阅读说明：本技术 用于制备塔格糖的组合物和利用其制备塔格糖的方法 (Composition for preparing tagatose and method for preparing tagatose using the same ) 是由 梁成才 赵显国 李英美 金成俌 于 2018-03-30 设计创作，主要内容包括：本发明提供一种包含果糖-6-磷酸-4-差向异构酶的用于制备塔格糖的组合物,以及利用其制备塔格糖的方法。(The present invention provides a composition for manufacturing tagatose comprising fructose-6-phosphate-4-epimerase, and a method for manufacturing tagatose using the same.)

1.A composition for preparing tagatose-6-phosphoric acid, comprising

A tagatose-bisphosphate aldolase, a microorganism expressing the tagatose-bisphosphate aldolase, or a culture of the microorganism.

2. The composition of claim 1, further comprising fructose-6-phosphate.

3. The composition of claim 1, wherein the composition comprises

One or more polypeptides consisting of SEQ ID NO: 1. 3, 5, 7 or 9, or a pharmaceutically acceptable salt thereof.

4. A composition for preparing tagatose comprising

A tagatose-bisphosphate aldolase, a microorganism expressing the tagatose-bisphosphate aldolase, or a culture of the microorganism; and

a tagatose-6-phosphate phosphatase, a microorganism expressing the tagatose-6-phosphate phosphatase, or a culture of the microorganism.

5. The composition of claim 4, further comprising fructose-6-phosphate.

6. The composition of claim 4, further comprising

Glucose-6-phosphate isomerase, a microorganism expressing said glucose-6-phosphate isomerase or a culture of said microorganism.

7. The composition of claim 6, further comprising

A phosphoglucomutase, a microorganism expressing the phosphoglucomutase, or a culture of the microorganism.

8. The composition of claim 7, further comprising

An alpha-glucan phosphorylase, starch phosphorylase, maltodextrin phosphorylase or sucrose phosphorylase, a microorganism expressing said alpha-glucan phosphorylase, starch phosphorylase, maltodextrin phosphorylase or sucrose phosphorylase, or a culture of said microorganism.

9. The composition of claim 6, further comprising

Glucokinase, a microorganism expressing said glucokinase, or a culture of said microorganism.

10. The composition of claim 9, further comprising

An alpha-amylase, pullulanase, isoamylase, glucoamylase or sucrase, a microorganism expressing said alpha-amylase, pullulanase, isoamylase, glucoamylase or sucrase, or a culture of said microorganism.

11.A method for preparing tagatose, which comprises

Contacting fructose-6-phosphate with tagatose-bisphosphate aldolase, a microorganism expressing the tagatose-bisphosphate aldolase, or a culture of the microorganism, to produce tagatose-6-phosphate.

12. The method of claim 11, further comprising

Contacting the prepared tagatose-6-phosphate with tagatose-6-phosphate phosphatase, a microorganism expressing the tagatose-6-phosphate phosphatase, or a culture of the microorganism to prepare tagatose.

13. The method of claim 11 or 12, further comprising

Contacting glucose-6-phosphate with glucose-6-phosphate-isomerase, a microorganism expressing said glucose-6-phosphate-isomerase, or a culture of said microorganism, to convert glucose-6-phosphate to fructose-6-phosphate.

14. The method of claim 13, further comprising

Contacting glucose-1-phosphate with a phosphoglucomutase, a microorganism expressing the phosphoglucomutase, or a culture of the microorganism to convert glucose-1-phosphate to glucose-6-phosphate.

15. The method of claim 14, further comprising

Contacting starch, maltodextrin, sucrose, or a combination thereof with an alpha-glucan phosphorylase, starch phosphorylase, maltodextrin phosphorylase, or sucrose phosphorylase, a microorganism expressing said alpha-glucan phosphorylase, starch phosphorylase, maltodextrin phosphorylase, or sucrose phosphorylase, or a culture of said microorganism to convert starch, maltodextrin, or sucrose to glucose-1-phosphate.

16. The method of claim 13, further comprising

Contacting glucose with glucokinase, a microorganism expressing said glucokinase, or a culture of said microorganism to convert glucose to glucose-6-phosphate.

17. The method of claim 16, further comprising

Contacting starch, maltodextrin, sucrose, or a combination thereof with an alpha-amylase, pullulanase, glucoamylase, sucrase, or isoamylase, a microorganism expressing the alpha-amylase, pullulanase, glucoamylase, sucrase, or isoamylase, or a culture of the microorganism, to convert starch, maltodextrin, or sucrose to glucose.

18. The method of claim 11 or 12, wherein

The contacting is performed at a pH of 5.0 to 9.0, at 40 ℃ to 80 ℃ and/or for 0.5 hours to 24 hours.

19. The method of claim 11 or 12, wherein

The tagatose-bisphosphate aldolase consists of SEQ ID NO: 1. 3, 5, 7 or 9, and

the tagatose-6-phosphate phosphatase is encoded by SEQ ID NO: 11, or a pharmaceutically acceptable salt thereof.

20. A method of making tagatose comprising contacting:

(a) starch, maltodextrin, sucrose, or a combination thereof; and

(b) (i) tagatose-6-phosphate phosphatase,

(ii) Tagatose-bisphosphate aldolase,

(iii) Glucose-6-phosphate-isomerase,

(iv) Glucose phosphoglucomutase or glucokinase,

(v) Phosphorylase, and

(vi) one or more of alpha-amylase, pullulanase, isoamylase, glucoamylase or sucrase; and

(c) a phosphate salt.

Technical Field

The present invention relates to a composition for preparing tagatose-6-phosphate comprising fructose-6-phosphate 4-epimerase, and a method for preparing tagatose using the same.

Background

Conventional tagatose preparation methods include a chemical method (catalytic reaction) and a biological method (isomerase reaction) using galactose as a main raw material (see korean patent No. 10-0964091). However, in the known production method, the base material of galactose as a main material is lactose, and the price of lactose is unstable depending on the production, supply and demand of raw milk and lactose, etc. in the global market. Therefore, the stable supply thereof is limited. In order to overcome such problems in the conventional tagatose preparation method, a method of preparing tagatose from inexpensive and stably supplied fructose (D-fructose) using hexuronic acid C4-epimerase (hexuronate C4-epimerase) has been reported (2011. applied biochem Biotechnol.163: 444-451; Korean patent No. 10-1550796), but it has a limitation that the conversion rate (conversion ratio) of the isomerization reaction is low.

It is known that tagatose-diphosphate aldolase (EC 4.1.2.40) can produce glycerophosphate (glycone phosphate) and D-glyceraldehyde3-diphosphate (D-glyceraldehyde3-diphosphate) as substrates of D-tagatose1, 6-diphosphate (D-tagatose1, 6-diphosphate) and participate in galactose metabolism as shown in [ equation 1 ]. However, there was no study as to whether the tagatose-bisphosphate aldolase had activity to produce an enzyme converting fructose-6-phosphate into tagatose-6-phosphate.

[ reaction formula 1] D-tagatose1,6-bisphosphate (glycerone phosphate + D-glyceraldehyde 3-bisphosphate)

Under this background, the present inventors have conducted extensive studies to develop an enzyme that can be used for the preparation of tagatose, and as a result, they have found that tagatose-bisphosphate aldolase (EC 4.1.2.40) has a function of converting glucose-6-phosphate (glucose-6-phosphate) into tagatose-6-phosphate, thereby completing the present invention.

Therefore, glucose-1-phosphate and glucose-6-phosphate (glucose-6-phosphate) can be sequentially prepared from glucose or starch as a raw material, glucose-6-phosphate is then converted into tagatose-6-phosphate (tagatose-6-phosphate) using the tagatose-bisphosphate aldolase of the present invention, tagatose is prepared using tagatose-6-phosphate phosphatase (tagatose-6-phosphate) which performs an irreversible reaction pathway, and the conversion rate from glucose or starch to tagatose can be significantly increased.

Disclosure of Invention

An object of the present invention is to provide a composition for preparing tagatose-6-phosphate, which comprises tagatose-bisphosphate aldolase, a microorganism expressing the tagatose-bisphosphate aldolase, or a culture of the microorganism.

It is another object of the present invention to provide a composition for producing tagatose, which comprises tagatose-bisphosphate aldolase, a microorganism expressing the tagatose-bisphosphate aldolase, or a culture of the microorganism; and tagatose-6-phosphate phosphatase, a microorganism expressing the tagatose-6-phosphate phosphatase, or a culture of the microorganism.

It is another object of the present invention to provide a method for preparing tagatose, which comprises contacting fructose-6-phosphate with tagatose-bisphosphate aldolase, a microorganism expressing the tagatose-bisphosphate aldolase, or a culture of the microorganism to convert fructose-6-phosphate into tagatose-6-phosphate, wherein the method may further comprise contacting tagatose-6-phosphate with tagatose-6-phosphate phosphatase, a microorganism expressing the tagatose-6-phosphate phosphatase, or a culture of the microorganism to convert tagatose-6-phosphate into tagatose.

Other objects and advantages of the present invention will be set forth in detail in the following detailed description and appended claims and drawings. Those not described in the present specification can be sufficiently recognized and derived by those skilled in the art or the like, and thus, the description thereof is omitted.

Drawings

FIGS. 1a to 1d are HPLC chromatography results showing that tagatose-bisphosphate aldolase (CJ _ KO _ F6P4E, CJ _ RM _ F6P4E, CJ _ RP _ F6P4E and CJ _ LP _ F6P4E) in one embodiment of the present invention has fructose-6-phosphate-4-epimerase activity.

FIGS. 2a and 2b are HPLC chromatography results showing that, in one embodiment of the present invention, tagatose-6-phosphate is converted into tagatose by treating tagatose-6-phosphate with tagatose-bisphosphate aldolase (CJ _ KO _ F6P4E and CJ _ RP _ F6P4E) and tagatose-6-phosphate phosphatase (CJ _ T4).

FIG. 3 is a result of HPLC chromatography, showing that the enzyme T4 has the activity of tagatose-6-phosphate phosphatase in one embodiment of the present invention.

FIG. 4 is a result of protein gel electrophoresis (SDS-PAGE) to analyze the molecular weight of an enzyme in a reaction pathway for preparing tagatose from starch, sucrose or glucose in an embodiment of the present invention, wherein M represents a protein size standard (sizemarker, Bio-RAD, USA).

FIG. 5 is a HPLC chromatography result showing that the enzyme TD1(CJ _ TD1_ F6P4E) prepared in one embodiment of the present invention has fructose-6-phosphate 4-epimerase activity.

Fig. 6 is a result of HPLC chromatography, showing that when all enzymes participating in a reaction pathway for preparing tagatose from maltodextrin were simultaneously added, tagatose was produced by a complex enzyme reaction in which TD1(CJ _ TD1_ F6P4E) was used as tagatose-bisphosphate aldolase.

Detailed Description

The present invention will be specifically described below. In addition, common matters in the descriptions and embodiments disclosed in the present invention can be applied to other descriptions and embodiments. In addition, combinations of the various elements disclosed in the present invention are within the scope of the invention. Moreover, the scope of the invention is not limited to the following detailed description.

To achieve an object of the present invention, the present invention provides in one aspect a composition for the production of tagatose-6-phosphate, comprising a tagatose-bisphosphate aldolase, a microorganism expressing the tagatose-bisphosphate aldolase, or a culture of the microorganism.

It is known that tagatose-bisphosphate aldolase (EC 4.1.2.40) can produce phosphoglyceridone (glycone phosphate) and D-glyceraldehyde 3-bisphosphate (D-glyceraldehyde3-diphosphate) as substrates by D-tagatose1,6-bisphosphate (D-tagatose1,6-bisphosphate), and participate in galactose metabolism. For example, the tagatose-bisphosphate aldolase is not limited as long as it can produce tagatose-6-phosphate using fructose-6-phosphate as a substrate.

Specifically, the tagatose-bisphosphate aldolase can be a polypeptide consisting of SEQ ID NO: 1. 3, 5, 7 or 9, or a polypeptide comprising an amino acid sequence identical to SEQ ID NO: 1. 3, 5, 7 and 9, or a polypeptide having at least 80%, 90%, 95%, 97% or 99% homology thereto. It is also clear that the sequences having the homology and exhibiting homology to SEQ ID NO: 1. 3, 5, 7 or 9 (i.e., the activity of epimerizing the 4 th carbon position of fructose in fructose-6-phosphate to convert fructose-6-phosphate to fructose-6-phosphate C4-epimerization of tagatose-6-phosphate) is included in the scope of the present invention even if the polypeptide has an amino acid sequence in which a part of the sequence is deleted, modified, substituted or added. In addition, a probe prepared based on a known nucleotide sequence, for example, a polypeptide encoded by a polynucleotide that can hybridize under stringent conditions to a complementary sequence of all or part of a nucleotide sequence encoding the polypeptide, may also be included without limitation so long as it has fructose-6-phosphate C4-epimerization activity. Accordingly, the composition for preparing tagatose-6-phosphate may further comprise fructose-6-phosphate. In addition, the composition may comprise one or more polypeptides consisting of SEQ ID NOs: 1. 3, 5, 7 or 9, or a pharmaceutically acceptable salt thereof.

The present invention discloses that "tagatose-bisphosphate aldolase" exhibits fructose-6-phosphate 4-epimerization activity, and fructose-6-phosphate is converted into tagatose-6-phosphate by epimerizing the 4 th carbon position of fructose-6-phosphate. Therefore, in the present invention, "tagatose-bisphosphate aldolase" can be used interchangeably with "fructose-6-phosphate C4-epimerase (fructose-6-phosphate C4-epimerase)".

As used herein, the term "stringent conditions" refers to conditions under which specific hybridization between polynucleotides can be achieved. These conditions depend on the length and degree of complementarity of the polynucleotides, these parameters are well known in the art and are described specifically in the literature (e.g., J.Sambrook et al, infra). Stringent conditions may include, for example, conditions in which genes having high homology hybridize with each other, genes having homology of 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more hybridize with each other, and homology is lower than that in which the above-mentioned genes do not hybridize with each other; or conventional washing conditions for Southern hybridization, i.e., conditions in which washing is performed once, specifically twice or three times, at a salt concentration and temperature of 60 ℃,1 XSSC, 0.1% SDS, specifically 60 ℃, 0.1 XSSC, 0.1% SDS, and more specifically 68 ℃, 0.1 XSSC, 0.1% SDS. The probe used in hybridization may be part of the complement of the nucleotide sequence. Such probes can be prepared by PCR using oligonucleotides based on known sequences as primers and DNA fragments containing these nucleotide sequences as templates. In addition, the temperature and the salt concentration of the washing solution may be adjusted by those skilled in the art according to factors such as the length of the probe, as needed.

As used herein, the term "homology" refers to the percentage of identity between two polypeptide moieties (moieity). Identity between sequences from one part to another can be determined by techniques known in the art. For example, homology can be determined using the following method: sequence information, such as parameters like score (score), identity (identity) and similarity (similarity), for two polypeptide molecules is aligned directly using computer programs that are readily available and that can align the sequence information (e.g., BLAST 2.0). In addition, the homology of polynucleotides can be determined as follows: polynucleotides are hybridized under conditions that form stable double strands between homologous regions, and the size of the digested fragments is then determined by digesting the hybridized strands with a single strand specific nuclease.

In a specific embodiment, the fructose-6-phosphate-4-epimerase of the present invention may be an enzyme derived from a thermophilic microorganism or a variant thereof, for example, an enzyme derived from Thermanothrix sp or a variant thereof, an enzyme derived from Kosmota sp or a variant thereof, an enzyme derived from Rhodothermus erythropolis (Rhodothermus sp) or a variant thereof, an enzyme derived from Limnochorda sp or a variant thereof, in particular, an enzyme derived from Thermanothrix daxensis, Kosmoga olearia, Rhodothermus marinus (Rhodothermus marinus), Rhodothermus profundi or Limnochorda pilosa, but is not limited thereto.

The fructose-6-phosphate-4-epimerase or the variant thereof of the present invention is characterized in that D-fructose-6-phosphate is converted into D-tagatose-6-phosphate by epimerizing the 4 th carbon position of D-fructose-6-phosphate (D-fructose-6-phosphate). The fructose-6-phosphate-4-epimerase of the present invention may be an enzyme known to have tagatose-bisphosphate aldolase activity, which produces phosphoglyceridone and D-glyceraldehyde 3-bisphosphate using D-tagatose1,6-bisphosphate as a substrate and participates in galactose metabolism. The invention discloses that tagatose-diphosphate aldolase has fructose-6-phosphate-4-epimerase activity for the first time. Accordingly, one embodiment of the present invention relates to a novel use of tagatose-bisphosphate aldolase, comprising using tagatose-bisphosphate aldolase as fructose-6-phosphate-4-epimerase in the production of tagatose-6-phosphate from fructose-6-phosphate. In addition, another embodiment of the present invention relates to a method for preparing tagatose-6-phosphate from fructose-6-phosphate using tagatose-bisphosphate aldolase as fructose-6-phosphate-4-epimerase.

In one embodiment, the fructose-6-phosphate-4-epimerase of the present invention may be an enzyme having high thermostability. Specifically, the fructose-6-phosphate-4-epimerase of the present invention may exhibit 50% to 100%, 60% to 100%, 70% to 100%, or 75% to 100% of its maximum activity at 50 ℃ to 70 ℃. More specifically, the fructose-6-phosphate-4-epimerase of the present invention may exhibit 80% to 100% or 85% to 100% of its maximum activity at 55 ℃ to 65 ℃, 60 ℃ to 70 ℃, 55 ℃, 60 ℃, or 70 ℃.

Furthermore, the polypeptide represented by SEQ ID NO: 1. 3, 5, 7 or 9 may consist of the amino acid sequence of SEQ ID NO: 2.4, 6, 8 or 10, but is not limited thereto.

The fructose-6-phosphate-4-epimerase or the variant thereof of the present invention can be obtained by: a microorganism such as Escherichia coli (Coli) is transformed with a DNA (e.g., SEQ ID NO: 2, 4, 6, 8 or 10) expressing the enzyme of the present invention or a variant thereof, the microorganism is cultured to obtain a culture, the culture is pulverized, and then purification using a chromatography column or the like is performed. The microorganism used for transformation may include Corynebacterium glutamicum (Corynebacterium glutamicum), Aspergillus oryzae (Aspergillus oryzae), or Bacillus subtilis (Bacillus subtilis), in addition to Escherichia coli. In a specific embodiment, the transformed microorganism may be Escherichia coli BL21(DE3)/CJ _ KO _ F6P4E, Escherichia coli BL21(DE3)/CJ _ RM _ F6P4E, Escherichia coli BL21(DE3)/CJ _ RP _ F6P4E, Escherichia coli BL21(DE3)/CJ _ LP _ F6P4E, or Escherichia coli BL21(DE3)/pBT7-C-His-CJ _ td 1. Escherichia coli BL21(DE3)/CJ _ KO _ F6P4E with accession number KCCM11999P (deposited date: 24/3 years 2017), Escherichia coli BL21(DE3)/CJ _ RM _ F6P4E with accession number KCCM12096P (deposited date: 11/8 months 2017), Escherichia coli BL21(DE3)/CJ _ RP _ F6P4E with accession number KCCM12097P (deposited date: 11/8 months 2017), Escherichia coli BL21(DE3)/CJ _ LP _ F6P4E with accession number KCCM12095P (deposited date: 11/8 months 2017), and Escherichia coli BL21(DE3)/pBT7-C-His-CJ _ td1 with accession number KCCM119 11995P (deposited date: 20 days 2017) according to the provisions of the Budapest treaty, the collection of Microorganisms deposited in the International centre of Microorganisms of Japan Collection, Certification of Cuora.

The fructose-6-phosphate-4-epimerase used in the present invention may be provided by a nucleic acid encoding the fructose-6-phosphate-4-epimerase.

As used herein, the term "nucleic acid" has a meaning encompassing DNA or RNA molecules, wherein the nucleotides that are the basic building blocks of nucleic acids may include not only natural nucleotides, but also Analogs having sugar or base modifications (see: Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, chemical reviews,90: 543-.

The nucleic acid of the invention may be a nucleic acid encoding a polypeptide of the invention consisting of SEQ ID NO: 1. 3, 5, 7 or 9, or a polypeptide encoding a fructose-6-phosphate-4-epimerase having at least 80%, 90%, 95%, 97% or 99% homology thereto and having fructose-6-phosphate-4-epimerase activity. For example, the coding sequence represented by SEQ ID NO: 1 can be a nucleic acid that hybridizes to the amino acid sequence of SEQ ID NO: 2 has at least 80%, 90%, 95%, 97%, 99% or 100% homology. In addition, the coding sequence represented by SEQ ID NO: 3. the nucleic acid of fructose-6-phosphate-4-epimerase consisting of the amino acid sequence of 5, 7 or 9 may be a nucleic acid which is identical to its corresponding amino acid sequence of SEQ ID NO: 4. 6, 8 or 10 has at least 80%, 90%, 95%, 97%, 99% or 100% homology. It is also clear that the nucleic acid of the invention may comprise nucleic acids which, due to codon degeneracy (codon degeneracy), translate into the fructose-6-phosphate-4-epimerase according to the invention and nucleic acids which, under stringent conditions, hybridize to the nucleotide sequence of SEQ ID NO: 2.4, 6, 8 or 10, and encodes a polypeptide having the fructose-6-phosphate-4-epimerase activity of the present invention.

The microorganism expressing fructose-6-phosphate-4-epimerase that can be used in the present invention may be a microorganism comprising a recombinant vector, wherein the recombinant vector comprises the nucleic acid.

The vector may be operably linked to the nucleic acid of the invention. As used herein, the term "operably linked" refers to operably linking a nucleotide expression regulating sequence and a nucleotide sequence encoding a protein of interest to each other in order to perform a general function, thereby affecting the expression of the encoding nucleotide sequence. Operable linkage to the vector can be achieved using genetic recombination techniques known in the art, and site-specific DNA cleavage and ligation can be achieved using restriction enzymes and ligases known in the art.

As used herein, the term "vector" refers to any medium used for cloning and/or transferring bases into an organism (e.g., a host cell). The vector may be a replicon (replicon) capable of replicating a combined fragment in which different DNA fragments are combined. Herein, the term "replicon" refers to any genetic unit (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as a self-replicating unit of DNA replication in vivo, i.e., capable of effecting replication through self-regulation. As used herein, the term "vector" may include viral and non-viral media for introducing bases into an organism (e.g., a host cell) in vitro, ex vivo, or in vivo, and may also include minicircle DNA, transposons such as Sleeping Beauty (Sleeping Beauty) (Izsvaket al., j.moi.biol.302:93-102(2000)), or artificial chromosomes. Examples of commonly used vectors may include natural or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, as phage vectors or cosmid vectors, pWE15, M13, MBL3, MBL4, xii, ASHII, APII, t10, t11, Charon4A, Charon21A and the like can be used; as the plasmid vector, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pET, and the like-based vectors can be used. The vector that can be used in the present invention is not particularly limited, and any known expression vector can be used. In addition, the vector may be a recombinant vector characterized by further comprising various antibiotic resistance genes. As used herein, the term "antibiotic resistance gene" refers to a gene that is resistant to an antibiotic, and cells having the gene can survive in an environment treated with the corresponding antibiotic. Therefore, antibiotic resistance genes are used as selectable markers in the production of large quantities of plasmids in E.coli. The antibiotic resistance gene in the present invention is not a factor that greatly affects the core technology (expression efficiency achieved by the optimal combination of vectors) of the present invention, and thus the antibiotic resistance gene, which is generally used as a selection marker, can be used without limitation. Specific examples may include resistance genes against ampicillin, tetracycline, kanamycin, chloramphenicol, streptomycin, neomycin, or the like.

The microorganism expressing fructose-6-phosphate-4-epimerase used in the present invention can be obtained using a method of introducing a vector comprising nucleic acid encoding the enzyme into a host cell, and the method of transforming the vector may be any method capable of introducing nucleic acid into a cell. Appropriate standard techniques known in the art may be selected and implemented. Electroporation, calcium phosphate coprecipitation, retroviral infection, microinjection, DEAE-dextran method, cationic liposome method, and heat shock method may be included, but are not limited thereto.

So long as the transformed gene is expressed in the host cell, it may be inserted into the chromosome of the host cell or may exist extrachromosomally. In addition, the gene includes DNA and RNA as a polynucleotide encoding a polypeptide, and any form can be used without limitation as long as it can be introduced into and expressed in a host cell. For example, the gene may be introduced into the host cell in the form of an expression cassette, which is a polynucleotide construct containing all the elements required for autonomous expression. Typically, an expression cassette can comprise a promoter operably linked to a gene, 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. Alternatively, the gene itself or in the form of a polynucleotide construct may be introduced into a host cell and operably linked to sequences required for expression in the host cell.

The microorganism of the present invention may include a prokaryotic microorganism or a eukaryotic microorganism as long as the microorganism can produce the fructose-6-phosphate-4-epimerase of the present invention by containing the nucleic acid or the recombinant vector of the present invention. For example, the microorganism may include a microorganism strain belonging to the genus Escherichia (Escherichia), Erwinia (Erwinia), Serratia (Serratia), Providencia (Providence), Corynebacterium (Corynebacterium) and Brevibacterium (Brevibacterium), specifically, but not limited to Escherichia coli (Escherichia coli) or Corynebacterium glutamicum (Corynebacterium glutamicum). Specific examples of the microorganism may include Escherichia coli BL21(DE3)/CJ _ KO _ F6P4E, Escherichia coli BL21(DE3)/CJ _ RM _ F6P4E, Escherichia coli BL21(DE3)/CJ _ RP _ F6P4E, Escherichia coli BL21(DE3)/CJ _ LP _ F6P4E, Escherichia coli BL21(DE3)/pBT7-C-His-CJ _ td1, and the like.

In addition to the introduction of the nucleic acid or vector, the microorganism of the present invention may include any microorganism capable of expressing the fructose-6-phosphate-4-epimerase or related enzyme of the present invention via various known methods.

A culture of the microorganism of the present invention can be prepared by culturing a microorganism capable of expressing tagatose-bisphosphate aldolase of the present invention or a related enzyme in a medium.

As used herein, the term "culturing" refers to growing a microorganism under controlled environmental conditions. The culturing process of the present invention can be performed according to an appropriate culture medium and culture conditions known in the art. The cultivation process can be easily adjusted by those skilled in the art according to the selected strain. The step of culturing the microorganism may be carried out by, but not particularly limited to, batch culture, continuous culture, fed-batch culture, or the like. As for the culture conditions, an appropriate pH (for example, pH5 to 9, specifically, pH7 to 9) can be adjusted using a basic compound (for example, sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (for example, phosphoric acid or sulfuric acid), but is not particularly limited thereto. In addition, an antifoaming agent such as fatty acid polyglycol ester may be added during the culture to suppress the generation of foam. In addition, oxygen or oxygen-containing gas may be injected into the culture to maintain the aerobic state of the culture; it is also possible to maintain the anaerobic or micro-aerobic state of the culture without injecting gas or with nitrogen, hydrogen or carbon dioxide gas. The culture temperature may be maintained at 25 ℃ to 40 ℃, specifically, 30 ℃ to 37 ℃, but is not limited thereto. The culturing may be continued until a desired amount of useful material is obtained, specifically, may be continued for about 0.5 hours to about 60 hours, but is not limited thereto. In addition, the medium used may include sugars and carbohydrates (e.g., glucose, sucrose, lactose, fructose, maltose, molasses, starch, and cellulose) as a carbon source, oils and fats (e.g., soybean oil, sunflower oil, peanut oil, and coconut oil), fatty acids (e.g., palmitic acid, stearic acid, and linoleic acid), alcohols (e.g., glycerol and ethanol), and organic acids (e.g., acetic acid), and the like. These may be used alone or in combination, but are not limited thereto. The nitrogen source may include nitrogen-containing organic compounds (e.g., peptone, yeast extract, meat extract, malt extract, corn steep liquor, soybean meal, and urea) or inorganic compounds (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate), and the like. These nitrogen sources may also be used alone or in combination, but are not limited thereto. The phosphorus source may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or their corresponding sodium-containing salts, and the like. These phosphorus sources may also be used alone or in combination, but are not limited thereto. The culture medium may contain necessary growth stimulants such as metal salts (e.g., magnesium sulfate or iron sulfate), amino acids, and vitamins.

Another aspect of the present invention provides a composition for producing tagatose, which comprises tagatose-bisphosphate aldolase, a microorganism expressing the tagatose-bisphosphate aldolase, or a culture of the microorganism; and tagatose-6-phosphate phosphatase, a microorganism expressing the tagatose-6-phosphate phosphatase, or a culture of the microorganism.

The description of the composition for preparing tagatose-6-phosphate can also be applied to the composition for preparing tagatose. The tagatose-6-phosphate of the present invention may be any protein without limitation as long as it has an activity of removing the phosphate group of tagatose-6-phosphate to convert tagatose-6-phosphate into tagatose. The tagatose-6-phosphate phosphatase of the present invention may be any enzyme derived from a heat-resistant microorganism, for example, an enzyme derived from Thermotoga maritima (Thermotoga sp.) or a variant thereof, and specifically, may be an enzyme derived from Thermotoga maritima (Thermotoga maritima) or a variant thereof.

According to an embodiment of the present invention, the tagatose-6-phosphate phosphatase of the present invention may be a protein consisting of SEQ ID NO: 11, or consists of an amino acid sequence identical to SEQ ID NO: 11 has a genetic homology of 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100%, or a genetic homology within a range defined by any two of the values. According to one embodiment of the invention, the polypeptide consisting of SEQ ID NO: 11 can consist of the amino acid sequence of SEQ ID NO: 12.

The composition for manufacturing tagatose of the present invention may further comprise glucose-6-phosphate isomerase (glucose-6-phosphate isomerase), a microorganism expressing the glucose-6-phosphate isomerase, or a culture of the microorganism. In the presence of the enzyme, glucose-6-phosphate may be isomerized to fructose-6-phosphate. The glucose-6-phosphate isomerase of the present invention may include any protein without limitation so long as it has an activity of isomerizing glucose-6-phosphate to fructose-6-phosphate. The glucose-6-phosphate isomerase of the present invention may be an enzyme derived from a thermotolerant microorganism, for example, an enzyme derived from Thermotoga maritima or a variant thereof, and specifically, may be an enzyme derived from Thermotoga maritima or a variant thereof. According to an embodiment of the present invention, the glucose-6-phosphate-isomerase of the present invention may be a protein consisting of SEQ ID NO: 13, or consists of an amino acid sequence identical to SEQ ID NO: 13 has a genetic homology of 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100%, or a genetic homology within a range defined by any two of the values. According to one embodiment of the invention, the polypeptide consisting of SEQ ID NO: 13 can consist of the amino acid sequence of SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof.

The composition for preparing tagatose of the present invention may further comprise phosphoglucomutase, a microorganism expressing the phosphoglucomutase, or a culture of the microorganism. The enzyme catalyzes a reversible reaction that converts glucose-1-phosphate to glucose-6-phosphate or glucose-6-phosphate to glucose-1-phosphate. The phosphoglucomutase of the present invention may include any protein without limitation as long as it has an activity of converting glucose-1-phosphate into glucose-6-phosphate or converting glucose-6-phosphate into glucose-1-phosphate. The phosphoglucomutase of the present invention may be an enzyme derived from a thermotolerant microorganism, for example, an enzyme derived from Thermotoga maritima or a variant thereof, and specifically, may be an enzyme derived from Thermotoga neoformans (Thermotoga neapolitana) or a variant thereof. According to an embodiment of the present invention, the phosphoglucomutase of the present invention may be a protein consisting of SEQ ID NO: 15, or consists of an amino acid sequence identical to SEQ ID NO: 15 has a genetic homology of 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100%, or a genetic homology within a range defined by any two of the values. According to one embodiment of the invention, the polypeptide consisting of SEQ ID NO: 15 can consist of the amino acid sequence of SEQ ID NO: 16.

The composition for manufacturing tagatose of the present invention may further comprise glucokinase (glucokinase), a microorganism expressing the glucokinase, or a culture of the microorganism. The glucokinase of the present invention may include any protein without limitation as long as it has an activity of phosphorylating glucose. The glucokinase of the present invention may be an enzyme derived from a heat-resistant microorganism, for example, an enzyme derived from Deinococcus sp, anoxydesmus sp, or a variant thereof, and specifically, may be an enzyme derived from moderately thermophilic bacterium geothermalis (Deinococcus geothermalis) or anoxydesmus thermophilus (anoxydesmus thermophila), or a variant thereof. The glucokinase of the present invention may be any protein without limitation so long as it has an activity of converting glucose into glucose-6-phosphate. In particular, the glucokinase of the present invention may be a phosphate-dependent glucokinase. According to one embodiment of the invention, the glucokinase of the invention may be a protein consisting of SEQ ID NO: 17 or 19, or consists of an amino acid sequence identical to SEQ ID NO: 17 or 19 has 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% genetic homology, or consists of a sequence having genetic homology within a range defined by any two of the numbers. According to one embodiment of the invention, the polypeptide consisting of SEQ ID NO: 17 can consist of the amino acid sequence of SEQ ID NO: 18, and the nucleotide sequence of SEQ ID NO: 19 can consist of the amino acid sequence of SEQ ID NO: 20 is encoded by the nucleotide sequence of seq id no.

The composition for preparing tagatose of the present invention may further comprise α -glucan phosphorylase (α -glucanphosphorylase), starch phosphorylase (starch phosphorylase), maltodextrin phosphorylase (maltodextrinphosphorylase) or sucrose phosphorylase (sucrose phosphorylase), a microorganism expressing the α -glucan phosphorylase, starch phosphorylase, maltodextrin phosphorylase or sucrose phosphorylase, or a culture of the microorganism. The phosphorylase may include any protein without limitation as long as it has an activity of converting starch, maltodextrin or sucrose into glucose-1-phosphate. The phosphorylase may be an enzyme derived from a thermotolerant microorganism, for example, an enzyme derived from Thermotoga maritima or a variant thereof, and specifically, may be an enzyme derived from Thermotoga maritima or a variant thereof. The phosphorylase of the present invention may be a protein consisting of SEQ ID NO: 21, or consists of an amino acid sequence identical to SEQ ID NO: 21 has a genetic homology of 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100%, or a genetic homology within a range defined by any two of the values. According to one embodiment of the invention, the polypeptide consisting of SEQ ID NO: 21 can consist of the amino acid sequence of SEQ ID NO: 22.

The composition for preparing tagatose of the present invention may further comprise alpha-amylase (alpha-amylase), pullulanase (pullulanase), glucoamylase (glucoamylase), sucrase (sucrase) or isoamylase (isoamylase); a microorganism expressing the amylase, pullulanase, glucoamylase, sucrase or isoamylase; or a culture of a microorganism expressing said amylase, pullulanase, glucoamylase, sucrase or isoamylase.

The composition for producing tagatose of the present invention may comprise two or more enzymes among the above-mentioned enzymes that can be used to produce tagatose alone, or transformants thereof, or transformants transformed with nucleotides encoding the two or more enzymes.

The composition for manufacturing tagatose of the present invention may further comprise 4- α -glucanotransferase (4- α -glucanotransferase), a microorganism expressing the 4- α -glucanotransferase, or a culture of the microorganism expressing the 4- α -glucanotransferase. The 4- α -glucanotransferase of the present invention may include any protein without limitation as long as it has an activity of converting glucose into starch, maltodextrin or sucrose. The 4- α -glucanotransferase of the present invention may be an enzyme derived from a thermotolerant microorganism, for example, an enzyme derived from Thermotoga maritima or a variant thereof, and specifically, may be an enzyme derived from Thermotoga maritima or a variant thereof. According to one embodiment of the invention, the 4- α -glucanotransferase of the invention may be a protein consisting of SEQ ID NO: 23, or consists of an amino acid sequence identical to SEQ ID NO: 23 has a genetic homology of 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100%, or a genetic homology within a range defined by any two of the values. According to one embodiment of the invention, the polypeptide consisting of SEQ ID NO: 23 can consist of the amino acid sequence of SEQ ID NO: 24.

Examples of microorganisms that can be used in the above-described embodiments may include Escherichia coli BL21(DE3)/pET21a-CJ _ ct1, Escherichia coli BL21(DE3)/pET21a-CJ _ ct2, Escherichia coli BL21(DE3)/pET21a-CJ _ tn1, Escherichia coli BL21(DE3)/pET21a-CJ _ tn2, and Escherichia coli BL21(DE3)/pET21a-CJ _ t4, and the like. The recombinant microorganisms were deposited in the Korean culture Collection on 3/20.2017 under accession numbers KCCM11990P (E.coli BL21(DE3)/pET21a-CJ _ ct1), KCCM11991P (E.coli BL21(DE3)/pET21a-CJ _ ct2), KCCM11992P (E.coli BL21(DE3)/pET21a-CJ _ tn1), KCCM11993P (E.coli BL21(DE3)/pET21a-CJ _ tn2) and KCCM11994P (E.coli BL21(DE3)/pET21a-CJ _ t4), respectively.

The composition for manufacturing tagatose of the present invention may further comprise a substance, ingredient or composition corresponding to each substrate of the above-mentioned enzyme.

The composition for preparing tagatose of the present invention may further comprise any suitable excipient generally used in the corresponding composition for preparing tagatose. Excipients may include, for example, but are not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizing agents, or isotonic agents and the like.

The composition for preparing tagatose of the present invention may further comprise a metal. In one embodiment, the metal of the present invention may be a metal comprising a divalent cation. Specifically, the metal of the present invention may be nickel, cobalt, aluminum, magnesium (Mg) or manganese (Mn). More particularly, toThe metal of the present invention may be a metal ion or a metal salt, and more specifically, the metal salt may be NiSO₄、MgSO₄、MgCl₂、NiCl₂、CoCl₂、CoSO₄、MnCl₂Or MnSO₄。

Yet another aspect of the present invention relates to a method for preparing tagatose-6-phosphate, which comprises contacting fructose-6-phosphate with tagatose-bisphosphate aldolase, a microorganism expressing the tagatose-bisphosphate aldolase, or a culture of the microorganism, to convert fructose-6-phosphate into tagatose-6-phosphate.

The description of the composition for preparing tagatose-6-phosphate can also be applied to the composition for preparing tagatose.

Another aspect of the present invention relates to a method for preparing tagatose, which comprises contacting fructose-6-phosphate with tagatose-bisphosphate aldolase, a microorganism expressing the tagatose-bisphosphate aldolase, or a culture of the microorganism, to convert fructose-6-phosphate into tagatose-6-phosphate. The method of preparing tagatose may further comprise contacting tagatose-6-phosphate with tagatose-6-phosphate phosphatase, a microorganism expressing the tagatose-6-phosphate phosphatase, or a culture of the microorganism to convert tagatose-6-phosphate into tagatose.

The method of the present invention may further comprise contacting glucose-6-phosphate with the glucose-6-phosphate-isomerase of the present invention, a microorganism expressing the glucose-6-phosphate-isomerase, or a culture of a microorganism expressing the glucose-6-phosphate isomerase to convert glucose-6-phosphate to fructose-6-phosphate.

The method of the present invention may further comprise contacting glucose-1-phosphate with a phosphoglucomutase of the present invention, a microorganism expressing the phosphoglucomutase, or a culture of a microorganism expressing the phosphoglucomutase to convert glucose-1-phosphate to glucose-6-phosphate.

The method of the present invention may further comprise contacting glucose with the glucokinase of the present invention, a microorganism expressing the glucokinase, or a culture of a microorganism expressing the glucokinase to convert glucose into glucose-6-phosphate.

The method of the present invention may further comprise contacting starch, maltodextrin, sucrose or a combination thereof with the α -glucan phosphorylase, starch phosphorylase, maltodextrin phosphorylase or sucrose phosphorylase of the present invention, a microorganism expressing the phosphorylase or a culture of a microorganism expressing the phosphorylase to convert starch, maltodextrin or sucrose to glucose-1-phosphate.

The methods of the present invention may further comprise contacting starch, maltodextrin, sucrose, or a combination thereof with an alpha-amylase, pullulanase, glucoamylase, sucrase or isoamylase, a microorganism expressing the alpha-amylase, pullulanase, glucoamylase, sucrase or isoamylase, or a culture of a microorganism expressing the alpha-amylase, pullulanase, glucoamylase, sucrase or isoamylase to convert starch, maltodextrin or sucrose to glucose.

The method of the present invention may further comprise contacting glucose with the 4- α -glucanotransferase of the present invention, a microorganism expressing the 4- α -glucanotransferase, or a culture of a microorganism expressing the 4- α -glucanotransferase to convert glucose to starch, maltodextrin, or sucrose.

The various contacts of the present method may be performed at a pH of 5.0 to 9.0, at a temperature of 30 ℃ to 80 ℃ and/or for a time of 0.5 hours to 48 hours. Specifically, the contacting of the present invention may be performed under the condition of pH 6.0 to pH 9.0 or pH 7.0 to pH 9.0. In addition, the contacting of the present invention can be performed under temperature conditions of 35 ℃ to 80 ℃, 40 ℃ to 80 ℃, 45 ℃ to 80 ℃, 50 ℃ to 80 ℃, 55 ℃ to 80 ℃, 60 ℃ to 80 ℃, 30 ℃ to 70 ℃, 35 ℃ to 70 ℃, 40 ℃ to 70 ℃, 45 ℃ to 70 ℃, 50 ℃ to 70 ℃, 55 ℃ to 70 ℃, 60 ℃ to 70 ℃, 30 ℃ to 65 ℃, 35 ℃ to 65 ℃, 40 ℃ to 65 ℃, 45 ℃ to 65 ℃, 50 ℃ to 65 ℃, 30 ℃ to 60 ℃, 35 ℃ to 60 ℃, 40 ℃ to 60 ℃, 45 ℃ to 60 ℃, 50 ℃ to 60 ℃, or 55 ℃ to 60 ℃. Further, the contacting of the present invention may be performed for 0.5 to 36 hours, 0.5 to 24 hours, 0.5 to 12 hours, 0.5 to 6 hours, 1 to 36 hours, 1 to 24 hours, 1 to 12 hours, 1 to 6 hours, 3 to 36 hours, 3 to 24 hours, 3 to 12 hours, 3 to 6 hours, 12 to 36 hours, or 18 to 30 hours.

In one embodiment, the contacting of the present invention may be carried out in the presence of a metal, metal ion or metal salt.

Another aspect of the present invention relates to a method for preparing tagatose, which comprises contacting the composition for preparing tagatose described herein with starch, maltodextrin, sucrose or a combination thereof, and polyphosphate (polyphosphate).

In a specific embodiment of the present invention, there is provided a method for preparing tagatose, comprising:

contacting glucose with a glucokinase of the invention, a microorganism expressing said glucokinase, or a culture of said microorganism to convert glucose to glucose-6-phosphate;

contacting glucose-6-phosphate with a glucose-6-phosphate-isomerase of the present invention, a microorganism expressing said glucose-6-phosphate-isomerase, or a culture of said microorganism to convert glucose-6-phosphate to fructose-6-phosphate;

contacting fructose-6-phosphate with a fructose-6-phosphate-4-epimerase of the present invention, a microorganism expressing the fructose-6-phosphate-4-epimerase, or a culture of the microorganism to convert fructose-6-phosphate into tagatose-6-phosphate; and

contacting tagatose-6-phosphate with the tagatose-6-phosphate phosphatase of the present invention, a microorganism expressing the tagatose-6-phosphate phosphatase, or a culture of the microorganism to convert tagatose-6-phosphate into tagatose.

The respective conversion reactions may be carried out sequentially or in situ (in situ) in the same reaction system. In the method, phosphate released from tagatose-6-phosphate by phosphatase can be used as a substrate for glucokinase for producing glucose-6-phosphate. Thus, phosphoric acid does not accumulate, and a high conversion can be obtained.

In the method, glucose may be produced by: for example, starch, maltodextrin, sucrose, or a combination thereof is contacted with an α -glucan phosphorylase, starch phosphorylase, maltodextrin phosphorylase, or sucrose phosphorylase of the present invention, a microorganism expressing the phosphorylase, or a culture of a microorganism expressing the phosphorylase to convert starch, maltodextrin, or sucrose to glucose. Thus, the method according to a specific embodiment may further comprise converting starch, maltodextrin or sucrose to glucose.

In another embodiment of the present invention, there is provided a method for preparing tagatose, which comprises:

contacting glucose-1-phosphate with a phosphoglucomutase of the invention, a microorganism expressing the phosphoglucomutase, or a culture of the microorganism to convert glucose-1-phosphate to glucose-6-phosphate;

contacting glucose-6-phosphate with a glucose-6-phosphate-isomerase of the present invention, a microorganism expressing said glucose-6-phosphate-isomerase, or a culture of said microorganism to convert glucose-6-phosphate to fructose-6-phosphate;

contacting fructose-6-phosphate with a fructose-6-phosphate-4-epimerase of the present invention, a microorganism expressing the fructose-6-phosphate-4-epimerase, or a culture of the microorganism to convert fructose-6-phosphate into tagatose-6-phosphate; and

contacting tagatose-6-phosphate with the tagatose-6-phosphate phosphatase of the present invention, a microorganism expressing the tagatose-6-phosphate phosphatase, or a culture of the microorganism to convert tagatose-6-phosphate into tagatose.

The respective conversion reactions may be carried out sequentially or in situ in the same reaction system.

In the method, glucose-1-phosphate can be produced as follows: for example, starch, maltodextrin, sucrose, or a combination thereof is contacted with an α -glucan phosphorylase, starch phosphorylase, maltodextrin phosphorylase, sucrose phosphorylase, a microorganism expressing the phosphorylase, or a culture of a microorganism expressing the phosphorylase of the present invention to convert starch, maltodextrin, or sucrose to glucose-1-phosphate. Thus, the method according to a specific embodiment may further comprise converting starch, maltodextrin or sucrose into glucose-1-phosphate. In this regard, phosphate released from tagatose-6-phosphate via phosphatase can be used as a substrate for phosphorylase for the production of glucose-1-phosphate. Therefore, phosphoric acid does not accumulate, and thus a high conversion rate can be obtained.

The method may further comprise purifying the prepared tagatose. The purification in the method may be a method generally used in the art to which the present invention pertains, without particular limitation. Non-limiting examples may include chromatography, fractional crystallization, and ion purification, among others. Purification may be performed using only one method, or two or more methods may be used. For example, the product tagatose can be purified by chromatography, and separation of the saccharide by chromatography can be performed using the difference in weak binding force between the saccharide to be separated and the metal ion attached to the ion resin.

In addition, the process of the present invention may further comprise a step of decolorizing, desalting or both decolorizing and desalting before or after the purification step of the present invention. By performing decolorization and/or desalting, purer tagatose free of impurities can be obtained.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for facilitating the understanding of the present invention, and the present invention is not limited to these examples.