The present application relates to a novel enzyme for dephosphorylating psicose-6-phosphate in order to produce psicose, a composition for producing psicose comprising the same, and a method for manufacturing psicose using the same.
[2]
background
[3]
Psychose-3-epimerase (D-psicose-3-epimerase, EC 5.1.3.30) and tagatose-3-epimerase (D-tagatose-3-epimerase, EC 5.1.3.31) are fructose (D- fructose) is known as an enzyme capable of producing D-psicose by 3-epimerization (3-epimerization, 3-carbon epimerization). When psicose is produced by a single enzymatic reaction using the enzyme, a certain level of reaction equilibrium exists between fructose as a substrate and psicose as a product (product/substrate = about 20% to 35%). Therefore, there is a problem in that an additional purification process is required to separate and remove a relatively high concentration of fructose from the result of the enzymatic reaction in order to produce high-purity Psychos. In addition, fructose is a relatively expensive raw material compared to starch or glucose, and when fructose is produced as a raw material, the cost ratio of Psychos and Tagatose increases. Accordingly, various studies on the production of allulose and tagatose through a reaction using relatively economical starch or glucose have been published. (KR 10-2018-0004023, WO2018-112139, WO2017-059278, WO2018-129275).
[4]
On the other hand, Chan et al. (2008. Biochemistry. 47:9608-9617) described 3- for fructose-6-phosphate and D-psicose-6-phosphate. D-ribulose-5-phosphate-3-epimerase (EC 5.1.3.1) from Streptococcus pyogenes capable of performing an epimerization reaction ; and E. coli- derived psicose-6-phosphate-3-epimerase (D-psicose 6-phosphate-3-epimerase, EC 5.1.3.-) has been reported, but these enzymes do not have heat resistance and are therefore industrially There is a problem that it cannot be used.
[5]
DETAILED DESCRIPTION OF THE INVENTION
technical challenge
[6]
The present inventors have endeavored to develop a method capable of increasing the conversion yield to allulose economically and industrially. As a result, after converting sucrose or starch (eg, maltodextrin), which is an economical raw material, into psicose-6-phosphate, the Psychos of the present application performs a specific and irreversible reaction pathway for the psicose-6-phosphate. When psicose is produced using -6-phosphate dephosphorylation enzyme (psicose-6-phosphate phosphatase), a simultaneous enzymatic reaction (one-pot enzymatic conversions) that can use a plurality of enzymes involved in the psicose production pathway at the same time This application was completed by confirming that it is possible and can significantly increase the conversion rate of psychosis. The enzyme of the present application is a more suitable advantage in the production of psychosis compared to the existing known dephosphorylation enzyme of psicose-6-phosphate (Panoramic view of a superfamily of phosphatases through substrate profiling, PNAS, 06.04. 2015, Huang, etc.) has
[7]
means of solving the problem
[8]
One object of the present application is to provide a dephosphorylation enzyme of psicose-6-phosphate.
[9]
Another object of the present application is to provide a nucleic acid encoding a dephosphorylation enzyme of psicose-6-phosphate.
[10]
Another object of the present application is to provide a transformant comprising a nucleic acid encoding a dephosphorylation enzyme of psicose-6-phosphate.
[11]
Another object of the present application is to provide a composition for production of psychosis including a phosphatic-6-phosphate dephosphorylation enzyme, a microorganism expressing the same, or a culture of the microorganism.
[12]
Another object of the present application is to contact psicose-6-phosphate dephosphorylation enzyme, a microorganism expressing the same, or a culture of the microorganism with psicose-6-phosphate to convert psicose-6-phosphate into psicose. It is to provide a method for producing a psycho course comprising the step of converting.
[13]
Effects of the Invention
[14]
Each of the novel enzymes and combinations thereof of the present application has heat resistance, so it is possible to industrially perform the route of converting psicose-6-phosphate to psicose, and economical raw materials such as glucose or starch (eg, maltodextrin) are used. This enables the progress of the psychosynthesis pathway, and enables the production of psychosis by the irreversible reaction pathway, psycho-6-phosphate dephosphorylation reaction, thereby significantly increasing the conversion rate to psychosis. Heat-resistant enzymes are commercially effective in preventing microorganisms and reacting with high-concentration substrates, so the effect is further added. In addition, the present manufacturing method can simplify or remove the separation and purification process including the high concentration of the reaction product including the high concentration of the psycho by increasing the psycho-conversion rate, so the manufacturing method is simple and economical. In particular, it is possible to avoid or minimize separation using SMB, thereby maximizing separation efficiency and yield.
[15]
Best mode for carrying out the invention
[16]
Hereinafter, the contents 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.
[17]
[18]
One aspect of the present application for achieving the above object is to provide a dephosphorylation enzyme of psicose-6-phosphate.
[19]
Specifically, in this application of course Im-6-phosphate dephosphorylation enzyme Arisa micro Bacillus ( Alicyclobacillus ) , ahmiko ratop sheath ( Amycolatopsis ) , know which reel Nea ( Anaerolinea ) , the booth (in ahreuke Ogle Archaeoglobus ), Bacillus ( Bacillus ), Caldicellulosiruptor ( Caldicellulosiruptor ), Caldilinea ), Caldithrix ( Caldithrix ), Carboxydocella ), Carboxydothermus ( Carboxydothermus ), Chloroflexi ( Chloroflexi ), Deflubitoga ( Defluviitoga), Deinococcus ( Deinococcus ), Desulfurococcus ( Desulfurococcus ), Dictyoglomus ( Dictyoglomus ), Effusibacillus ( Effusibacillus ), Pervidobacterium ( Fervidobacterium ), Geobacillus ( Geobacillus ), Halo ( Halococcus ), Hydrogenivirga ( Hydrogenivirga ), Hydrogenobacter ( Hydrogenobacter ), Hyperthermus ( Hyperthermus ), Kosmotoga ( Kosmotoga ), Marinitoga ( Marinitoga ), Meiothermus ( Meiothermus ), Mesotho Ga ( Mesotoga ), Metallosphaera ( Metallosphaera ), Metanocella ( Methanocella ), meta furnace kokoyi des ( Methanococcoides ), in meta-angry emptying ( Methanohalobium ), meta furnace booth ( Methanolobus ), meta labor LE key ( Methanosarcina ), meta nosseo mousse ( Methanothermus ), Petro toga ( Petrotoga ), a peak filler's ( Picrophilus ), pseudo-no carboxylic Dia ( Pseudonocardia ), Pyrococcus ( Pyrococcus ), fatigue Dick tium ( Pyrodictium ), also written Moose ( Rhodothermus ), Sligo Kea ( Slackia ), moose (as a star blood Staphylothermus ), installed in captivity Bus ( Sulfolobus ), Thermanaerothrix ( Thermanaerothrix )), Thermoanaerobacter ( Thermoanaerobacter ), Thermoanaerobacterium ( Thermoanaerobacterium ), Thermobifida ( Thermobifida ), Thermococcus ( Thermococcus ), Thermocrinis ( Thermocrinis ), Thermoflexus ( Thermoflexus ), Thermotoga ( Thermotoga ), sseomeoseu ( Thermus ), or true-Blow ( Truepera ) in the origin may be a dephosphorylation enzyme of course Im-6-phosphate, more specifically, Arisa micro Bacillus know sword Darius ( Alicyclobacillus acidocaldarius ), Arisa micro Bacillus Tenchon Gensis ( Alicyclobacillus tengchongensis ), Amycolatopsis thermoflava ( Amycolatopsis thermoflava )), Anaerolinea thermolimosa , Anaerolinea thermophila , Archaeoglobus fugidus , Archaeoglobus fugidus , Archaeoglobus profundus , Archaeoglobus profundus , Robus Beneficus ( Archaeoglobus veneficus ), Bacillus licheniformis , Caldicellulosiruptor bescii , Caldilinea aerophila , Caldithrix abyssi , Caldithrix abyssi ), Carboesophageal sp. ULO1 ( Carboxydocella sp. ULO1 ), Carboxydothermus ferrireducens ), Chloroplexibacterium 54-19 ( 54-19 bacterium Chloroflexi ), to Tunisia during the flu Vito N-Sys ( Defluviitoga tunisiensis ), Day-no caucus ah Sagittarius ( Deinococcus aerius ), Day-no caucus sick Chen System ( Deinococcus apachensis ), Day-no caucus aka Antilles ( Deinococcus aquatilis ), Deinococcus geothermalis ( Deinococcus geothermalis ), Deinococcus hopiensis ( Deinococcus hopiensis ), Deinococcus maricopensis ( Deinococcus maricopensis ), Deinococcus murrayi ( Deinococcus murrayi ), Deinococcus reticulithermic ( Deinococcus reticulitermitis ), Deinococcus wulumuqiensis ( Deinococcus wulumuqiensis ), Daytococcus sp. Leaf326 ( Deinococcus sp. Leaf326), Deinococcus phoenicis ( Deinococcus phoenicis ), Deinococcus proteolyticus ( Deinococcus proteolyticus ), Deinococcus sp. 17bor-2 ( Deinococcus sp. 17bor-2 ), Deinococcus sp. NW-56 ( Deinococcus sp. NW-56 ), Deinococcus sp. RL ( Deinococcus sp. RL ), Deinococcus sp. YIM 77 859 ( Deinococcus SP. YIM 77 859 ), desul furosemide Hancock Syracuse Temuco Saskatchewan ( Desulfurococcus mucosus ), Dick Moose Tours jidum to Tio posts ( Dictyoglomus turgidum ), Espoo City Bacillus poly kid ( Effusibacillus pohliae ), Fernand Non-Gaming Te Solarium gondeu and Nence ( Fervidobacterium gondwanense ), Fervidobacterium islandicum ( Fervidobacterium islandicum )), Fervidobacterium nodosum ( Fervidobacterium nodosum ), Fervidobacterium pennivorans ( Fervidobacterium pennivorans ), Geobacillus sp. ( Geobacillus sp. ), Geobacillus stearothermophilus ), Halococcus Sarifodinae ( Halococcus salifodinae ), Hydrogenivir sp. R1-1-128-5 ( Hydrogenivirga sp. 128-5-R1-1 ), Gino dihydro bakteo hydrogenocarbonate pillar's ( Hydrogenobacter hydrogenophilus ), Gino dihydro bakteo Thermo filler's ( Hydrogenobacter thermophilus ), Hi FER Terre mousse butynyl Lee coarse ( Hyperthermus butylicus ), Kosmotoga arenicorallina ( Kosmotoga arenicorallina ), Kosmotoga olearia ( Kosmotoga olearia ), Marinitoga piezophylla ( Marinitoga piezophila ), Meiothermus cerbereus , Meiothermus chliarophilus , Meiothermus ruber , Meiothermus Silvanus , Meiothermus Taiwanensis ( Meiothermus taiwanensis ), Meiothermus timidus ( Meiothermus timidus ), Meiothermus rufus ( Meiothermus rufus ), Mesotoga infera , Metallosphaera sedula , Meta Nocella conradii ( Methanocella conradii ), Methanococcoides methylutens ( Methanococcoides methylutens ), Methanohalobium evestigatum ( Methanohalobium evestigatum )), Metanorobus tindarius ( Methanolobus tindarius ), Methanosarcina sicilia ( Methanosarcina sicilia ), Methanothermus fervidus ( Methanothermus fervidus ), Petrotoga mobilis ( Petrotoga mobilis ), Pycrophilus toridus ( Picrophilus torridus ), Pseudonocardia thermophila ), Pyrococcus furiosus , Pyrococcus furiosus , Pyrodictium occultum ), Rhodothermus marinus ) Trini reducens ( Slackia heliotrinireducens ), Staphylothermus marinus ( Staphylothermus marinus ), Sulfolobus acidocaldarius ( Sulfolobus acidocaldarius )) (Matrix with the write mana daksen cis Thermanaerothrix daxensis ), written together Nero bakteo sp. ( Thermoanaerobacter sp .), Written together Nero bakteo Thermo hydrosulfite Fury kusu ( Thermoanaerobacter thermohydrosulfuricus ), written together Gary (a Nero bakteo non Thermoanaerobacter wiegelii ), Thermoanerobacterium silanolyticum ( Thermoanaerobacterium xylanolyticum ), Thermobifida halotolerans ( Thermobifida halotolerans ), Thermococcus celer ( Thermococcus celer ), Thermococcus litoralis ( Thermococcus litoralis ), Thermococcus litoralis Propondus ( Thermococcus profundus ), Thermocrinis minervae ( Thermocrinis minervae ), Thermocrinis ruber ( Thermocrinis ruber ), Thermopraxus hugenholt paper ( Thermoflexus hugenholtzii ), Thermotoga lettingae ( Thermotoga lettingae ), Thermotoga neapolitana ( Thermotoga neapolitana ), Thermotoga petrophilia ( Thermotoga petrophilia ), Somers amyloliquefaciens ( Thermus amyloliquefaciens ), Somers philiformis ( Thermus filiformis ), Thermophilus thermophilus ( Thermus thermophilus ), or Truepera radiovictrix ( Truepera radiovictrix ) It may be derived from, but is not limited thereto.
[20]
[21]
In the present application, "psicose-6-phosphate" is also known as allulose 6-phosphate, "psicose-6-phosphate dephosphorylation enzyme" is thy It refers to an enzyme that dephosphorylates the phosphate group of course-6-phosphate to produce psicose.
[22]
Psychos-6-phosphate dephosphorylation enzyme of the present application can be used to produce Psychos with high efficiency by complexly combining with starch processing enzymes and phosphate sugar converting enzymes in manufacturing Psychose by decomposing high-concentration starch. can
[23]
[24]
Psychos-6-phosphate dephosphorylation enzyme of the present application may be an amino acid sequence of any one of SEQ ID NOs: 1 to 222, or may include an amino acid sequence having at least 70% identity with the amino acid sequence.
[25]
In addition, SEQ ID NOs: 1, 6, 9, 12, 26, 29,38 to 43, 45 to 53,56, 57,59, 60, 64 to 66, 69, 70, 72, 76, 80, 81, 91 to 93, 95, 99 to 103, 113, 114, 116,117, 131, 134, 136, 142, 145, 146, 148, 164, 167, 169, 172, 177, 184 to 187, 189, 191, 192, 211, 217, and any one of the amino acid sequence of 221, or may include an amino acid sequence having at least 70% identity to the amino acid sequence, but is not limited thereto.
[26]
More specifically, the amino acid sequence of any one of SEQ ID NOs: 26, 29, 53, 56, 60, 70, 76, 80, 81, 116, 117, 131, 134, 145, 167, 185, 186, and 191; , or may include an amino acid sequence having at least 70% identity with the amino acid sequence, but is not limited thereto.
[27]
In addition, sequences having the same activity as the amino acid sequence may be included without limitation. In addition, it may include an amino acid sequence of any one of SEQ ID NOs: 1 to 222 or an amino acid sequence having 80% or more 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: 1 to 222 and the sequence. Or it may include amino acids having the same identity. In addition, it is obvious that proteins having amino acid sequences in which some sequences are deleted, modified, substituted or added are also 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.
[28]
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.
[29]
[30]
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.
[31]
The terms homology and identity can often be used interchangeably.
[32]
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.
[33]
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) 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.
[34]
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.
[35]
[36]
Psychos-6-phosphate dephosphorylation enzyme of the present application may be an enzyme that selectively dephosphorylates psicose-6-phosphate. Specifically, psicose-6-phosphate dephosphorylation enzyme is psicose-6-phosphate and glucose-1-phosphate (D-glucose-1-phosphate), glucose-6-phosphate (D-glucose-6-phosphate), or fructose When D-fructose-6-phosphate is mixed, it may be an enzyme that dephosphorylates psicose-6-phosphate. As an example, psicose-6-phosphate dephosphorylation enzyme produces the same amount of psicose-6-phosphate, glucose-1-phosphate, glucose-6-phosphate, and fructose-6-phosphate when mixing The dephosphorylation rate may be 1% or more, 10% or more, or 30% or more. Due to the selective activity of the psychoses-6-phosphate dephosphorase of the present application as described above, it is possible to exhibit a high psycho-conversion rate in a one-pot enzymatic conversion using a plurality of enzymes and substrates at the same time.
[37]
[38]
The enzyme itself or the DNA expressing it of the present application may be transformed into a strain, cultured to obtain a culture, crushed, and purified through a column or the like. As the transformation strain, Escherichia coli , Corynebacterium glutamicum , Aspergillus oryzae , Saccharomyces cerevisiae , Yarrowia Lee Politica ( Yarrowia lipolytica ), Pichia pastoris ( Pichia pastoris ) or Bacillus subtilis ( Bacillus subtilis ), but is not limited thereto, there may be a possibility of transformation in a GRAS (Generally Recognized as Safe) strain in the future .
[39]
[40]
Another aspect of the present application is to provide a nucleic acid encoding the dephosphorylation enzyme of the psycho-6-phosphate, or a vector including the nucleic acid.
[41]
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 (References : Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).
[42]
The nucleic acid encoding the enzyme of the present application may be a DNA or RNA sequence in which nucleotides as a unit are covalently linked, and specifically, when DNA conversion of the amino acid sequence of SEQ ID NOs: 1 to 222 (amino acids are modified with 61 codons) ) can be any one nucleotide sequence of all possible numbers, and more specifically, 90% or more, 95 % or more, 97% 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 the amino acid sequence of any one of SEQ ID NOs: 1 to 222, 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 be composed of all the number of DNA codons that can be translated into SEQ ID NOs: 1 to 222 amino acid sequence, but is not limited thereto.
[43]
[44]
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. .
[45]
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 with 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 wash conditions for normal Southern hybridization at 60 °C , 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.
[46]
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.
[47]
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 depending on the purpose.
[48]
The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementarity, and the parameters are well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).
[49]
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 allow expression of a target 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 a suitable host cell, the vector can replicate or function independently of the host genome, and can be integrated into the genome itself.
[50]
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 a phage vector or a cosmid vector, and pBR-based, pUC-based, pBluescriptII-based plasmid vectors may be used as plasmid vectors. , pGEM-based, pTZ-based, pCL-based, 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.
[51]
[52]
Another aspect of the present application provides a transformant comprising a vector comprising a nucleic acid encoding each enzyme of the present application or a nucleic acid encoding the enzyme of the present application.
[53]
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 psicose-6-phosphate dephosphorylation enzyme of the present application is It may refer to a microorganism recombinant to be expressed. For example, it is transformed with a vector containing a nucleic acid encoding a psicose-6-phosphate dephosphorylation enzyme, or a nucleic acid encoding a psicose-6-phosphate dephosphorylation enzyme, and the psicose-6-phosphate dephosphorylation enzyme is transformed. It refers to a host cell or microorganism capable of expressing a phosphorylation enzyme. For the purpose of the present application, the psicose-6-phosphate dephosphorylation enzyme expressed by the transformant may be composed of the amino acid sequence of any one of SEQ ID NOs: 1 to 222, but is not limited thereto.
[54]
[55]
In the present application, the term "transformation" refers to introducing a vector containing a nucleic acid encoding a dephosphorylation enzyme of psycho-6-phosphate 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 make 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 dephosphorylation enzyme of psycho-6-phosphate of the present application. As long as the nucleic acid can be introduced and expressed into a host cell, it may be introduced in any form. 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 operably linked to the nucleic acid, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication. In addition, the nucleic acid may be introduced into 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.
[56]
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
[57]
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 the 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 ? may be used. In an environment treated with a selective agent, only the cells expressing the selectable marker survive or exhibit other expression traits, so that the transformed cells can be selected.
[58]
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, and lithium acetate-DMSO method, but is not limited thereto.
[59]
As the host cell, it is preferable to use a host having a 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, and 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.
[60]
More specifically, the transformants of the present application are E. coli BL21(DE3)/pET-CJ-ap1 to E. coli BL21(DE3)/pET-CJ-ap222, a total of 222.
[61]
[62]
Another aspect of the present application is to provide a composition for production of psychosis including a phosphatic-6-phosphate dephosphorylation enzyme, a microorganism expressing the same, or a culture of the microorganism. Specifically, the composition may further include a phosphate sugar, and more specifically, may further include psicose-6-phosphate as a substrate, but is not limited thereto.
[63]
The composition for production of psychos of the present application includes a psychophysic-6-phosphate dephosphorylation enzyme, a microorganism expressing the enzyme, or the enzyme, which dephosphorylates Psychos-6-phosphate to produce Psychos. By including a culture of microorganisms, it may be to produce a psicose through dephosphorylation of psicose-6-phosphate.
[64]
[65]
In addition, the composition for production of psychos of the present application is an enzyme and/or substrate [(i) starch, maltodextrin, sucrose or a combination thereof involved in the production pathway of psychosis of the present application; (ii) phosphate or polyphosphate or other phosphorylated compounds; (iii) fructose-6-phosphate-3-epimerase; (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, alpha-glucano transferase, polyphosphate glucokinase or sucrase]; Microorganisms expressing an enzyme involved in the psycho-manufacturing pathway; Or it may further include a culture of microorganisms expressing an enzyme involved in the production pathway of the psycho. However, this is an example, and if it is possible to produce psicose using the psicose-6-phosphate dephosphorylation enzyme 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
[66]
Specifically, the fructose-6-phosphate-3-epimerase may include any protein as long as it has an activity to convert fructose-6-phosphate to psicose-6-phosphate. The glucose-6-phosphate-isomerase may include any protein as long as it has an activity of converting glucose-6-phosphate into fructose-6-phosphate. The phosphoglucomutase (EC 5.4.2.2) may include any protein as long as it has an activity to convert glucose-1-phosphate to glucose-6-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 (isoamylase, EC 3.2.1.68), 4-α-glucanotrens Perase (4-α-glucanotransferase, EC 2.4.1.25) and glucoamylase (EC 3.2.1. 3) may include any protein as long as it has an activity to convert starch or maltodextrin into debranched maltooligosaccharide or glucose. The sucrase (EC 3.2.1.26) may include any protein as long as it has an activity of converting sucrose into glucose. Polyphosphate glucokinase (polyphosphate glucokinase, EC 2.7.1.63) may include any protein as long as it has an activity to convert phosphate of polyphosphate to glucose to glucose-6-phosphate.
[67]
Psychos-6-phosphate dephosphorase, α-glucan phosphorylase, phosphoglucomutase (or phosphomannomutase), glucose-6-phosphate isomerase contained in the composition for production of psicose of the present application , psicose-6-phosphate-3-epimerase (or ribulose-5-phosphate-3-epimerase), pullulanase (or isoamylase), 4-α-glucanotransferase, Polyphosphate glucose kinase and the like may have little or no side reaction to the final product, Psychose.
[68]
[69]
The composition for production of psycho of the present application may appropriately include a plurality of enzymes and substrates thereof for producing psycho, as well as a dephosphorylation enzyme of psicose-6-phosphate, and even in an environment in which several enzymes exist, the present application The dephosphorylation enzyme of psicose-6-phosphate has the effect of selectively and irreversibly producing psicose from psicose-6-phosphate.
[70]
[71]
The composition for production of Psychos of the present application may further include any suitable excipients commonly used in the composition for production of Psychos. Such excipients may include, but are not limited to, for example, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizing agents and isotonic agents.
[72]
The composition for producing psycho 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.
[73]
[74]
Another aspect of the present application is to contact the psychic-6-phosphate dephosphorylation enzyme, a microorganism expressing it, or a culture of the microorganism with psicose-6-phosphate to convert psicose-6-phosphate into psicose. It is to provide a method for manufacturing psychosis, including the step of converting.
[75]
Specifically, the method for producing psychos of the present application may be to produce psychos by contacting a psychoses-6-phosphate dephosphorylation enzyme, a microorganism expressing the same, or a culture of the microorganisms with psychos-6-phosphate, but , but not limited thereto.
[76]
[77]
In the method of the present application, before the step of converting psicose-6-phosphate to psicose, fructose-6-phosphate to fructose-6-phosphate, 3-epimerase, the fructose-6- By contacting a culture of a microorganism expressing a phosphate-3-epimerase or a microorganism expressing the fructose-6-phosphate-3-epimerase, the fructose-6-phosphate is converted into psicose-6-phosphate. It may additionally include the step of
[78]
In the method of the present application, before the step of converting fructose-6-phosphate to psicose-6-phosphate, glucose-6-phosphate-isomerase to glucose-6-phosphate, the glucose-6 -Contacting a culture of a microorganism expressing a phosphate-isomerase or a microorganism expressing the glucose-6-phosphate-isomerase to convert the glucose-6-phosphate into fructose-6-phosphate. can
[79]
In the method of the present application, before the step of converting glucose-6-phosphate into fructose-6-phosphate, phosphoglucomutase and the phosphoglucomutase are expressed in glucose-1-phosphate. It may further include the step of converting the glucose-1-phosphate into glucose-6-phosphate by contacting the microorganism or a culture of a microorganism expressing the phosphoglucomutase.
[80]
In the method of the present application, before the step of converting glucose-6-phosphate into fructose-6-phosphate, a polyphosphate glucose kinase on glucose, a microorganism expressing the polyphosphate glucose kinase, or the polyphosphate glucose phosphorylation The method may further comprise converting the glucose into glucose-6-phosphate by contacting a culture of a microorganism expressing the enzyme, and polyphosphate.
[81]
The method of the present application includes α-glucan phosphorylase, starch phosphorylase, maltodextrin in starch, maltodextrin, sucrose or a combination thereof before the step of converting glucose-1-phosphate into glucose-6-phosphate. 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.
[82]
Before the step of converting the starch, maltodextrin, sucrose or a combination thereof into glucose-1-phosphate, the method of the present application includes α-amylase, pullulanase, isoamylase, starch, maltodextrin, sucrose or a combination thereof, 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.
[83]
Psychos-6-phosphate dephosphorase, 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-glucanotransferase, poly Phosphate glucose kinase and the like may have little or no side reaction to the final product, Psychose.
[84]
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 a maximum of 8 types of enzymes are combined to secure the maximum productivity of Psychos. can be used by
[85]
First, glucan phosphorylase (glycogen phosphorylase, EC 2.4.1.1), an enzyme that breaks down starch and produces glucose-1-phosphate, specifically binds α-1,4-bound starch to glucose-1 -Produces phosphoric acid (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, utilize ribulose-5-phosphate-3-epimerase or psicose-6-phosphate-3-epimerase, which is an enzyme that converts fructose-6-phosphate to psicose-6-phosphate, as described above. Thus, it is possible to generate up to psycho-6-phosphate of a reversible reaction. Although it is impossible to produce more than a certain amount by a reversible reaction from starch to psicose-6-phosphate, high yield of psicose when using psicose-6-phosphate phosphatase included in the present invention production is possible
[86]
Additionally, in order to increase starch utilization, pullulanase (EC 3.2.1.41) or isoamylase to break down the branch bonds of α-1,6 other than α-1,4 bonds of amylopectin (isoamylase EC 3.2.1.68) The enzyme is used together, and glucanotransferase (4-alpha-glucanotransferase, EC 2.4.1.25) is used to increase the 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 from the decomposed glucose after using starch, thereby securing the maximum psicose conversion rate.
[87]
[88]
In addition, in the manufacturing method 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.
[89]
In the manufacturing method 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.
[90]
In the manufacturing method of the present application, the contacting of the present application may be carried out for 2 hours to 24 hours, specifically 6 to 24 hours to 120 hours.
[91]
In the manufacturing method of the present application, the contacting 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 to 120 hours.
[92]
The 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 alone, or two or more methods may be performed together. For example, it is possible to purify the reaction product to produce psychosis 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.
[93]
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.
[94]
Modes for carrying out the invention
[95]
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.
[96]
[97]
In the present application, amino acids may be represented by the following abbreviations or amino acid names:
[98]
[Table 1]
[99]
[100]
Example 1: Preparation of recombinant expression vectors and transformed microorganisms for each enzyme
[101]
Heat-resistant genes were selected to provide each enzyme required for the psychic production pathway of the present application, and summarized in Table 2 below.
[102]
[Table 2]
[103]

[104]

[105]

[106]

[107]

[108]

[109]
[110]
The genes of the selected amino acids are amplified using polymerase chain reaction (PCR) for each gene from gene synthesis or genomic DNA of each pre-distribution strain, and each amplified DNA is restriction enzymes NdeI and XhoI or Sal I was inserted into the E. coli expression plasmid vector pET21a (Novagen) to prepare a recombinant expression vector. 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.
[111]
Specifically, psycho-6-phosphate dephosphorylation of SEQ ID NOs: 26, 29, 53, 56, 60, 70, 76, 80, 81, 116, 117, 131, 134, 145, 167, 185, 186, and 191 Enzymes were transformed into E. coli BL21(DE3) strains, respectively, and E.coil BL21(DE3)/pET-CJ-ap26, E.coil BL21(DE3)/pET-CJ-ap29, E.coil BL21 (DE3)/pET-CJ-ap53, E.coil BL21(DE3)/pET-CJ-ap56, E.coil BL21(DE3)/pET-CJ-ap60, E.coil BL21(DE3)/pET-CJ- ap70, E.coil BL21(DE3)/pET-CJ-ap76, E.coil BL21(DE3)/pET-CJ-ap80, E.coil BL21(DE3)/pET-CJ-ap81, E.coil BL21(DE3) )/pET-CJ-ap116, E.coilBL21(DE3)/pET-CJ-ap117, E.coil BL21(DE3)/pET-CJ-ap131, E.coil BL21(DE3)/pET-CJ-ap134, E.coil BL21(DE3)/pET-CJ -ap145, E.coil BL21(DE3)/pET-CJ-ap167, E.coil BL21(DE3)/pET-CJ-ap185, E.coil BL21(DE3)/pET-CJ-ap186, and E.coil BL21 A transformed microorganism named (DE3)/pET-CJ-ap191 was prepared.
[112]
The transformed microorganisms prepared above were deposited at the Korea Center for Conservation of Microorganisms (KCCM), an international depository organization, as of November 14, 2018, and KCCM12390P ( E.coil BL21(DE3)/pET-CJ-ap26), KCCM12391P ( E.coil BL21) (DE3)/pET-CJ-ap29), KCCM12392P( E.coil BL21(DE3)/pET-CJ-ap53), KCCM12393P( E.coil BL21(DE3)/pET-CJ-ap56), KCCM12394P( E.coil BL21(DE3)/pET-CJ-ap60), KCCM12395P( E.coil BL21(DE3)/pET-CJ-ap70), KCCM12396P( E.coil BL21(DE3)/pET-CJ-ap76), KCCM12397P( E. coil BL21(DE3)/pET-CJ-ap80), KCCM12398P( E.coil BL21(DE3)/pET-CJ-ap81), KCCM12399P( E.coil BL21(DE3)/pET-CJ-ap116), KCCM12400P( E .coilBL21(DE3)/pET-CJ-ap117), KCCM12401P( E.coil BL21(DE3)/pET-CJ-ap131), KCCM12402P( E.coil BL21(DE3)/pET-CJ-ap134), KCCM12403P( E. coil BL21(DE3)/pET-CJ-ap145), KCCM12404P( E.coil BL21(DE3)/pET-CJ-ap167), KCCM12405P( E.coil BL21(DE3)/pET-CJ-ap185), KCCM12406P( E .coil BL21(DE3)/pET-CJ-ap186) and KCCM12407P ( E.coil BL21(DE3)/pET-CJ-ap191) were each given an accession number.
[113]
[114]
Example 2: Preparation of Recombinant Enzyme
[115]
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 broth, 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 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.
[116]
[117]
Example 3: Analysis of phosphate dephosphorylation activity and psicose-6-phosphate dephosphorylation activity
[118]
After preparing psicose-6-phosphate from fructose-6-phosphate, the activity of psicose production was confirmed by psicose-6-phosphate dephosphorylation enzyme, or by adding 3 enzymes additionally with glucose-1-phosphate as the initial substrate to psicose. was detected and the activity was measured. Here, the three enzymes are phosphoglucomutase (EC 2.7.5.1) or phosphomannomutase (EC 5.4) that converts glucose-1-phosphate to glucose-6-phosphate. .2.8) and glucose-6-phosphate isomerase (EC 5.3.1.9), which converts glucose-6-phosphate to fructose-6-phosphate. Mixing ribulose-5-phosphate-3-epimerase or psicose-6-phosphate-3-epimerase, which is an enzyme that converts phosphoric acid to psicose-6-phosphate, with each of 222 dephosphorylation enzymes of the present application It was confirmed by qualitative and quantitative evaluation of glucose, fructose, and psycho, which are general sugars produced by the
[119]
[120]
Specifically, 50 mM fructose-6-phosphate or 20 mM glucose-1-phosphate was mixed with 50 mM Tris-HCl (pH 7.0) or 50 mM Sodium-posphate (pH 6-7) or 50 mM Potassium-posphate (pH 6- 7) After suspending in a buffer solution, phosphoglucomutase or phosphomannomutase, glucose-6-phosphate isomerase and ribulose-5-phosphate-3-epimerase or psicose-6-phosphate-3 -Epimerase and 222 kinds of recombinant psicose-6-phosphate dephosphorase prepared in Example 2 were each added 0.1 unit/ml, 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 the HPLC analysis was performed using an 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 , and it was detected with a Refractive Index Detector (RID).
[121]
[122]
Then, in the prepared mixture of glucose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate and psicose-6-phosphate, the dephosphorylation activity of the dephosphorylation enzyme of psicose-6-phosphate of the present application and The rate of course-6-phosphate specific (selective) dephosphorylation was determined.
[123]
Specifically, 0.1 unit/ml of each of the dephosphorylation enzyme of psycho-6-phosphate of the present application and 5 mM MgCl 2 (or MgSO 4 ) 1% (w/v) glucose-6-phosphate, glucose-1 -Phosphoric acid, fructose-6-phosphoric acid, and psicose-6-phosphoric acid were added to a mixture and reacted at 50° C. for 12 hours, and then the reaction product was analyzed using HPLC. HPLC analysis was performed using an Aminex HPX-87C (Bio-RAD) column at 80 °C while flowing as a mobile phase at a flow rate of 0.6 ml/min. Psychos and other sugars (fructose and glucose) generated by Refractive Index Detector were detected. did.
[124]
[125]
As a result, high selective dephosphorylation activity was confirmed for psicose-6-phosphate in 69 of all 222 enzymes, and weak selective dephosphorylation activity of psicose-6-phosphate was confirmed in 45 enzymes. In 108 enzymes, selective psicose-6-phosphate dephosphorylation titers could not be confirmed. However, dephosphorylation activity was confirmed in 221 enzymes except for one out of 222 enzymes, of which 113 enzymes showed high dephosphorylation activity, and low dephosphorylation activity was confirmed in 108 enzymes. On the other hand, the enzyme of SEQ ID NO: 54, which was confirmed to have no dephosphorylation titer, is known as a dephosphorylation enzyme, but was actually confirmed to have no dephosphorylation activity. The results are summarized in Table 3 below.

Claims
[Claim 1]
Alicyclobacillus, Amycolatopsis, Anaerolinea, Archaeoglobus, Bacillus, Caldicellulosiruptor, Caldilinea, Caldithrix, Carboxydocella, Carboxydothermus, Chloroflexi, Defluviitoga , Deinococcus, Hydrolococcus, Hydrolocusibacter, Geobacurococcus, Dictyoglomus, Effusibacother, Hydroga Mesotoga, Metallosphaera, Methanocella, Methanococcoides, Methanohalobium, Methanolobus, Methanosarcina, Methanothermus, Petrotoga, Picrophilus, Pseudonocardia, Pyrococcus, Pyrodictium, Rhodothermus, Slackia, Staphylothermus, Sulferoanabus, Thermifanaerocoanabus, Thermifanaerocoanabus, Thermus, and TrueperaA dephosphorylation enzyme of psycho-6-phosphate derived from any one selected from the group consisting of the genus.
[Claim 2]
The method of claim 1, wherein the enzyme is Alicyclobacillus acidocaldarius; ULO1, Carboxydothermus ferrireducens, Chloroflexi bacterium 54-19, Defluviitoga tunisiensis, Deinococcus aerius, Deinococcus apachensis, Deinococcus aquatilis, Deinococcus geothermalis, Deinococcus hopiensis, Deinococcus hopiensis, Deinococcus spic Leaf326, Deinococcus phoenicis, Deinococcus proteolyticus, Deinococcus sp. 17bor-2, Deinococcus sp. NW-56, Deinococcus sp. RL, Deinococcus sp. YIM 77859, Desulfurococcus mucosus, Dictyoglomus turgidum, Effusibacillus pohliae, Fervidobacterium gondwanense, Fervidobacterium islandicum, Fervidobacterium nodosum, Fervidobacterium pennivorans, Geobacillus sp., Geobacillus stearothermophilus, Halococcus salifodinae, Hydrogenivirga sp. 128-5-R1-1, Hydrogenobacter hydrogenophilus, Hydrogenobacter thermophilus, Hyperthermus butylicus, Kosmotoga arenicorallina, Kosmotoga olearia, Marinitoga piezophila, Meiothermus cerbereus, Meiothermus chliarophilus, Meiothermus ruber, Meiothermus silvanus, Meiothermus tiwana in a fers, Meiothermus ruber, Meiothermus silvanus, Meiothermus silvanus, Meiothermus silvanus, Meiothermus Metallosphaera sedula, Methanocella conradii, Methanococcoides methylutens, Methanohalobium evestigatum, Methanolobus tindarius, Methanosarcina sicilia, Methanothermus fervidus, Petrotoga mobilis, Picrophilus torridus, Pseudonocardia thermococcaAnd Truepera radiovictrix will be derived from any one selected from the group consisting of, the enzyme.
[Claim 3]
The enzyme of claim 1, wherein the enzyme comprises an amino acid sequence having at least 70% identity to the amino acid sequence of any one of SEQ ID NOs: 1-222.
[Claim 4]
4. The method of claim 3, wherein the enzyme is SEQ ID NO: 1, 6, 9, 12, 26, 29, 38 to 43, 45 to 53,56, 57,59, 60, 64 to 66, 69, 70, 72, 76 , 80, 81, 91 to 93, 95, 99 to 103, 113, 114, 116,117, 131, 134, 136, 142, 145, 146, 148, 164, 167, 169, 172, 177, 184 to 187, 189 , 191, 192, 211, 217, and an amino acid sequence having at least 70% identity to the amino acid sequence of any one of 221.
[Claim 5]
The enzyme according to claim 1, wherein the enzyme exhibits psicose-6-phosphate selective activity.
[Claim 6]
Any one of claims 1 to 5 nucleic acid encoding the psychophysic-6-phosphate dephosphorylation enzyme.
[Claim 7]
A transformant comprising the nucleic acid of claim 6.
[Claim 8]
Claims 1 to 5 of any one of claims 1 to 5 of the Psycos-6-phosphate dephosphorylation enzyme, a microorganism expressing the same, or a composition for producing psychosis comprising a culture of the microorganism.
[Claim 9]
The composition of claim 8, wherein the composition further comprises a phosphate sugar.
[Claim 10]
Claims 1 to 5 of any one of claims 1 to 5 of the Psycos-6-phosphate dephosphorylation enzyme, a microorganism expressing it, or a culture of the microorganism by contacting the culture of the microorganism with psicose-6-phosphate to reduce psicose-6-phosphate. Including the step of switching to the course, Psychos manufacturing method.
[Claim 11]
11. The method of claim 10, wherein the method is before the step of converting psicose-6-phosphate to psicose, fructose-6-phosphate to fructose-6-phosphate-3-epimerase, By contacting the expressing microorganism or a culture of the microorganism, further comprising the step of converting the fructose-6-phosphate to psicose-6-phosphate, Psychos manufacturing method.
[Claim 12]
According to claim 11, wherein the method is before the step of converting the fructose-6-phosphate to psicose-6-phosphate, glucose-6-phosphate-isomerase to glucose-6-phosphate; Contacting a microorganism or a culture of the microorganism expressing this, further comprising the step of converting the glucose-6-phosphate to fructose-6-phosphate, Psychos manufacturing method.
[Claim 13]
The method according to claim 12, wherein, before the step of converting glucose-6-phosphate into fructose-6-phosphate, phosphoglucomutase in glucose-1-phosphate, a microorganism expressing the same Or by contacting the culture of the microorganism, further comprising the step of converting the glucose-1-phosphate to glucose-6-phosphate, Psychos manufacturing method.
[Claim 14]
The method according to claim 12, wherein, before the step of converting glucose-6-phosphate into fructose-6-phosphate, a polyphosphate glucose kinase on glucose, a microorganism expressing the same, or a culture of the microorganism, and By contacting the polyphosphate, further comprising the step of converting the glucose to glucose-6-phosphate, Psychos manufacturing method.
[Claim 15]
14. The method of claim 13, wherein the method comprises α-glucan phosphorylase, starch phosphorylase in starch, maltodextrin, sucrose or a combination thereof prior to the step of converting glucose-1-phosphate to glucose-6-phosphate. ase, maltodextrin phosphorylase or sucrose phosphorylase; microorganisms expressing it; Or by contacting the culture of the microorganism, and phosphate, the starch, maltodextrin, sucrose or a combination thereof further comprising the step of converting to glucose-1-phosphate, Psychos manufacturing method.
[Claim 16]
16. The method of claim 15, wherein the step of converting the starch, maltodextrin, sucrose or a combination thereof into glucose-1-phosphate comprises α-amylase, pullulanase, isoamylase, α-glucanotransferase, glucoamylase or sucrase; microorganisms expressing it; Or, further comprising the culture of the microorganism, further comprising the step of converting the starch, maltodextrin, sucrose or a combination thereof into maltooligosaccharide or glucose, Psychos manufacturing method.
[Claim 17]
In starch, maltodextrin, sucrose, or a combination thereof, and phosphate (a) the psicose-6-phosphate dephosphorylation enzyme of claim 1; fructose-6-phosphate-3-epimerase; glucose-6-phosphate-isomerase; phosphoglucomutase or glucose kinase; and α-glucan phosphorylase, starch phosphorylase, maltodextrin phosphorylase, sucrose phosphorylase, α-amylase, pullulanase, isoamylase, glucoamylase, α-glucanotransferase, polyphosphate glucokinase or sucrase; Or (b) comprising the step of contacting a microorganism or a culture of the microorganism expressing the enzyme of the item (a), Psychos manufacturing method.
[Claim 18]
According to any one of claims 11 to 17, wherein the contact is pH 5.0 to 9.0, 40 ℃ to 80 ℃ temperature, and / or carried out for 2 hours to 24 hours or 120 hours, Psychos manufacturing method.