-phosphate to fructose-6-phosphate. Psychos-6-phosphate dephosphorylation enzyme of the present application may include any protein as long as it is a protein having an activity to convert psicose-6-phosphate into psicose. More specifically, the psicose-6-phosphate dephosphorylation enzyme may be a protein having an activity of irreversibly converting psicose-6-phosphate into psicose. The glucose-6-phosphate-isomerase of the present application may include any protein as long as it has an activity to convert glucose-6-phosphate to fructose-6-phosphate. Psychos-6-phosphate dephosphorylation enzyme of the present application may include any protein as long as it is a protein having an activity to convert psicose-6-phosphate into psicose. More specifically, the psicose-6-phosphate dephosphorylation enzyme may be a protein having an activity of irreversibly converting psicose-6-phosphate into psicose. The glucose-6-phosphate-isomerase of the present application may include any protein as long as it has an activity to convert glucose-6-phosphate to fructose-6-phosphate. Psychos-6-phosphate dephosphorylation enzyme of the present application may include any protein as long as it is a protein having an activity to convert psicose-6-phosphate into psicose. More specifically, the psicose-6-phosphate dephosphorylation enzyme may be a protein having an activity of irreversibly converting psicose-6-phosphate into psicose.
[162]
The enzyme included in the composition for producing psycho of the present application may exhibit a high conversion rate of psycho in one-pot enzymatic conversion using a plurality of enzymes and substrates at the same time.
[163]
[164]
The composition for production of psychosis of the present application may further include any suitable excipients commonly used in the composition for production of psychosis. Such excipients may include, but are not limited to, for example, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizing agents, and isotonic agents.
[165]
The composition for production of psychosis of the present application may further include a metal ion or a metal salt. In one embodiment, the metal ion may be a divalent cation, and specifically, may be one or more metal ions selected from the group consisting of Ni, Mg, Ni, Co, Mn, Fe, and Zn. More specifically, the composition for production of psychosis of the present application may further include a metal salt, and more specifically, the metal salt is NiSO 4 , MgSO 4 , MgCl 2 , NiCl 2 , CoSO 4 , CoCl 2 , MnCl 2 , MnSO 4 , FeSO 4 and ZnSO 4 It may be at least one selected from the group consisting of.
[166]
[167]
Another aspect of the present application provides a method for producing psychoses comprising the step of contacting fructose-6-phosphate with ribulose-phosphate 3-epimerase, a microorganism expressing the same, or a culture of the microorganism .
[168]
[169]
Specifically, the Psychos manufacturing method of the present application is
[170]
Contacting a culture of a microorganism expressing the ribulose-phosphate 3-epimerase to fructose-6-phosphate, the ribulose-phosphate 3-epimerase, or the microorganism expressing the ribulose-phosphate 3-epimerase to convert the fructose-6-phosphate to psicose-6-phosphate; and
[171]
Psychos-6-phosphate dephosphorylation enzyme in the psycho-6-phosphate, a microorganism expressing it, or a culture of the microorganism is contacted with psycho-6-phosphate to convert psycho-6-phosphate to psycho It may include the steps sequentially, but is not limited thereto.
[172]
[173]
In addition, before the step of converting the fructose-6-phosphate to psicose-6-phosphate, the method for producing psicose of the present application is glucose-6-phosphate-isomerase to glucose-6-phosphate (Glucose-6-phosphate). , by contacting a culture of a microorganism expressing the glucose-6-phosphate-isomerase or a microorganism expressing the glucose-6-phosphate-isomerase to convert the glucose-6-phosphate into fructose-6-phosphate It may include additional steps.
[174]
Before the step of converting the glucose-6-phosphate to fructose-6-phosphate, the method for producing psicose of the present application is phosphoglucomutase to glucose-1-phosphate, and the phosphoglucomutase. The method may further include converting the glucose-1-phosphate into glucose-6-phosphate by contacting a microorganism expressing the enzyme or a culture of the microorganism expressing the phosphoglucomutase.
[175]
Before the step of converting the glucose-6-phosphate to fructose-6-phosphate, the method for producing Psychos of the present application is a polyphosphate glucose kinase in glucose, a microorganism expressing the polyphosphate glucose kinase, or the poly The method may further include contacting a culture of a microorganism expressing a phosphate glucose kinase and polyphosphate to convert the glucose into glucose-6-phosphate.
[176]
Before the step of converting the glucose-1-phosphate to glucose-6-phosphate, the method for producing psychosis of the present application includes α-glucan phosphorylase, starch phosphorylase in starch, maltodextrin, sucrose or a combination thereof. , maltodextrin phosphorylase or sucrose phosphorylase; a microorganism expressing the phosphorylase; Alternatively, the method may further include converting the starch, maltodextrin, sucrose, or a combination thereof into glucose-1-phosphate by contacting a culture of a microorganism expressing the phosphorylase and phosphate.
[177]
Before the step of converting the starch, maltodextrin, sucrose, or a combination thereof into glucose-1-phosphate, the method for producing psicose of the present application includes α-amylase, pullulanase, starch, maltodextrin, sucrose or a combination thereof, isoamylase, glucoamylase or sucrase; a microorganism expressing the α-amylase, pullulanase, glucoamylase, sucrase or isoamylase; or contacting a culture of a microorganism expressing the α-amylase, pullulanase, glucoamylase, sucrase or isoamylase to convert the starch, maltodextrin, sucrose or a combination thereof into glucose. can do.
[178]
Ribulose-phosphate 3-epimerase, psicose-6-phosphate dephosphorase, α-glucan phosphorylase, phosphoglucomutase (or phosphomannomutase) used in the method for producing psicose of the present application ), glucose-6-phosphate isomerase, psicose-6-phosphate-3-epimerase (or ribulose-5-phosphate-3-epimerase), pullulanase (or isoamylase), 4 -α-glucanotransferase, polyphosphate glucose kinase, etc. may have little or no side reaction to the final product, Psychose.
[179]
The Psychos manufacturing method of the present application may be to decompose high-concentration starch to produce the optimum/maximum Psychos in a complex combination with phosphate-converting enzymes, and combine up to 8 types of enzymes to secure the maximum productivity of Psychos can be used by
[180]
First, glucan phosphorylase (glycogen phosphorylase, EC 2.4.1.1), an enzyme that decomposes starch and produces glucose-1-phosphate, specifically binds α-1,4-bound starch to glucose-1 -Produces glucose-1-phosphate. Second, phosphoglucomutase (EC 2.7.5.1) or phosphomannomutase (EC) that converts glucose-1-phosphate thus produced into glucose-6-phosphate 5.4.2.8) is used for the intermediate complex enzymatic reaction. The third enzyme used is glucose-6-phosphate isomerase (EC 5.3.1.9), which converts glucose-6-phosphate to fructose-6-phosphate. Fourth, it is possible to generate even psycho-6-phosphate in a reversible reaction by using the fructose-6-phosphate-3-epimerase, which is an enzyme that converts fructose-6-phosphate to psycho-6-phosphate of the present application. .
[181]
Additionally, in order to increase starch utilization, pullulanase (EC 3.2.1.41) or isoamylase to break down branch bonds of α-1,6 other than α-1,4 bonds of amylopectin (isoamylase EC 3.2.1.68) enzyme is used together, and glucanotransferase (4-alpha-glucanotransferase, EC 2.4.1.25) can be used to increase starch utilization of glucan phosphorylase. By binding oligosaccharides in the form of α-1,4 bonds to maltose or other oligosaccharides, which are relatively low activity substrates, the utilization rate of granular starch matrix can be increased. Additionally, polyphosphate-glucose phosphotransferase (EC 2.7.1.63) is used to produce additional psicose through a complex enzymatic reaction in the decomposed glucose after using starch.
[182]
[183]
In addition, in the method for producing psychosis of the present application, the contacting of the present application may be carried out at pH 5.0 to 9.0, specifically pH 6.0 to 8.0.
[184]
In the method for producing psychosis of the present application, the contacting of the present application may be carried out at a temperature of 40 °C to 80 °C, specifically 40 °C to 60 °C or 50 °C to 60 °C temperature.
[185]
In the method of manufacturing psychosis of the present application, the contact of the present application may be carried out for 2 hours to 24 hours, specifically 6 to 24 hours.
[186]
In the preparation method of the course of the present application, the contact of the present application may be carried out for a pH of 5.0 to 9.0, a temperature of 40°C to 80°C, and/or 2 hours to 24 hours. Specifically, the contacting may be carried out for a pH of 6.0 to 8.0, a temperature of 40°C to 60°C or 50°C to 60°C, and/or 6 hours to 24 hours.
[187]
Psychos manufacturing method of the present application may further include the step of purifying the Psychos. The purification of the present application is not particularly limited, and methods commonly used in the technical field of the present application may be used. Non-limiting examples include chromatography, fractional crystallization and ionic purification. The purification method may be performed only one, or two or more methods may be performed together. For example, the reaction product to produce psychosis can be purified through chromatography, and the separation of sugars by chromatography can be performed using the difference in weak binding force between the sugar to be separated and the metal ion attached to the ion resin. have.
[188]
In addition, the present application may further include performing bleaching, desalting, or both before or after the purification step of the present application. By performing the decolorization and / or desalting, it is possible to obtain a more purified psicose reactant without impurities.
[189]
Modes for carrying out the invention
[190]
Hereinafter, the present application will be described in detail with reference to examples, but this is only to help the understanding of the present application, and the scope of the present application is not limited thereto.
[191]
[192]
In the present application, amino acids may be represented by the following abbreviations or amino acid names:
[193]
[194]
[Table 1]
[195]
[196]
Example 1: Preparation of a recombinant expression vector for each enzyme and a transforming microorganism
[197]
[198]
In order to provide each enzyme required for the psychic production route of the present application, the gene of the heat-resistant enzyme was selected, and an enzyme having an amino acid sequence of SXMC (SEQ ID NO: 4) or GXXXXF (SEQ ID NO: 5) for additional high-activity epimerization activity their genes were selected (Table 2).
[199]
[200]
The genes of the selected amino acids are amplified using polymerase chain reaction (PCR) for each gene from gene synthesis or chromosomal DNA (genomic DNA) of each distribution strain, and each amplified DNA using DNA assembly methods It was inserted into a plasmid vector pET21a (Novagen) for E. coli expression, and a recombinant expression vector was prepared. The expression vector can be prepared by a conventional transformation method [Sambrook et al. 1989] to prepare a transformed microorganism by transforming each E. coli BL21 (DE3) strain.
[201]
[202]
Specifically, psicose-6-phosphate 3-epimerase of SEQ ID NOs: 6 to 26 was transformed into E. coli BL21 (DE3) strains, respectively. Among them, the transformed microorganisms of SEQ ID NOs: 19, 20, and 22 were deposited with the international depository organization, the Korea Microorganism Conservation Center (KCCM) on April 16, 2019 through the method prepared above, and KCCM12494P ( E.coil BL21(DE3)/ pET-CJ-fep19), KCCM12495P ( E.coil BL21(DE3)/pET-CJ-fep20), and KCCM12496P ( E.coil BL21(DE3)/pET-CJ-fep22) were assigned accession numbers, respectively.
[203]
[204]
Example 2: Preparation of Recombinant Enzyme
[205]
[206]
In order to prepare the recombinant enzyme, each transformed microorganism prepared in Example 1 was inoculated into a culture tube containing 5 ml of LB liquid medium, and seed culture was performed in a shaking incubator at 37 ° C until the absorbance at 600 nm became 2.0. did This seed culture was inoculated into a culture flask containing an LB liquid medium, and the main culture was performed. When the absorbance at 600 nm became 2.0, 1 mM IPTG was added to induce expression production of the recombinant enzyme. The stirring speed during the incubation process was 180 rpm and the culture temperature was maintained at 37 °C. The culture medium was centrifuged at 8,000×g at 4° C. for 20 minutes, and the cells were recovered. The recovered cells were washed twice with 50 mM Tris-HCl (pH 8.0) buffer, suspended in the same buffer, and then cells were disrupted using an ultrasonic cell disrupter. The cell lysate was centrifuged at 13,000×g at 4° C. for 20 minutes, and only the supernatant was taken. The recombinant enzyme was purified from the supernatant using His-tag affinity chromatography, dialyzed against a 50 mM Tris-HCl (pH 8.0) buffer, and then used for the reaction.
[207]
[208]
[209]
Example 3: Psychos-6-phosphate 3-epimerase modeling, psycho 3-epimerization activity confirmation and sequence comparison
[210]
[211]
The amino acid sequence of SEQ ID NO: 9, known as ribulose-phosphate-3-epimerase, was input to I-TASSER, Phyre2, and Galaxyweb server to analyze the protein structure.
[212]
As a result, it was confirmed that the enzyme has a TIM-barrel fold in which the β-sheet is centered and the α-helix structure surrounds it. barrel fold) and motifs I (VDG) and III (MXX-X'-PG) were selected (FIG. 1, Blue dotted circle (motif I), Red dotted circle (motif III) , example structure-model (SEQ ID NO: 9) )).
[213]
[214]
On the other hand, the previously known psicose-6-phosphate 3-epimerization enzymes (ADL69228, WP_034772999, WP_029098887; WO2018/129275, WO2018/112139) exhibit the activity of epimerizing psicose into fructose, thus producing psicose. will reduce the yield.
[215]
[216]
The present inventors predicted that structurally (MXXDPG) aspartic acid (D) has a negative charge and will affect the 3-epimerization activity of Psychos, thereby affecting the 3-epimerization activity of Psychos. Among motifs III (MXX-X'-PG) that are not expected to give, enzymes having N/K in which X' has a different charge from aspartic acid were selected (WP_085113038, PKM55438, WP_117016900).
[217]
[218]
As a result, it was confirmed that the psychocosm 3-epimerization activity may vary depending on the type of a specific motif in the enzyme.
[219]
[220]
Example 4: Confirmation of ribulose-phosphatase-3-epimerase psicose-6-phosphate conversion activity
[221]
[222]
The activity of ribulose-phosphate-3-epimerase, an enzyme that converts psicose-6-phosphate of the present application, is determined by mixing 50 mM fructose-6-phosphate or 20 mM glucose-1-phosphate with 50 mM Tris-HCl (pH 7.0) or 50 mM Sodium-posphate (pH 6-7) or 50 mM Potassium-posphate (pH 6-7) buffer, followed by phosphoglucomutase or phosphomannomutase and glucose-6-phosphate 0.1 unit/ml of isomerase, psicose-6-phosphate dephosphorase, and the recombinant ribulose-phosphate-3-epimerase prepared in Example 2 were each added, and at 45°C to 70°C for 1 to 24 hours. reacted.
[223]
[224]
To analyze the activity of psicose 3-epimerase, psicose was dissolved in 50 mM Tris-HCl (pH 7.0) or 50 mM Sodium-posphate (pH 6-7) or 50 mM Potassium-posphate (pH 6-7) buffer. After suspension at a concentration of 1% (w/v), 0.1 unit/ml of ribulose-phosphate-3-epimerase was added, and reacted at 45° C. to 70° C. for 1 to 24 hours. Using HPLC, the generation of glucose, fructose, and psicose was analyzed, and HPLC analysis was performed using a SP_0810 (Shodex) column and an Aminex HPX-87C (Bio-RAD) column at 80° C. as a mobile phase at a flow rate of 0.6 ml/min. It was carried out while flowing with a Refractive Index Detector (RID), and was confirmed by qualitative and quantitative evaluation of glucose, fructose, and psycho, which are general sugars produced by mixing with the above enzymes, respectively. For quantitative evaluation, the tolerance of the initial psychoses concentration ratio fructose conversion rate was set within 5% in consideration of LC sensitivity, rapid substrate conversion rate, and naturally occurring experimental error according to substrate concentration.
[225]
[226]
Table 2 distinguishes enzymes without psychoses 3-epimerization activity based on the presence or absence of MXXN/KPG and VDG motifs among several ribulose-phosphate-3-epimerization enzymes exhibiting psycho6-phosphate 3-epimerization activity From this, an enzyme containing a specific motif, specifically, the motif of SEQ ID NO: 1, does not include the motif of SEQ ID NO: 2, and instead contains the motif of SEQ ID NO: 3 at the same site is specific for psycho 6-phosphate It was confirmed that it has high epimerization activity. In addition, as confirmed in sequences A and B, it was confirmed that when Motif I and III are included, it can have high epimerization activity specifically for psicose 6-phosphate.
[227]
[228]
[Table 2]
[229]
[230]
In addition, it was confirmed that the enzyme having the amino acid sequence of SEQ ID NO: 20 of the present application has significantly lower fructose production compared to the conventionally known psicose-6-phosphate 3-epimerase (FIG. 2).
[231]
[232]
This suggests that the presence or absence of the motif plays an important role in the yield of allulose production.
[233]
[234]
In addition, in the case of the sequence comprising SEQ ID NO: 4 and/or SEQ ID NO: 5, it was confirmed that the phosphate 3-epimerization activity of psicose 6 was increased.
[235]
[236]
Example 4: Analysis of Psychos Production Activity through Complex Enzyme (Multi Enzyme) Reaction
[237]
[238]
Glucan phosphorylase, pullulanase, 4-alpha-glucanotransferase, phosphoglucomutase, glucose-6-phosphate isomerase, psicose-6-phosphate to produce psicose from maltodextrin The dephosphorylation enzyme and the fructose-6-phosphate-3-epimerase of the present application were reacted simultaneously (one-pot).
[239]
[240]
Specifically, 0.1 unit/ml of each of the seven enzymes was added to a solution containing 5% (w/v) maltodextrin in 1-5 mM MgCl 2 , 10-50 mM sodium phosphate pH 7.0. and reacted at a temperature of 50° C. for 12 hours.
[241]
After the reaction was completed, psychosis was analyzed in the reaction result using HPLC. HPLC analysis was performed using an Aminex HPX-87C (Bio-RAD) column at 80° C. as a mobile phase at a flow rate of 0.6 ml/min, and detection was performed with a Refractive Index Detector. As a result, it was confirmed that psychoses were generated from maltodextrin by a complex enzymatic reaction.
[242]
[243]
[Table 3]
[244]
[245]
From the above description, those skilled in the art to which the present application pertains will be able to understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present application should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description and equivalent concepts thereof.
[246]
[247]
accession number
[248]
Name of depositary institution: Korea Microorganism Conservation Center
[249]
Accession number: KCCM12494P
[250]
Deposit date: 20190416
[251]
[252]
Name of depositary institution: Korea Microorganism Conservation Center
[253]
Accession number: KCCM12495P

WE CLAIMS

A ribulose-phosphate 3-epimerase comprising 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.
[Claim 2]
According to claim 1, wherein the enzyme does not have the activity of converting psicose to fructose, or within 5%, the enzyme.
[Claim 3]
The enzyme according to claim 1, wherein the enzyme further comprises a motif consisting of the amino acid sequence of SEQ ID NO: 4 or 5.
[Claim 4]
The enzyme according to claim 1, wherein the enzyme comprises motif I at positions 173 to 184 from the N-terminal amino acid.
[Claim 5]
The enzyme according to claim 1, wherein the enzyme comprises motif III at positions 136 to 150 from the N-terminal amino acid.
[Claim 6]
The enzyme of claim 1, wherein the enzyme has a 3-epimerization activity of psicose-6-phosphate at a temperature of 50°C to 90°C.
[Claim 7]
The method of claim 1, wherein the enzyme is keusso grandma eggplant ( Chthonomonas ), geo Bacillus ( Geobacillus ), e HeLa ( Mahella ), Thermo unloading area tumefaciens ( Thermoanaerobacterium ), te the blood Nero bakteo ( Tepidanaerobacter ), I'll Aden urticae O ( Ardenticatenia ), Firmicutes ( Firmicutes ), Aribacillus ( Aeribacillus ), Ipulopiscium ( Epulopiscium ), and Thermoflavimicrobium ( Thermoflavimicrobium ) Enzyme from any one selected from the group consisting of.
[Claim 8]
The method of claim 1, wherein the enzyme is keusso grandma eggplant potassium di to Seah ( Chthonomonas calidirosea ) T49, geo Bacillus 8 ( Geobacillus sp . 8), geo Bacillus Thermo category Cronulla tooth ( Geobacillus thermocatenulatus ), e HeLa brother host Raleigh N-Sys ( Mahella australiensis ) 50-1 BON, Thermoanaerobacterium genus PSU-2 ( Thermoanaerobacterium sp . PSU-2), Thermoanaerobacterium thermosaccharolyticum ( Thermoanaerobacterium thermosaccharolyticum ), Tepidanerobacter syntropicus ( Tepidanaerobacter syntrophicus ), Ardenticatenia bacterium , Firmicutes bacterium HGW-Firmicutes-5 ( Firmicutes bacterium )HGW-Firmicutes-5), Aeribacillus pallidus , Ipulopicium genus SCG-B05WGA-EpuloA1 ( Epulopiscium sp. SCG-B05WGA-EpuloA1 ), and Thermoflavimic microbium dichotomicum ( Thermoflavimicrobium ) The enzyme that is derived from any one selected from the group consisting of
[Claim 9]
According to claim 1, wherein the enzyme is any one sequence selected from the group consisting of the amino acid sequence of SEQ ID NOs: 15 to 26 and an amino acid sequence having at least 26% identity with a region excluding motifs I and III among the amino acid sequence of which enzymes are composed.
[Claim 10]
A nucleic acid encoding the ribulose-phosphate 3-epimerase of any one of claims 1 to 9.
[Claim 11]
A transformant comprising the nucleic acid of claim 10.
[Claim 12]
In the composition for producing ribulose-phosphate 3-epimerase, a microorganism expressing the same, or a culture of the microorganism for psycho-6-phosphate, the ribulose-phosphate 3-epimerase is SEQ ID NO: 1 A composition for producing psycho-6-phosphate comprising a motif III consisting of the amino acid sequence of Motif I and SEQ ID NO: 3 consisting of the amino acid sequence of.
[Claim 13]
To fructose-6-phosphate (fructose-6-phosphate) to ribulose-phosphate 3-epimerase, a microorganism expressing it, or a method for producing psicose-6-phosphate comprising the step of contacting a culture of the microorganism In the following, the ribulose-phosphate 3-epimerase enzyme comprises the motif I consisting of the amino acid sequence of SEQ ID NO: 1 and the motif III consisting of the amino acid sequence of SEQ ID NO: 3 Psychos-6-phosphate production method.
[Claim 14]
Ribulose-phosphate 3-epimerase, a microorganism expressing the same, or a culture of the microorganism, and psicose-6-phosphate dephosphorylation enzyme, a microorganism expressing the same, or Psychos production comprising a culture of the microorganism In the composition for use, the ribulose-phosphate 3-epimerase enzyme comprises 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 The composition for producing psychosis.
[Claim 15]
15. The method of claim 14, wherein the composition comprises glucose-6-phosphate-isomerase, phosphoglucomutase, polyphosphate glucose kinase, α-glucan phosphorylase, starch phosphorylase, maltodextrin phosphorylase. or sucrose phosphorylase, α-amylase, pullulanase, isoamylase, α-glucanotransferase, glucoamylase, sucrase, and psicose-6-phosphate dephosphorylation enzyme selected from the group consisting of one or more enzymes; microorganisms expressing it; Or, the composition for producing Psychos, which further comprises a culture of the microorganism.
[Claim 16]
Contacting fructose-6-phosphate with ribulose-phosphate 3-epimerase, a microorganism expressing the same, or a culture of the microorganism; And Psychos-6-phosphoric acid prepared from the fructose-6-phosphate in the psychocos-6-phosphate dephosphorylation enzyme, a microorganism expressing the same, or a method for producing psycho comprising the step of contacting the culture of the microorganism In the following, the ribulose-phosphate 3-epimerase is a method for producing psychosis comprising a 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.
[Claim 17]
The method according to claim 16, wherein the manufacturing method further comprises obtaining a psicose prepared from psicose-6-phosphoric acid.