KR20150015670A - Novel compound Luteolin-7-O-N-acetyl-D-glucosaminuronic acid and method for producing the same - Google Patents

Abstract

The present invention relates to a method for synthesizing Luteolin-7-O-N-acetyl-D-glucosaminuronic acid which is a novel compound in Luteolin by expressing an Antirrhinum majus UDP-dependent glycosyltransferase (AmUGT) gene and an UDP-N-acetylglucosamine dehydrogenase (UGlcNAcDH) gene derived from Bacillus cereus subsp. in colon bacterium whose metabolism is controlled.

Description

7-O-N-acetyl-D-glucosamine uronic acid compound and a method for producing the same.

The present invention is a novel luteolin -7 -ON - acetyl -D- glucosamine-uronic acid compounds and relates to a production method thereof, and more particularly, snapdragon-derived sugar transferase AmUGT (Antirrhinum majus dependent UDP-glycosyltransferase) genes and Bacillus celebrity Bacillus cereus subsp . ) -7 -ON the UGlcNAcDH (UDP- N -acetylglucosamine dehydrogenase) gene derived by expressing the E. coli metabolic regulation in De ruthenate in luteolin-acetyl -D- glucosamine-uronic acid (Luteolin-7 -ON -acetyl-D- glucosaminuronic acid. < / RTI >

In general, natural products can be enzymatically modified by introducing genes useful for microorganisms such as E. coli, which are well established in culture methods and genetic engineering techniques. This method is called biotransformation. Bioconversion has the advantage of saving costly cofactors in the middle of the reaction.

Flavonoids, which have attracted much attention as new drug candidates, are one of the many secondary metabolites produced by plants. Flavonoids have been reported to have various physiological activities such as antioxidant, anti-cancer, anti-inflammatory and anti-allergic effects. The glycosylation of these flavonoids is known to increase the solubility and at the same time increase the water absorption. Such natural structural changes cause changes in physiological activity. Typical structural modifications involve the use of glycosyltransferases, which transfer a molecule of glucose to the hydroxyl group (-OH) .

Luteolin is an excellent antioxidant and an excellent free radical scavenger for flavonoids present in fruits and vegetables. In addition, enzymatic lipid peroxidation, non-enzymatic lipid peroxidation and CCl ₄ - inhibits all-induced lipid peroxidation. Luteolin has a beneficial effect on cardiovascular and may prevent the development of atherosclerosis. In particular, the anticancer effect of luteolin is evident from the significant antiproliferative activity against various human tumor cell lines. In addition, luteolin has been reported for its anti-inflammatory, antiviral, antibacterial and radiation protection properties and its prophylactic effect on the development of diabetic cataract as an inhibitor of the enzyme aldose reductase has also been reported.

The transglycosylase utilizes a nucleotide sugar as a donor. The present invention analyzes the nucleic acid sugar metabolism pathway of E. coli to make a new structure of the nucleic acid sugar in E. coli, Bacillus sereus subspecies ( Bacillus cereus subsp . ) UGlcNAcDH (UDP- N- acetylglucosamine 6-dehydrogenase) gene was introduced into E. coli. UGlcNAcDH gene, UDP- N-acetylglucosamine using a (acetylglucosamine) UDP- N-to a substrate by a uronic acid glucosamine transferase per-acetyl-glucosamine-uronic acid creates a (glucosaminuronic acid), thus created UDP- N -acetyl It is saccharified. The present invention was used as a substrate, luteolin, luteolin -7 -ON novel compound through the reaction as above-acetyl -D- glucosamine-uronic acid (Luteolin-7 -ON -acetyl-D -glucosaminuronic acid) synthesis Respectively.

These substances do not exist in nature, and it is almost impossible to synthesize them using existing chemical methods. The bioconversion method (enzymatic synthesis method) has several advantages over the chemical synthesis method because it has position selectivity and chiral selectivity which are difficult to be modified by a chemical synthesis method. Therefore, the bioconversion method using the two genes ( AmUGT and UGlcNAcDH ) of the present invention using E. coli could be a new method for producing luteolin-7- ON -acetyl-D-glucosamine uronic acid.

Korean Patent No. 0716797 discloses a process for preparing a derivative of a precursor compound using the enzyme and a derivative prepared therefrom. Korean Patent Laid-Open No. 2001-0085971 discloses a process for preparing a derivative of luteolin and its derivative 7- ON -acetyl-D-glucosamine uronic acid compound of the present invention and a method for producing the same are not disclosed.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and the present inventors have found that the present invention provides a transgenic transferase AmugT ( Antirrhinum majus UDP-dependent glycosyltransferase) genes and subspecies of Bacillus cereus (Bacillus cereus subsp . ) Derived UGlcNAcDH (UDP- N -acetylglucosamine dehydrogenase) and expressing the gene in the E. coli metabolic regulation, raised in a novel and a luteolin using the transformed E. coli as the substrate compound ruthenate -7 -ON - acetyl -D- glucosamine Uronic acid, thereby completing the present invention.

In order to solve the above problems, the present invention relates to novel luteolin -7 -ON - provides acetyl -D- glucosamine-uronic acid (Luteolin-7 -ON -acetyl-D -glucosaminuronic acid) compound.

In addition, the present invention derived from snapdragon sugar transferase AmUGT (Antirrhinum majus UDP-dependent glycosyltransferase gene and UGlcNAcDH (UDP- N- acetylglucosamine dehydrogenase) gene derived from Bacillus cereus subsp . Or snapdragon provides expression vectors and Bacillus cereus subspecies derived UGlcNAcDH composition for preparing the novel compounds containing an expression vector containing the gene derived from transition per including AmUGT enzyme gene.

In addition,

a) transferase per snapdragon-derived AmUGT (Antirrhinum majus UDP-dependent glycosyltransferase) genes and subspecies of Bacillus cereus (Bacillus cereus subsp . An expression vector comprising UGlcNAcDH (UDP- N- acetylglucosamine dehydrogenase) gene; Or snapdragon to prepare an expression vector, and Bacillus cereus subspecies UGlcNAcDH derived expression vector containing the gene derived from transition per including AmUGT enzyme gene;

b) transforming the expression vectors into E. coli; And

c) adding the luteolin to the substrate while culturing the transformed E. coli.

In addition, the present invention derived from snapdragon sugar transferase AmUGT (Antirrhinum majus UDP-dependent glycosyltransferase gene and UGlcNAcDH (UDP- N- acetylglucosamine dehydrogenase) gene derived from Bacillus cereus subsp . Or an expression vector containing a glycosyltransferase- transferring enzyme AmUGT gene and an expression vector comprising a UGlcNAcDH gene derived from Bacillus subtilis subspecies to produce the novel compound.

The present invention also provides a food composition comprising the novel luteolin-7- ON -acetyl-D-glucosamine uronic acid compound as an active ingredient.

The present invention relates to the synthesis of novel compounds by expressing the transferase gene AmUGT and Bacillus cereus subspecies UGlcNAcDH derived gene derived from E. coli per snapdragon. As can be seen from the present invention, a novel compound, luteolin-7- ON -acetyl-D-glucosamine uronic acid, was produced from luteolin as a substrate through the E. coli system of the present invention. The compound of the present invention, luteolin-7- ON -acetyl-D-glucosamine uronic acid, can be usefully used in various foods, medicines and cosmetics due to the functional properties of the respective components constituting the compound.

1 is a schematic illustration of synthesis of a novel compound, luteolin-7- ON -acetyl-D-glucosamine uronic acid, from a substrate luteolin.
Fig. 2 is a graph showing the effect of the sugar chain transferase AmugT And UGlcNAcDH gene derived from Bacillus cereus subtype were transformed with luteolin to a substrate, and analyzed by high performance liquid chromatography (HPLC).
FIG. 3 is a mass spectrometry (MS) result of a reaction product obtained by biotransformation of luteolin to a substrate using metabolically controlled E. coli transformed with the transglycosylation enzyme AmUGT gene and the UGlcNAcDH gene derived from Bacillus cereus subsp .
Fig. 4 is a graph showing the effect of the sugar chain transferase AmugT Gene and the UGlcNAcDH gene derived from Bacillus cereus subsp. ≪ / RTI > gene. Product; Luteolin-7- ON -acetyl-D-glucosamine uronic acid, Substrate; Luteolin.
5 is by varying the temperature and time on the protein expression induced culture conditions in the metabolism of E. coli was transformed with pCDFDuet vector comprising a transferase AmUGT gene and Bacillus cereus subspecies derived UGlcNAcDH gene per snapdragon-derived compare the production of the novel compounds This is a result.
6 is a result of comparing the production of the novel compounds by the cell concentration of the protein expression induced culture conditions in the metabolism of E. coli was transformed with pCDFDuet vectors per snapdragon-derived transferase include AmUGT gene and Bacillus cereus subspecies derived UGlcNAcDH gene . Product; Luteolin-7- ON -acetyl-D-glucosamine uronic acid.
Fig. 7 is a graph showing the effect of the sugar chain transferase AmugT 7- ON -acetyl-D-glucosamine uronic acid compound was produced by culturing the metabolically-regulated Escherichia coli transformed with an expression vector containing the gene and the UGlcNAcDH gene derived from Bacillus subtilis subsp . The result is confirmed. P; Luteolin-7- ON -acetyl-D-glucosamine uronic acid, S; Luteolin.

According to an aspect of the invention, the invention is to luteolin -7 -ON of formula - provides acetyl -D- glucosamine-uronic acid (Luteolin-7 -ON -acetyl-D -glucosaminuronic acid) compound.

In addition, the present invention derived from snapdragon sugar transferase AmUGT (Antirrhinum majus UDP-dependent glycosyltransferase gene and UGlcNAcDH (UDP- N- acetylglucosamine dehydrogenase) gene derived from Bacillus cereus subsp . Or snapdragon provides a composition for preparing the compounds which contain the expression vector and Bacillus cereus subspecies UGlcNAcDH derived expression vector containing the gene derived from transition per including AmUGT enzyme gene.

The composition according to one embodiment of the present invention may be introduced into one expression vector or introduced into separate expression vectors, respectively, in which the AmUGT gene and the UGlcNAcDH gene are introduced.

In the composition according to an embodiment of the present invention, the sugarcane- derived transgene-derived AmugT gene may be a nucleotide sequence of SEQ ID NO: 1. In addition, homologues of the nucleotide sequences are included within the scope of the present invention. Specifically, the gene has a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more, with the nucleotide sequence of SEQ ID NO: 1 . "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

Also, in the composition according to an embodiment of the present invention, the UGlcNAcDH gene derived from Bacillus cereus subtype may be composed of the nucleotide sequence of SEQ ID NO: 2. In addition, homologues of the nucleotide sequences are included within the scope of the present invention, and the details thereof are as described above.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector ". The term "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

The vector of the present invention can typically be constructed as a vector for cloning or expression. In addition, the vector of the present invention can be constructed using prokaryotic cells as hosts. For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (e.g., pL? Promoter, trp promoter, lac promoter, T7 promoter, tac promoter, etc.) It is common to include a ribosome binding site and a transcription / translation termination sequence for initiation of translation. When E. coli is used as a host cell, the promoter and operator site of the E. coli tryptophan biosynthesis pathway and the left promoter of the phage lambda (pL 貫 promoter) can be used as a regulatory region.

The vectors that can be used in the present invention include plasmids such as pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pGEX series, pET series and pUC19 which are frequently used in the art, phages such as λgt4 · λB ,? -charon,?? z1, and M13), or a virus (e.g., SV40, etc.).

The vector of the present invention may be a selection marker and may include an antibiotic resistance gene commonly used in the art, for example, ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, And resistance genes for tetracycline.

In addition, the composition according to an embodiment of the present invention may further include E. coli, and more preferably, it may include E. coli lacking a phosophoglucumutase, but the present invention is not limited thereto.

In addition,

The transgenic enzyme AmugT ( Antirrhinum majus UDP-dependent glycosyltransferase) genes and subspecies of Bacillus cereus (Bacillus cereus subsp . An expression vector comprising UGlcNAcDH (UDP- N- acetylglucosamine dehydrogenase) gene; Or snapdragon to prepare an expression vector, and Bacillus cereus subspecies UGlcNAcDH derived expression vector containing the gene derived from transition per including AmUGT enzyme gene;

Transforming the expression vectors into Escherichia coli; And

7- ON -acetyl-D-glucosamine uronic acid, comprising the step of adding luteolin to the substrate while culturing the transformed E. coli.

In the method according to one embodiment of the present invention, the A. tumefaciens AmugT gene derived from Snapdragon can be a base sequence of SEQ ID NO: 1, and the UGlcNAcDH gene derived from Bacillus subtilis may be a base sequence of SEQ ID NO: 2. In addition, homologues of the nucleotide sequences are included within the scope of the present invention, and the details thereof are as described above.

The method of delivering the vector of the present invention into a host cell can be carried out by a CaCl ₂ method, a Han method (Hanahan, 1983, J. Mol. Biol. 166: 557-580) and an electroporation method when the host cell is a prokaryotic cell . ≪ / RTI >

In the method according to one embodiment of the present invention, the E. coli transformed with the expression vectors may be, but is not limited to, Escherichia coli lacking a phosophoglucutase.

In the method according to another embodiment of the present invention, the protein expression inducing culture conditions of the transformed E. coli for producing luteolin-7- ON -acetyl-D-glucosamine uronic acid are at a temperature of 15 to 30 DEG C, a culture time of 12 To 72 hours and a cell concentration OD _{600 of} 2 to 10, preferably at a temperature of 17 to 19 ° C, a culturing time of 45 to 55 hours, a cell concentration of 600 nm = 4 to 5, most preferably 18 ° C , 48 hours culture and cell concentration OD ₆₀₀ = 5, but is not limited thereto.

The present invention also relates to a method for producing a sugar chain- transferring enzyme AmugT ( Antirrhinum majus UDP-dependent glycosyltransferase gene and UGlcNAcDH (UDP- N- acetylglucosamine dehydrogenase) gene derived from Bacillus cereus subsp . Or snapdragon is transformed with the expression vector and Bacillus cereus subspecies expression vector containing the gene derived UGlcNAcDH derived per transition including enzyme gene AmUGT luteolin -7 -ON - service coli producing acetyl -D- glucosamine-uronic acid do.

In one embodiment of the present invention, the E. coli is preferably E. coli deficient in a phosophoglucumutase, but is not limited thereto.

The present invention also provides a food composition comprising the novel luteolin-7- ON -acetyl-D-glucosamine uronic acid compound as an active ingredient. Foods containing the compound of the present invention are not limited, but include cold confectionery products such as ice cream, sherbet and ice cream; Beverages such as milk drinks, lactic acid beverages, soft drinks (including juice), carbonated drinks, vegetable / fruit drinks, sports drinks, powdered drinks; Alcoholic drinks such as liqueur; Coffee beverages such as coffee beverages and tea beverages; Soups such as consommé and soup; Puddings such as custard pudding, milk pudding, juice-containing pudding, desserts such as jelly, babaloa and yogurt; Chewing gum or balloon gum (stick gum, sugar coated gum ball); Chocolate such as chocolate coated with berries such as strawberry chocolate, blueberry chocolate and melon chocolate in addition to coated chocolate such as marble chocolate; Such as hard candy (including sweets, butter balls, marbles, etc.), soft candy (including caramel, nougat, gummy candy, marshmallow, etc.) caramels such as drop and taffy; Baked confectionery such as hard biscuits, cookies, okaki (rice crackers), sembei (rice crackers); Sauces such as pickles, kimchi, separate dressings, oil-free dressings, ketchup, dips and sauces; Jams such as strawberry jam, blueberry jam, marmalade, apple jam, apricot jam, and preserves; Fruit wine such as red wine; Fruit for processing syrup, cherry, apricot, apple, strawberry, and pear; Meat products such as ham, sausage, and roast pork; Fish meat ham, fish sausage, fish fillet, fish cake; Dairy products such as cheese; Noodles such as udon, cold noodle, somen, buckwheat noodle, Chinese buckwheat noodle, spaghetti, macaroni, rice bifun, bean curd and mandarin; And various kinds of processed foods such as various kinds of corrosion.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  1. Snapdragon Transglycosylase  Gene Cloning  And Escherichia coli Expression Vector Production

Isolation of Transgene Enzyme Gene from Snapdragon

Snapdragon flowers were finely ground with liquid nitrogen and total RNA was isolated. CDNA was synthesized by reverse transcription using oligo-dT as a primer using the separated total RNA. (5'-GC CATATG GAGGACACTATCGTTCTCTACG-3 '; SEQ ID NO: 3, underlined; NdeI (SEQ ID NO: 3)) which was prepared by inserting NdeI and XhoI restriction enzyme sites based on the nucleotide sequence obtained from the genome of the Snapdragon (5'-GC CTCGAG TTAAGAAACCACCATATCAACA-3 '; SEQ ID NO: 4, underlined; XhoI PCR primer (polymerase chain reaction) was performed using primers.

The PCR was carried out by repeating the cycle of 94 ° C. for 60 seconds, 55 ° C. for 60 seconds, and 72 ° C. for 90 seconds by repeating 40 times of Taq polymerase with the above cDNA and primers. As a result, about 1.3 kbp of pertussis transferase AmugT ( Antirrhinum majus UDP-dependent glycosyltransferase gene. The amplified DNA was transferred to the E. coli cloning vector pGEMT-easy to determine the nucleotide sequence.

AmUGT Vector generation for gene expression

In order to express the cloned transglycosylated glycosyltransferase gene, the NdeI and XhoI restriction sites were inserted into AmugT The secondary PCR was carried out using forward and reverse primers (SEQ ID NO: 3 and SEQ ID NO: 4) of the glycosyltransferase gene and pfu Taq polymerase. The PCR products were then treated with restriction enzymes and then ligated with pACYCDuet, pCDFDuet and pETDuet vector Novagen, Germany).

Example  2. UGlcNAcDH Synthesis of genes and Escherichia coli expression vector production

UGlcNAcDH Synthesis of genes

Bacillus cereus subsp. Cytotoxis NVH391-98 ( Bacillus cereus subsp. cytotoxis NVH391-98) derived UGlcNAcDH The gene was synthesized in biona (Korea) based on the nucleotide sequence of a previously published paper (Gu et al., 2010, J. Biol. Chem. 285: 24825-24833) Lt; / RTI > Also UGlcNAcDH In the synthesis of the gene, the EcoRI site was inserted at the 5 'end and the NotI site was inserted at the 3' end.

UGlcNAcDH Vector generation for gene expression

To express the synthesized UGlcNAcDH gene, DNA synthesized with EcoRI and NotI restriction enzyme was treated, and AmUGT Were inserted into pACYCDuet, pCDFDuet and pETDuet vectors in which the gene was inserted.

Example  3. AmUGT Gene and UGlcNAcDH  Generation of new substances using recombinant E. coli and bioconversion

The E. coli BL21 (DELTA pgm), which is deficient in the phosphoglucurcomutase, And an expression vector containing the UGlcNAcDH gene were transformed. AmUGT The transformed E. coli expressing the gene and the UGlcNAcDH gene were cultured in LB medium supplemented with antibiotics of 50 占 퐂 / ml of chloramphenicol (pACYCDuet vector), 50 占 퐂 / ml of spectinomycin (pCDFDuet vector) and 50 占 퐂 / ml of ampicillin (pETDuet vector) . The overnight cultured Escherichia coli strain was inoculated into fresh LB medium and cultured at 600 nm to an absorbance value of 0.8. IPTG was added to the cultured Escherichia coli at a final concentration of 1 mM, and the protein was expressed at 18 ° C for 20 hours. The cultured Escherichia coli was centrifuged and the supernatant was discarded. The recovered Escherichia coli contained 50 μg / ml of chloramphenicol (pACYCDuet vector), 50 μg / ml of spectinomycin (pCDFDuet vector), 50 μg / ml of ampicillin (pETDuet vector) % Glucose was added to the M9 medium at an absorbance of 600 nm to a value of 5.0. Luteolin, a substrate for the final concentration of 100 μM, was added thereto, followed by incubation at 30 ° C. for 24 hours with shaking. The supernatant was extracted using boiling. The reactants were used for high performance liquid chromatography (HPLC) analysis.

As a result, the generation of a new compound from luteolin was confirmed (Fig. 2), and the molecular weight of the substance was confirmed by mass spectrometry. As a result, it was confirmed that the substance had a size of 502.3-Da (Fig.

Example  4: Luteolin-7 by Escherichia coli Expression Vector -O-N -Acetyl-D- Glucosamine uronic acid  Comparison of production

Luteolin -7 -ON - acetyl -D- glucosamine-uronic acid (luteolin-7 -ON -acetyl-D -glucosaminuronic acid) of comparing the difference between the E. coli expression vector for the expression of specific production UGlcNAcDH genes for optimization of production conditions saw. The types of expression vectors used are pACYCDuet, pCDFDuet and pETDuet vectors. Production of a new compound was confirmed in the same manner as in Example 3 above. Each vector-specific reactant was analyzed using high performance liquid chromatography.

As a result, when the same amount of luteolin was added to the substrate, the amount of luteolin-7- ON -acetyl-D-glucosamine uronic acid, which is a novel compound, was the highest in the E. coli strain using the pCDFDuet vector (FIG.

Example  5. Comparison of yield of new compounds by optimizing culture conditions for inducing protein expression of transformed E. coli

Using the optimal expression vector, AmUGT And E. coli BL21 ( Δpgm ) expressing the UGlcNAcDH gene were cultured on LB medium supplemented with antibiotics. E. coli strain cultured overnight was inoculated into fresh LB medium. Escherichia coli was cultured at an absorbance of 600 nm to a value of 0.8. IPTG was added to the cultured Escherichia coli at a concentration of 1 mM and the protein was incubated at 18 ° C, 25 ° C and 30 ° C for 1 day (24 hours) and 2 days (48 hours) Lt; / RTI > The cultured Escherichia coli was centrifuged and the supernatant was discarded. The recovered Escherichia coli was adjusted to 2.5 at an absorbance of 600 nm on M9 medium supplemented with an antibiotic and 2% glucose. The substrate luteolin was added to the final concentration of 100 μM, and the mixture was incubated at 30 ° C. for 24 hours with shaking, and then the supernatant was extracted using the continuous method. The reactants were used for high performance liquid chromatography analysis.

As a result, the production of new compounds was good at the temperature condition of 18 ° C rather than 25 ° C and 30 ° C in the protein expression inducing culture condition, and the protein expression was observed for 2 days (48 hours) 7- ON -acetyl-D-glucosamine uronic acid (Fig. 5).

Example  6. Comparison of the production of new compounds by optimizing cell concentration conditions

AmUGT Escherichia coli BL21 (△ pgm) carrying the gene and UGlcNAcDH gene was cultured on LB medium supplemented with antibiotics. Escherichia coli strain cultured overnight was inoculated into fresh LB medium, and E. coli was cultured to an absorbance of 600 nm to 0.8. IPTG was added to the cultured Escherichia coli at 1 mM, and the protein was expressed at 18 ° C for 1 day (24 hours). The cultured E. coli was centrifuged and the supernatant was discarded. The recovered E. coli was cultured on M9 medium supplemented with antibiotics and 2% glucose at an absorbance of 600 nm at 1.0, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, And 10, respectively. The substrate luteolin was added thereto to a final concentration of 100 μM and incubated at 30 ° C for 24 hours with shaking. The supernatant was extracted using the continuous method and the reactants were used for high performance liquid chromatography analysis.

As a result, it was confirmed that the production amount of the new compound was increased depending on the cell concentration until the cell concentration reached the value 5 at the absorbance 600 nm, and the production amount of the new compound was again decreased from the case where the value exceeded 5 at the absorbance 600 nm 6).

As a result, the vector for expression of UGlcNAcDH gene was pCDFDuet, and the transgene enzyme AmUGT In a protein expression inducing condition in which E. coli BL21 (DELTA pgm) having the gene and UGlcNAcDH gene is cultured at 18 DEG C for 2 days (48 hours) while adjusting the cell concentration so that the value is 4 to 5 at an absorbance of 600 nm, It was confirmed that the production of luteolin-7- ON -acetyl-D-glucosamine uronic acid compound was optimized.

<110> Konkuk University Industrial Cooperation Corp <120> Novel compound Luteolin-7-O-N-acetyl-D-glucosaminuronic acid and          method for producing the same <130> PN13210 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 1374 <212> DNA <213> Antirrhinum majus <400> 1 atggaggaca ctatcgttct ctacgcttca gcagagcacc ttaactccat gctactactc 60 ggcaaactca tcaacaaaca ccaccccaca atctccgtcg ccattatcag caccgcccca 120 aacgccgccg ctagttccgt cgccgacgtg gcggccatat cttatcagca actcaaaccg 180 gccactctcc cttcggatct aaccaaaaac ccaatcgagc tcttcttcga aatcccacgt 240 ctacataatc ctaacttgct cgaagcgctg gaagaactgt cactaaaatc aaaagtaagg 300 gcatttgtga tagatttctt ttgcaatccc gcatttgagg tttcgactag cttgaacata 360 cccacttact tctatgtcag cagcggcgcg tttgggctat gcgggttctt gcattttccg 420 acaatcgacg aaactgtcga aaaagacatc ggtgaactga acgatatctt ggagatcccg 480 ggttgccccc cggttttgtc ctcggatttt ccgaaaggta tgttctttcg caagagtaac 540 acttacaagc attttttaga cacggcgaaa aacatgagga gagcgaaagg gatcgtggtg 600 aacgccttcg acgcgatgga gttccgagct aaagaagccc tcgtcaacaa tctgtgcgta 660 cccaattcgc caactccccc agttttctta gtcggcccat tggtcggagc aagcacaact 720 acgaaaacca caaacgaaca gcacgaatgc ttgaaatggc tggacgtgca gccagacaga 780 agcgtgatct tcttatgttt cggtaggagg ggtttgttct ccgcagacca attgaaggaa 840 atcgcaattg gtctggagaa cagcggccac aggttcctgt ggtccgtgcg ttgcccacca 900 agtaagccta actcttataa cactgatccg gacctggacg agctcctgcc cgaggggttt 960 ttgtccagga ccgagacccg gggtttcgtg atcaagtcgt gggcgcctca gaaggaggtg 1020 ctgagccatg gcgcggttgg agggttcgtg acgcactgtg ggaggagttc gatattggaa 1080 gcggtgtcgt ttggggtgcc gatgatcggg tggccgatat acgcggagca gaggatgaat 1140 agggtgttca tggtggagga gatgaaggtg gcgttgcagt tggatgaggt ggaggaaggg 1200 ttcgtggcgg cggtggaatt ggagaagaga gtgaaggagt tgatggattc gaagaatggg 1260 agagcggtta ggcagagagt gaaggagatg aaagtggcgg ctgaggtggc ggttgaaaag 1320 ggtggttcgt cagttgtggc gttgcaacgc tttgttgata tggtggtttc ttaa 1374 <210> 2 <211> 1287 <212> DNA <213> Bacillus cereus subsp. <400> 2 gaattcgatg gaaaaagaga aaggtgaaga aaaaatgaac gattctcgca aaatcactgt 60 tatcggattg ggctatgttg gcctgccgct ggcgatccat tttgcggaac ggggctataa 120 cgtcgtcggc ttagataagg acaaacgcaa aattgaacgc attgaaaaag gagattctta 180 cataccagac gtttcctcga acgttttaaa agatttggta caaaacaaac agttggtagt 240 ttatacacct cataccggga tagaagagtt tcagaatagt gaatatgtga tcgtgacggt 300 cccgacgcca atcaataaac agaaagaacc tgatttatcg gctctcattg cagcgtcaca 360 ctatataaaa cagaatcttc aggcaggaca gaccttcatt tttgaaagtt ccacatatcc 420 gggtacactt gaagaggtca tcattccgat tattgcccaa tcaggtaagg aagttggaaa 480 agattattat attggctaca gccctgagcg tattgatccg gccaatcaac agtacacagt 540 tcagaccatt ccaaaagtga tttcgggtca aacagagaga tgtaagcagc aagtccagaa 600 attgtatagc accatttttg acaccgtcgt tccagtgagt tccccaaaag tagcagagat 660 gtgtaaactg ttcgaaaata tacagcgtct ggttaacata tccctggtaa atgagttaaa 720 catcctttgc gaaaagctgg ggattgactt tcgggaggct cttgaagccg cggccaccaa 780 accattcggt tttacgccat actggccggg cccaggtata gggggacatt gtattccggt 840 tgatccctta tactttcagt ggaaagcccg tcagctaggg caatcatcac agctgatcga 900 agtggctcac atgattaatg agaagatgcc gcaacaaatc gtaacgcagg tgaaggagct 960 gagtgctccc ccagggactg tcttcctgat cggcattgcg tacaaaaaag acgtgaatga 1020 tttacgagag tctcctgcac tcccgattat cgagctgctc gtgaacgagg gttataaagt 1080 gcaatatcac gatccctaca ttagctctgc gaaaatcggt gacaaaatat acgattctat 1140 cccccttaaa aagaaaaccc tggaaaaggc agattgcatt ctaattgtaa cggaccatag 1200 caatatagac tggaatatat gcaagggaat gaagcatgta atcgatactc gcggtgtatt 1260 gaagaaggtt agcgcataag cggccgc 1287 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gccatatgga ggacactatc gttctctacg 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gcctcgagtt aagaaaccac catatcaaca 30

Claims (13)

To luteolin -7 -ON of formula 1-acetyl -D- glucosamine-uronic acid (Luteolin-7 -ON -acetyl-D -glucosaminuronic acid) compound.
[Chemical Formula 1]

The transgenic enzyme AmugT ( Antirrhinum majus UDP-dependent glycosyltransferase) genes and subspecies of Bacillus cereus (Bacillus cereus subsp . An expression vector comprising UGlcNAcDH (UDP- N- acetylglucosamine dehydrogenase) gene; Or an expression vector comprising a P. persicae transposase enzyme AmUGT gene and an expression vector comprising a UGlcNAcDH gene derived from Bacillus subtilis subspecies. The method of claim 2, wherein the snapdragon-derived AmUGT Wherein the gene has the nucleotide sequence of SEQ ID NO: 1. 3. The composition according to claim 2, wherein the Bacillus subtilis UGlcNAcDH gene has the nucleotide sequence of SEQ ID NO: 2. 3. The composition according to claim 2, wherein the composition further comprises E. coli. a) transferase per snapdragon-derived AmUGT (Antirrhinum majus UDP-dependent glycosyltransferase) genes and subspecies of Bacillus cereus (Bacillus cereus subsp . Producing an expression vector comprising an expression vector comprising UGlcNAcDH (UDP- N- acetylglucosamine dehydrogenase) gene or an expression vector comprising a sugar chain-derived glycosyltransferase AmUGT gene and a UGlcNAcDH gene derived from Bacillus subtilis subspecies;
b) transforming the expression vectors into E. coli; And
c) adding the luteolin to the substrate while culturing the transformed E. coli. 7. The method of claim 6 wherein the snapdragon-derived AmUGT The method according to claim 1, wherein the gene has the nucleotide sequence of SEQ ID NO: 1. [Claim 7] The method according to claim 6, wherein the UGlcNAcDH gene derived from Bacillus cereus subtype has the nucleotide sequence of SEQ ID NO: 2. [Claim 7] The method according to claim 6, wherein the Escherichia coli is Escherichia coli lacking a phosophoglucumutase. [7] The method according to claim 6, wherein the culture conditions for inducing protein expression of the transformed E. coli in step c) are 17 to 19 DEG C for a period of 45 to 55 hours and a cell concentration is 4 to 5 for a cell concentration of 600 nm. Lt; RTI ID = 0.0 &gt; 1. &Lt; / RTI &gt; The transgenic enzyme AmugT ( Antirrhinum majus UDP-dependent glycosyltransferase) genes and subspecies of Bacillus cereus (Bacillus cereus subsp . An expression vector comprising UGlcNAcDH (UDP- N- acetylglucosamine dehydrogenase) gene; Or snapdragon are transfected with the expression vector and Bacillus cereus subspecies expression vector containing the gene derived UGlcNAcDH conversion originating party comprises a transition AmUGT enzyme gene of E. coli to produce a compound of claim 1. 12. The Escherichia coli strain according to claim 11, wherein the Escherichia coli is deficient in a phosophoglucumutase. A food composition comprising the compound of claim 1 as an active ingredient.

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