KR20120049684A - A novel compound, quercetin 3-o-n-acetylglucosamine, gene for producing the compound, and method for producing the compound - Google Patents

Abstract

PURPOSE: A gene producing quercetin 3-O-N-cetyl glucosamine and a method thereof are provided to be applied to various food or pharmaceutical products. CONSTITUTION: A quercetin-3-O-N-acetyl glucosamine compound is a conjugate of quercetin and N-acetytl glucosamine. A gene mediating quercetin and N-acetyl glucosamine has a base sequence of sequence number 1. A method for preparing the compound comprises: a step of preparing an expression vector containing the gene; a step of transforming the expression vector to a host cell; a step of adding quercetin to the host cell.

Description

A novel compound, quercetin 3-O-N-Acetylglucosamine, gene for producing the compound, and method for producing the compound

The present invention relates to a quercetin 3-O-N-acetylglucosamine, a novel compound, and a method for producing the compound.

In recent years, it has been known that oxidative stress caused by free radicals and free radicals causes various diseases including lifestyle diseases. While oxygen is an essential molecule in order to produce energy for living, it is also true that excess oxygen is converted into highly reactive active oxygen, which damages the living body. Examples of active oxygen include superoxide anion radicals (? O2-), hydrogen peroxide (H2O2), OH radicals (? OH), and single oxygen ( ¹ O ₂ ), which are excitation molecule species. Organisms originally have the ability to prevent the oxidative disturbances caused by these free radicals, which are in charge of vitamin E or antioxidant enzymes. However, if the ability to prevent oxidative disorders is reduced by factors such as aging, or when free radicals exceeding the protective ability are generated by factors such as extreme exercise or stress load, free radicals oxidized the target molecule. As a result, the biological components are disturbed and aging occurs.

Therefore, efficient intake of antioxidants scavenging free radicals or free radicals and defense from oxidative stress are considered to be very important in view of the prevention or treatment of various diseases. In particular, it is thought that by actively ingesting a defensive functional ingredient against oxidative disorders from food, it is possible to control and eliminate such disorders more efficiently. Therefore, attention has been focused on various food components having an antioxidant action.

Flavonoids (flavonoids) are included in a variety of forms in the daily intake of food, is known to have a strong antioxidant activity. However, the antioxidant activity is effective in vitro, but due to low oral absorption, it is not sufficient to eliminate free radicals and free radicals in vivo. Therefore, a method of enhancing the absorbency by combining sugars with flavonoids (hesperidine, diosmin, naringin, neohesperidine) has been proposed (Japanese Patent Laid-Open No. 2000-78956). Reference). In addition, it has been reported that the α-glycosyl rutin obtained by transfer to rutin contained in buckwheat improves its absorbency compared to the routine (Japanese Patent Laid-Open No. 2004-59522). And Shimoi K. et al., J Agric Food Chem., 51,2785-2789, 2003).

Quercetin, Rugly's aglycone, is a potent antioxidant (Middlton), 3,3 ', 4', 5,7-pentahydroxyflavone (3,3 ', 4', 5,7-pentahydroxyflavone). In addition to EJ. Et al., Pharmacol Rev., 52, 673-751, 2000), it is known to have various physiological functions such as inhibiting aggregation and adhesion of platelets, vasodilation, and anticancer. Also about this quercetin, quercetin glycoside (Quercetin-4'-β-D-glucoside), quercetin-3,4'-β-D-glucoside (Quercetin) contained in a lot of onion -3,4'-β-D-glucoside) have been reported to have good absorption (Hollman PC et al., Arch Toxicol Suppl., 20, 237-248, 1998). Similarly, glucose-binding isokercitrin (Quercetin-3-β-D-glucoside) at the 3-position of quercetin has been reported to be more absorbent than quercetin and rutin. (Morand C et al., Free Raf Res., 33, 667-676, 2000).

Amino sugars, on the other hand, are generally found as monomer residues in complex oligosaccharides and polysaccharides. Glucosamine is an amino derivative of glucose, a monosaccharide. N-acetylglucosamine is an acetylated derivative of glucosamine. Glucosamine, N-acetylglucosamine and other amino sugars are important components of many natural polysaccharides. For example, polysaccharides comprising amino sugars can form structural materials of cells similar to structural proteins.

Glucosamine is produced as a functional food for the treatment of osteoarthritis diseases, among other diseases of animals and humans. N-acetylglucosamine is also a valuable pharmacological component that can treat a wide range of diseases. N-acetylglucosamine is

No side effects are known. N-acetylglucosamine is a valuable and important component of biosynthetic protein synthesis that has a positive effect on tissue regeneration, resulting in gastritis, food allergies, inflammatory bowel disease (IBD), diverticulitis and metabolic disorders of bone joint tissue It has therapeutic potential in preventing and / or treating a wide range of diseases such as acute and chronic rheumatoid arthritis and osteoarthritis, as well as pathological disorders.

The present invention has been made in view of the above problems, and the object of the present invention is that the compounds of various physiological activities such as antioxidant, anti-inflammatory, anti-cancer, quercetin and cartilage health, gastritis, moisturizing, etc. It is to provide a combination of N-acetylglucosamin (acetylglucosamin) known to be functional.

Another object of the present invention is to provide a method for preparing the compound.

Another object of the present invention is to provide a gene for preparing the compound.

Still another object of the present invention is to provide a method of preparing a gene for preparing the compound.

Another object of the present invention is to provide a protein for preparing the compound.

In order to achieve the above object, the present invention provides a quercetin-3-O-N-acetylglucosamine compound which is a combination of quercetin and N-acetylglucosamin.

The present invention also provides a gene that mediates the binding reaction between quercetin and N-acetylglucosamin to produce the quercetin-3-O-N-acetylglucosamine compound of the present invention.

In one embodiment of the present invention, the gene has a nucleotide sequence of SEQ ID NO: 1, for example, the mutation of one or more substitutions, deletions, additions, etc. in the nucleotide sequence described in SEQ ID NO: 1 to the present invention Genes encoding all polypeptides having the activity of the invention are also included in the scope of the present invention, and all genes having at least 80% homology with these sequences are included in the scope of the present invention in consideration of degeneracy of the genetic code. do.

In another aspect, the present invention comprises the steps of preparing an expression vector comprising the gene of the present invention; b) transforming the expression vector into a host cell; and c) adding a quercetin as a substrate to the host cell. The method provides a method of preparing the quercetin-3-ON-acetylglucosamine compound of the present invention. .

In one embodiment of the invention, the host cell is preferably one that is missing a pgm gene involved in UDP-glucose synthesis metabolism, but is not limited thereto.

The present invention also provides a composition comprising the compound of the present invention as an active ingredient.

The present invention also provides an enzyme that mediates the binding reaction between quercetin and N-acetylglucosamin to produce the quercetin-3-O-N-acetylglucosamine compound of the present invention.

In one embodiment of the invention, the enzyme has the amino acid sequence of SEQ ID NO: 2, having one or more substitutions, deletions, additions, etc. mutations in the amino acid sequence of SEQ ID NO: 2 having the enzyme activity of the present invention All polypeptides are included within the scope of the present invention.

Hereinafter, the present invention will be described in more detail.

The transgenic gene of the present invention is isolated from the genome sequence of Arabidopsis. First, chromosomal DNA is obtained from the Arabidopsis with the gene transfer gene. As a method for acquiring chromosomal DNA, a known method can be used. In the present invention, cDNA is obtained from total RNA, and the cDNA obtained here is subjected to PCR with a template to obtain a gene of the present invention. Various methods used herein and methods for separating and taking DNA fragments may be known methods such as Molecular Cloning and Cold Spring Harbor Laboratory.

As the vector, a phage vector or plasmid vector capable of autonomous propagation in host microorganisms is used. As a phage vector or a cosmid vector, pWE15, M13, (lambda) EMBL3, (lambda) EMBL4, (lambda) FIXII, (lambda) DASHII, (lambda) ZAPII, (lambda) gt10, (lambda) gt11, Charon4A, Charon21A, etc. are mentioned, for example, pBR system, pUC system, etc. , pBluescriptII system, pGEM system, pTZ system, pET system and the like. In addition, various shuttle vectors such as vectors capable of autonomous propagation in a plurality of host microorganisms such as E. coli and Pseudomonas can also be used. Also for such a vector, desired fragments can be obtained by cleaving with a suitable restriction enzyme as described above.

The linkage between the chromosomal DNA fragment and the vector fragment may be performed by using a DNA ligase, for example, by using a ligation kit (manufactured by Takara Co., Ltd.). In this way, the chromosomal DNA fragment and the vector fragment are linked to produce a mixture of recombinant plasmids containing various DNA fragments (hereinafter referred to as "gene library"). Herein, in the production of gene libraries, in addition to using chromosomal DNA fragments of appropriate length, extracting and purifying mRNA from Bacillus licheniformis strain, and synthesizing cDNA fragments using reverse transcriptase (Molecular Cloning, Cold Spring Harbor Laboratory) , 1982). In addition, the gene library can be transformed or transduced into E. coli once, and then a large amount of the gene library can be amplified (Molecular Cloning, Cold Spring Harbor Laboratory, 1982).

Introduction of the recombinant vector into a host microorganism is performed by a well-known method. For example, if the host microbe is Escherichia coli, the Calcium Chloride Method (Vol. 53, p. 159, 1970), the Method of Enzymology (Vol. 68, p. 253)

1979) or the Electroporation Act (Current Protocols in Molecular Biology, Vol. 1, p. 184, 1994). In addition, the in vitro packaging method (Current Protocols in Molecular Biology, Vol. 1, p. 571, 1994) can be used for transduction in the case of using a cosmid vector or a phage vector. In addition, a method by junction transfer is also available.

In addition, a primer for obtaining a DNA fragment containing the sugar transfer gene of the present invention in Arabidopsis is prepared. The base sequence of the sugar transfer gene of the present invention designs an oligonucleotide from the base sequence as described in SEQ ID NO: 1. These oligonucleotides can be synthesized using, for example, a custom synthesis service of Amarsham-Pharmacia Biotech.

Next, a polymerase chain reaction (hereinafter referred to as "PCR") is performed using the designed oligonucleotide as a primer, and the Arabidopsis-derived DNA as a template to amplify the sugar transfer gene of the present invention.

The DNA fragment containing the sugar transfer gene of the present invention was recovered by recovering the plasmid from the Escherichia coli selected by any of the above methods using an alkaline method (Current Protocols in Molecular Biology, Vol. 1, p. 161, 1994). You can get it. The base sequence of the DNA fragment can be determined by, for example, a Sanger's sequencing method (Molecular Cloning, Vol. 2, p. 133, 1989) and the like. The sequencer 377A (Perkin Elmer) etc. can be used by the diprimer method or the diterminator method.

After determining the entire nucleotide sequence by the above method, the DNA fragment prepared by any method, such as chemical synthesis, PCR using chromosomal DNA as a template, or digestion by restriction enzymes of the DNA fragment having the nucleotide sequence. By hybridizing with a probe, it is possible to obtain the gene of the present invention.

The base sequence of the sugar transfer gene of the present invention in SEQ ID NO: 1 shows the amino acid sequence encoded by the gene in SEQ ID NO: 2, as described above, as long as the polypeptide having the amino acid sequence has the activity of the present invention, There may be variations, such as deletion, substitution, addition, etc., of one or several amino acids of the branch. The gene of the present invention also includes decondensers encoding the same polypeptides which differ only in degenerate codons, in addition to those having a nucleotide sequence encoding the amino acid represented by SEQ ID NO: 2. Herein, mutations such as deletions, substitutions, and additions can be introduced by site mutation introduction methods (Current Protocols in Molecular Biology, Vol. 1, p. 811, 1994) and the like.

The transformed microorganism of the present invention is obtained by introducing the recombinant vector of the present invention into a host suitable for the expression vector used when producing the recombinant vector. As a host, the genus Esherichia, Pseudomonas, Ralstonia, Alcaligenes, Comamonas, Burkholderia, Agrobacte Genus Agrobacterium, Genus Flabobacterium, Genus Vibrio, Genus Enterobacter, Genus Rhizobium, Genus Gluconobacter, Genus Acinetobacter , Genus Moraxella, genus Nitrosomonas, genus Aeromonas, genus Paracoccus, genus Bacillus, genus Clostridium, genus Lactobacillus Genus, Corynebacterium genus, Arthrobacter genus, Achromobacter genus, Micrococcus genus, Mycobacterium genus, Streptococcus genus , Streptomyces Genus, Actinomyces genus, Nocardia genus, Methylobacterium genus and the like various kinds of bacteria can be mentioned. In addition to the above bacteria, yeasts such as Saccharomyces genus and Candida genus, as well as various molds, may be mentioned as hosts.

For example, when a bacterium such as Escherichia coli is used as a host, the recombinant vector according to the present invention can autonomously replicate in the host, and at the same time, a promoter, a DNA containing the sugar transfer gene of the present invention, and a transcription termination sequence It is preferable to have what is necessary for expression of these. Although pGEX was used as the expression vector used in the present invention, any expression vector satisfying the above requirements can be used.

Any promoter can be used as long as it can be expressed in a host. For example, a trp promoter, a trc promoter, a tac promoter, a lac promoter, a PL promoter, a PR promoter, a T7 promoter, a T3 promoter, or the like can be used. Derived promoters can be used. As a method of introducing recombinant DNA into bacteria, the calcium chloride method, electroporation method and the like described above can be used.

When yeast is used as a host, for example, YEp13, YCp50, pRS-based, pYEX-based vectors and the like can be used as expression vectors. As a promoter, a GAL promoter, an AOD promoter, etc. can be used, for example. As a method of introducing recombinant DNA into yeast, for example, the electroporation method (Method Enzymol., 194, 182-187 (1990)), the spheroplast method (Proc. Natl. Acad. Sci. USA, 84, 1929-1933 (1978)), lithium acetate method (J. Bacteriol., 153, 163-168 (1983)), and the like.

In the recombinant vector, fragments for suppressing expression having various functions for suppressing or amplifying or inducing expression, markers for selection of transformants, resistance genes against antibiotics, or secreted out of the cells It is also possible to have a gene for encoding a signal for the purpose of.

Production of the sugar transfer enzyme according to the present invention, for example, culture the transformant obtained by transforming the host with a recombinant vector having a gene encoding the same, the gene product in culture (cultured culture or culture supernatant) Phosphorus transferase is produced and accumulated, and the sugar transferase is obtained from the culture.

As a method for culturing the transformant of the present invention, any conventional method used for culturing a host may be used.

As the culture method, any method used for culturing ordinary microorganisms, such as batch type, flow batch type, continuous culture, and reactor type, can be used. Transformants obtained by using bacteria such as Escherichia coli as a host As a medium for culturing, medium or synthetic medium such as LB medium, M9 medium and the like can be given. In addition, the culture temperature is cultured in the range of the temperature mentioned in the examples of the present invention, such that the sugar transfer enzyme of the present invention accumulates in the cells and recovered.

The carbon source is required for the growth of microorganisms, and for example, sugars such as glucose, fructose, sucrose, maltose, galactose, and starch; Lower alcohols such as ethanol, propanol and butanol; Polyhydric alcohols such as glycerol; Organic acids such as acetic acid, citric acid, succinic acid, tartaric acid, lactic acid and gluconic acid; Fatty acids such as propionic acid, butanoic acid, pentanic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid and dodecanoic acid can be used.

Examples of the nitrogen source include ammonium salts such as ammonia, ammonium chloride, ammonium sulfate and ammonium phosphate, as well as those derived from natural products such as peptone, meat juice, yeast extract, malt extract, caseinate and corn steep liquor. Moreover, as an inorganic substance, 1 potassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, etc. are mentioned, for example. You may add antibiotics, such as kanamycin, ampicillin, tetracycline, chloramphenicol, and streptomycin, to a culture liquid.

In addition, when the promoter cultures the microorganism transformed using an inducible expression vector, an inducer suitable for the type of promoter may be added to the medium. For example, isopropyl- (beta) -D-thiogalactopyranoside (IPTG), tetracycline, indole acrylic acid (IAA), etc. are mentioned as an inducer.

Acquisition and purification of the sugar transfer enzyme of the present invention by centrifuging the cells or supernatant from the obtained culture, cell disruption, extraction, affinity chromatography, cation or anion exchange chromatography, gel filtration alone Or it can carry out by combining suitably.

Confirmation that the obtained purified substance is the target enzyme can be performed by a conventional method, for example, SDS-polyacrylamide gel electrophoresis, western blotting or the like.

In addition, the method of culturing the transformant using the microorganism as a host, the production of the sugar transfer enzyme of the present invention by the transformant and the accumulation in the cells, and the recovery of the sugar transfer enzyme of the present invention from the cells are the aforementioned methods. It is not limited to.

The present invention also relates to compositions such as pharmaceuticals, food and cosmetics containing quercetin 3-O-N-acetylglucosamine.

The quercetin used in the present invention is preferably one obtained by hydrolyzing rutin. A commercial item of quercetin may be used, and chemically synthesized may be used.

In addition, in order to prevent the decomposition of quercetin during the reaction of the present invention, it is preferable to carry out under shading or anaerobic conditions as long as possible.

Quercetin 3-ON-acetylglucosamine of the present invention is a drug for treating diseases, vitamin P enhancers, natural yellow coloring agents with high safety, antioxidants, deodorants, stabilizers, quality improvers, antibacterial agents, preventive agents, ultraviolet absorbers and the like. It can be advantageously used in pharmaceuticals, food, preferences, feed, cosmetics, plastic products and the like.

In addition, since the quercetin 3-ON-acetylglucosamine of the present invention has chemical / enzymatic stability, it is usually used in general foods, symbols, such as seasonings, confectionery, confectionery, beverages, spreads, pastes, pickles, bottles and canned foods, meat products. It is widely used in fish products, milk and egg products, vegetable products, fruit products, grain products, etc., and vitamin P fortification or palatability improvement in feed for animals such as livestock, poultry, bees, silkworms and fish. It can also mix and use for the purpose.

Other cigarettes, toroches, cod liver oil drops, multivitamins, laxatives, deadweight coolants, deadweight tablets, gagle, landscape nutrition, herbal medicines, oral medicines, injections, dentifrices, lipsticks, lip balms, sunburn, etc. And it can also be advantageously used in combination with cosmetics, such as a liquid preference, a preventive agent, a therapeutic agent for susceptible diseases, that is, an anti-sensitive disease agent, a skin beautifier, a skin colorant, a hair restorer, etc., furthermore, UV absorbers, antidegradants, etc. It can also be advantageously used in combination with plastic products.

In addition, the term "sensitive disease" as used herein refers to a disease that is prevented or treated with quercetin 3-ON-acetylglucosamine, for example, viral diseases, bacterial diseases, traumatic diseases, immune diseases, rheumatoid arthritis, circulatory diseases, and malignant tumors. , Neurological diseases, etc., but is not limited thereto.

Quercetin 3-O-N-acetylglucosamine sensitive disease prophylactic and / or therapeutic agents can be freely selected according to their purpose. Examples include liquid preparations such as sprays, eye drops, nasal drops, gargles, and injections, and pastes and powders such as ointments, poultices and creams, solids such as granules, capsules, and tablets.

In formulating, it is also possible to use together with 1 type (s) or 2 or more types from other components, such as a therapeutic agent, a bioactive substance, an antibiotic auxiliary agent, an extender, a stabilizer, a coloring agent, and a flavoring agent, as needed.

The dosage of prophylactic and / or therapeutic agents for susceptible diseases can be adjusted appropriately according to the content of quercetin 3-ON-acetylglucosamine, route of administration and frequency of administration. The range of about 0.001 to 10.0 g per day is suitable.

And in the case of cosmetics in general, according to the aforementioned preventive and / or therapeutic agents, the quercetin 3-O-N-acetylglucosamine of the present invention can be used.

As the method of using quercetin 3-O-N-acetylglucosamine, well-known methods, such as mixing, kneading, dissolving, dipping, infiltration, dispersion, application, spraying, and injection, are appropriately selected in the process until the completion of these products.

Hereinafter, the present invention will be described in detail.

The present inventors have developed a system capable of producing quercetin 3- O- N-Acetylglucosamine using sugar transferase. quercetin is a compound known for various physiological activities such as antioxidant, anti-inflammatory and anti-cancer, and N-acetylglucosamin is a substance known to be functional for joint and cartilage health and skin moisturization. The system was constructed using E. coli produced by the technique.

In the present invention, Arabidopsis thaliana (Arabidopsis The gene sequence of the sugar transferase cloned in thaliana ) is provided, and the metabolic regulation of Escherichia coli used in its cloning method and production method is provided.

The inventors have isolated the glycogene from the genome sequence of Arabidopsis. The isolated gene was used pGEX Escherichia coli expression vector system modified by gene recombination. The newly recombined gene was introduced into E. coli. E. coli was used to produce quercetin 3- O- N-Acetylglucosamine as a substrate. In addition, the production of quercetin 3- O -N-Acetylglucosamine was increased by the metabolic regulation of Escherichia coli.

As can be seen through the present invention, the present invention can synthesize a quercetin 3- O -N-Acetylglucosamine from Quercetin by expressing a sugar transferase cloned in plants in Escherichia coli, a novel substance Quercetin 3- O -N -Acetylglucosamine has an effect that can be applied to various foods or medicines due to the functionality of each component of the compound.

1 is a diagram illustrating the reaction mediated by the gene of the present invention.
Figure 2a is a graph showing the HPLC flow chart of the analysis of the expression of the AtUGT78D2 gene of the present invention in Escherichia coli BL21DE3 bioconversion to the substrate;
FIG. 2B is a graph showing an HPLC flow chart of the AtUGT78D2 gene of the present invention expressed in BL21DE3 pgi Escherichia coli and subjected to bioconversion with quercetin as a substrate. FIG.
2C is a graph showing an HPLC flow chart of the AtUGT78D2 gene of the present invention expressed in BL21DE3 Escherichia coli and subjected to bioconversion using quercetin as a substrate.
FIG. 2D is a graph showing an HPLC flow chart of the AtUGT78D2 gene of the present invention expressed in BL21DE3 pgm Escherichia coli and subjected to bioconversion of quercetin to a substrate.
Figure 3 is a diagram showing the NMR data of the sample Quercetin-3 -O-N-acetyl glucosamine.

Hereinafter, the present invention will be described in more detail through non-limiting examples. However, the following examples are set forth to illustrate the present invention and the scope of the present invention is not to be construed as being limited by the following examples.

Example  One : AtUGT78D2  Isolation of genes

Total RNA isolated from the entire plant of Arabidopsis larvae grown for about 20 days and reverse transcription using oligo dT as a primer was synthesized cDNA. PCR was performed using two primers prepared based on the nucleotide sequence obtained from the genome database of Arabidopsis with the synthesized cDNA as a template. The base sequence used at this time is as follows.

AtUGT78D2

1.5'-AAGAATTCATGACCAAACCCTCCGACCC-3 '(SEQ ID NO: 3)

2. 5'-AACTCGAGCATTCAAATAATGTTTACAACTGC-3 '(SEQ ID NO: 4)

PCR was performed by mixing Taq DNA polymerase with the above reverse transcript and primers at 94 ° C for 1 min. 55 ° C. 1 min. 72 ° C. 1 min. The cycle of 30 sec was repeated 40 times to amplify the AtUGT78D2 gene corresponding to 1,383 bp. The amplified PCR product was cloned into pGETM-easy, which is an E. coli cloning vector, and its nucleotide sequence was determined.

Example  2 : AtUGT78D2 Vector construction to express genes

In order to express the glycotransferase cloned in Example 1, it was transferred to pGEX, an E. coli expression vector. For cloning, PCR was performed using primers of the AtUGT78D2 glycotransferase gene and pyrobest taq polymerase prepared by inserting EcoR I and Xho I restriction enzyme sites, and the PCR reaction was purified by PCR purification kit and then EcoRI and XhoI restriction enzyme. And then cloned into the same restriction site of the pGEX vector. This was used to confirm the production of new products when Quercetin was used as a substrate.

Example  3: AtUGT78D2 Production of New Substances Using Expression and Bioconversion

Escherichia coli BL21 with AtUGT78D2 was inoculated into 2ml LB medium with 50 ㎍ / ml empicillin and seed cultured overnight. Seed cultured E. coli is inoculated in a new 50ml LB medium and incubated in a 37 ° C shacking incubator. Escherichia coli is incubated until absorbance reaches 0.6 at 600 nm. When the cell density reached 0.6 at 600 nm, IPTG was added to the final concentration of 0.1 mM in Escherichia coli, and the protein was expressed at 25 ° C. for 15 hours. The cultured E. coli was centrifuged to discard the supernatant, and the recovered E. coli was adjusted to a cell density of 3 at an absorbance of 600 in M9 medium containing 50 ㎍ / ml empicillin and 2% glucose. The substrate is added to a final concentration of 500 uM and shaken at 30 ° C. for 24 hours. The supernatant is mixed well with the same amount of ethyl acetate and extracted twice over a period of time. The extracted reactant is completely dried using speed vac. The completely dried reactant was again dissolved in 100 uM DMSO and used for HPLC analysis.

Biotransformation of Quercetin into the substrate using E. coli transformed with AtUGT78D2 produced two new reactants. The molecular weights of the two materials were analyzed using the negative mode of LC-MS / MS and found to be 463-Da and 504-Da. The first reaction was compared with the standard and NMR analysis showed that the product was quercetin 3- O -glucoside. Since the second material was not able to use the standard material, the analysis was performed using NMR, and it was found that quercetin-3- O- N-Acetylglucosamine (see FIG. 3).

AtUGT78D2 is a glycosyltransferase that requires sugar-donor and sugar-acceptor. In the present invention, the sugar-acceptor was quercetin and the sugar-donor was considered to be UDP-glucose and UDP-N-Acetylglucosamine in view of the product structure. As a result of confirming the metabolic pathway to make UDP-N-acetylglucosamine in Escherichia coli, the gene pgi (phosphoglucose isomerase; Mol Syst Biol. Pp 1-11, 2006) produces UDP-N-Acetylglucosamine from D-glucose-6-phosphate. It was found that the gene is the first step.

Deletion of the pgi (phosphoglucose isomerase) gene in E. coli was produced using the FRT-PGK-gb2-neo-FRT kit from GENE BRIDGES (Germany) and the following two primers. It was confirmed that E. coli was not produced quercetin 3- O -N-Acetylglucosamine.

phosphoglucose isomerase (pgi) primer sequences

5'-TACAATCTTCCAAAGTCACAATTCTCAAAATCAGAAGAGTATTGCTAATGaattaaccctcactaaagggcg-3 '(SEQ ID NO: 5)

5'-GCCTTATCCGGCCTACATATCGACGATGATTAACCGCGCCACGCTTTATAtaatacgactcactatagggctc-3 '(SEQ ID NO: 6)

Example  4: using biotransformation quercetin -3-O-N- Acetylglucosamine Production

Biotransformation was used to regulate the metabolism of E. coli to increase the production of quercetin-3-O-N-Acetylglucosamine. Synthetic pathway of UDP-glucose and UDP-N-Acetylglucosamine is based on D-glucose-6-phosphate as a substrate, and the synthesis of UDP-N-Acetylglucosamine is initiated by the gene pgi, whereas pgm (phosphoglucomutase; Mol Syst Biol. Pp 1 -11, 2006) initiates the synthesis of UDP-glucose

Therefore, in order to increase the production of UDP-N-Acetylglucosamine, the pgm gene involved in UDP-glucose synthesis metabolism was deleted. The deletion method was prepared using the FRT-PGK-gb2-neo-FRT kit of GENE BRIDGES (Germany) and the following two primers.

pgm (phosphoglucomutase) primer sequence;

 5'-GAAGGTTTGCGGAACTATCTAAAACGTTGCAGACAAAGGACAAAGCAATGaattaaccctcactaaagggcg-3 '(SEQ ID NO: 7)

5'-TGCGACCGCCCTTTTTTTATTAAATGTGTTTACGCGTTTTTCAGAACTTCtaatacgactcactatagggctc-3 '(SEQ ID NO: 8)

As a result, the flow of UDP-N-Acetylglucosamine biosynthesis was greatly improved, and the production of quercetin-3- O -N-Acetylglucosamine was also greatly increased. The yield was about 8 times improved in the pgm mutant compared to the wild type (see Figure 2d).

<110> Konkuk University Industrial Cooperation Corp. <120> A novel compound, quercetin 3-O-N-Acetylglucosamine, gene for          producing the compound, and method for producing the compound <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 1383 <212> DNA <213> Artificial Sequence <220> <223> AtUGT78D2 <400> 1 atgaccaaac cctccgaccc aaccagagac tcccacgtgg cagttctcgc ttttcctttc 60 ggcactcatg cagctcctct cctcaccgtc acgcgccgcc tcgcctccgc ctctccttcc 120 accgtcttct ctttcttcaa caccgcacaa tccaactctt cgttattttc ctccggtgac 180 gaagcagatc gtccggcgaa catcagagta tacgatattg ccgacggtgt tccggaggga 240 tacgtgttta gcgggagacc acaggaggcg atcgagctgt ttcttcaagc tgcgccggag 300 aatttccgga gagaaatcgc gaaggcggag acggaggttg gtacggaagt gaaatgtttg 360 atgactgatg cgttcttctg gttcgcggct gatatggcga cggagataaa tgcgtcgtgg 420 attgcgtttt ggaccgccgg agcaaactca ctctctgctc atctctacac agatctcatc 480 agagaaacca tcggtgtcaa agaagtaggt gagcgtatgg aggagacaat aggggttatc 540 tcaggaatgg agaagatcag agtcaaagat acaccagaag gagttgtgtt tgggaattta 600 gactctgttt tctcaaagat gcttcatcaa atgggtcttg ctttgcctcg tgccactgct 660 gttttcatca attcttttga agatttggat cctacattga cgaataacct cagatcgaga 720 tttaaacgat atctgaacat cggtcctctc gggttattat cttctacatt gcaacaacta 780 gtgcaagatc ctcacggttg tttggcttgg atggagaaga gatcttctgg ttctgtggcg 840 tacattagct ttggtacggt catgacaccg cctcctggag agcttgcggc gatagcagaa 900 gggttggaat cgagtaaagt gccgtttgtt tggtcgctta aggagaagag cttggttcag 960 ttaccaaaag ggtttttgga taggacaaga gagcaaggga tagtggttcc atgggcaccg 1020 caagtggaac tgctgaaaca cgaagcaacg ggtgtgtttg tgacgcattg tggatggaac 1080 tcggtgttgg agagtgtatc gggtggtgta ccgatgattt gcaggccatt ttttggggat 1140 cagagattga acggaagagc ggtggaggtt gtgtgggaga ttggaatgac gattatcaat 1200 ggagtcttca cgaaagatgg gtttgagaag tgtttggata aagttttagt tcaagatgat 1260 ggtaagaaga tgaaatgtaa tgctaagaaa cttaaagaac tagcttacga agctgtctct 1320 tctaaaggaa ggtcctctga gaatttcaga ggattgttgg atgcagttgt aaacattatt 1380 tga 1383 <210> 2 <211> 460 <212> PRT <213> Artificial Sequence <220> <223> AtUGT78D2 <400> 2 Met Thr Lys Pro Ser Asp Pro Thr Arg Asp Ser His Val Ala Val Leu   1 5 10 15 Ala Phe Pro Phe Gly Thr His Ala Ala Pro Leu Leu Thr Val Thr Arg              20 25 30 Arg Leu Ala Ser Ala Ser Pro Ser Thr Val Phe Ser Phe Phe Asn Thr          35 40 45 Ala Gln Ser Asn Ser Ser Leu Phe Ser Ser Gly Asp Glu Ala Asp Arg      50 55 60 Pro Ala Asn Ile Arg Val Tyr Asp Ile Ala Asp Gly Val Pro Glu Gly  65 70 75 80 Tyr Val Phe Ser Gly Arg Pro Gln Glu Ala Ile Glu Leu Phe Leu Gln                  85 90 95 Ala Ala Pro Glu Asn Phe Arg Arg Glu Ile Ala Lys Ala Glu Thr Glu             100 105 110 Val Gly Thr Glu Val Lys Cys Leu Met Thr Asp Ala Phe Phe Trp Phe         115 120 125 Ala Ala Asp Met Ala Thr Glu Ile Asn Ala Ser Trp Ile Ala Phe Trp     130 135 140 Thr Ala Gly Ala Asn Ser Leu Ser Ala His Leu Tyr Thr Asp Leu Ile 145 150 155 160 Arg Glu Thr Ile Gly Val Lys Glu Val Gly Glu Arg Met Glu Glu Thr                 165 170 175 Ile Gly Val Ile Ser Gly Met Glu Lys Ile Arg Val Lys Asp Thr Pro             180 185 190 Glu Gly Val Val Phe Gly Asn Leu Asp Ser Val Phe Ser Lys Met Leu         195 200 205 His Gln Met Gly Leu Ala Leu Pro Arg Ala Thr Ala Val Phe Ile Asn     210 215 220 Ser Phe Glu Asp Leu Asp Pro Thr Leu Thr Asn Asn Leu Arg Ser Arg 225 230 235 240 Phe Lys Arg Tyr Leu Asn Ile Gly Pro Leu Gly Leu Leu Ser Ser Thr                 245 250 255 Leu Gln Gln Leu Val Gln Asp Pro His Gly Cys Leu Ala Trp Met Glu             260 265 270 Lys Arg Ser Ser Gly Ser Val Ala Tyr Ile Ser Phe Gly Thr Val Met         275 280 285 Thr Pro Pro Pro Gly Glu Leu Ala Ala Ile Ala Glu Gly Leu Glu Ser     290 295 300 Ser Lys Val Pro Phe Val Trp Ser Leu Lys Glu Lys Ser Leu Val Gln 305 310 315 320 Leu Pro Lys Gly Phe Leu Asp Arg Thr Arg Glu Gln Gly Ile Val Val                 325 330 335 Pro Trp Ala Pro Gln Val Glu Leu Leu Lys His Glu Ala Thr Gly Val             340 345 350 Phe Val Thr His Cys Gly Trp Asn Ser Val Leu Glu Ser Val Ser Gly         355 360 365 Gly Val Pro Met Ile Cys Arg Pro Phe Phe Gly Asp Gln Arg Leu Asn     370 375 380 Gly Arg Ala Val Glu Val Val Trp Glu Ile Gly Met Thr Ile Ile Asn 385 390 395 400 Gly Val Phe Thr Lys Asp Gly Phe Glu Lys Cys Leu Asp Lys Val Leu                 405 410 415 Val Gln Asp Asp Gly Lys Lys Met Lys Cys Asn Ala Lys Lys Leu Lys             420 425 430 Glu Leu Ala Tyr Glu Ala Val Ser Ser Lys Gly Arg Ser Ser Glu Asn         435 440 445 Phe Arg Gly Leu Leu Asp Ala Val Val Asn Ile Ile     450 455 460 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 aagaattcat gaccaaaccc tccgaccc 28 <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 aactcgagca ttcaaataat gtttacaact gc 32 <210> 5 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 tacaatcttc caaagtcaca attctcaaaa tcagaagagt attgctaatg aattaaccct 60 cactaaaggg cg 72 <210> 6 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gccttatccg gcctacatat cgacgatgat taaccgcgcc acgctttata taatacgact 60 cactataggg ctc 73 <210> 7 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gaaggtttgc ggaactatct aaaacgttgc agacaaagga caaagcaatg aattaaccct 60 cactaaaggg cg 72 <210> 8 <211> 73 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 tgcgaccgcc ctttttttat taaatgtgtt tacgcgtttt tcagaacttc taatacgact 60 cactataggg ctc 73

Claims (8)

Quercetin-3-O-N-acetylglucosamine compound that is a combination of quercetin and N-acetylglucosamin. A gene that mediates the binding reaction between quercetin and N-acetylglucosamin to produce the quercetin-3-O-N-acetylglucosamine compound of claim 1. The gene according to claim 2, wherein the gene has a nucleotide sequence of SEQ ID NO: 1. a) preparing an expression vector comprising the gene of claim 2 or 3;
b) transforming the expression vector into a host cell: And
c) A method for preparing the quercetin-3-ON-acetylglucosamine compound of claim 1, comprising adding quercetin as a substrate to the host cell. The method of claim 4, wherein the host cell is a deletion of the phosphoglucomutase gene involved in UDP-glucose synthesis metabolism (1) characterized in that the quercetin-3-O-N-acetylglucosamine compound of claim 1. A composition comprising the compound of claim 1 as an active ingredient. An enzyme that mediates the binding reaction between quercetin and N-acetylglucosamin to produce the quercetin-3-O-N-acetylglucosamine compound of claim 1. 8. The enzyme according to claim 7, wherein the enzyme has an amino acid sequence as set forth in SEQ ID NO: 2.

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