KR20180105387A - Novel ginsenosidase protein and use thereof - Google Patents

Abstract

The present invention relates to a ginsenosidase protein consisting of amino acid sequences represented by sequence number 1. The Lactobacillus casei GFC 1 strain (Accesion No.: KCTC18333P)-derived ginsenosidase protein of the present invention is capable of bioconverting gypenoside XVll into ginsenoside compound K (C-K) or ginsenoside F2 in a form with easy body absorption at a high yield.

Description

NOVEL GINSENOSIDASE PROTEIN AND USE THEREOF <br> <br> <br> Patents - stay tuned to the technology NEW GINSENOSIDASE PROTEIN AND USES THEREOF

The present invention relates to novel ginsenosidase proteins and uses thereof.

Ginseng ( Panax ginseng CA Meyer ) is a herb that belongs to the genus Ogawa and Ginseng. It has been used in Korea, China and Japan for over 2,000 years and has been used to prevent disease and prolong its life. The effects of ginseng on the central nervous system, anticancer activity, anti-cancer activity, immune function regulation, anti-diabetic effect, hepatic function hyperactivity, cardiovascular disorder improvement, anti-arteriosclerosis effect, blood pressure control effect, , Effects on osteoporosis, anti-stress action, anti-fatigue action, antioxidant activity, and inhibition of aging.

Ginseng contains more than 40 kinds of ginsenosides (glycosides) as physiologically active substances of non saponin type, including polyacetylene, phenolic compounds, acid polysaccharides, peptides ), And alkaloids (Alkaloids).

Ginsenoside, a representative physiologically active ingredient of ginseng, refers to saponin contained in ginseng, and ginseng contains various kinds of ginsenosides. Ginsenosides are distributed evenly in the ground and underground of ginseng. Especially, it is known that not only ginsenoside content but also composition is different depending on the parts such as ginseng roots, ginseng leaves, and ginseng fruit.

The ginsenosides of ginseng constitute about 5% of the ginseng dry matter. The Ginsenosides of the ginseng contain about 5% of the ginseng in the Triterpenoid series of Dammarane skeleton containing glucose (Glucose), Arabinose, Xylose, (Rhamnose) and the like. (3, 6, 12 positions) Protopanaxadiol (PPD) saponins, 3 individuals (positions 3, 6 and 12) in the case of two individuals (positions 3 and 12) (Protopanaxatriol; PPT) family saponins. Glucose, Arabinose, Xylose, Rhamnose and the like are bonded to -OH in the R1, R2 and R3 positions of PPD and PPT. More than 30 species of ginsenosides have been isolated from ginseng. However, when extracting ginseng, six of Rb1, Rb2, Rc, Rd, Re and Rg1 account for more than 90% of total saponins, low.

Ginsenosides are not absorbed in the body as they are, Bacteriophage des (Bacteriodes), It is degraded by microorganisms such as Lactobacillus , Fusobacterium , Eubacterium and Provetella , and the metabolism is absorbed. In the case of ginsenoside Rb1, intestinal microorganisms switch sequentially to ginsenoside Rd, compound K and protopanaxadiol. Rb1, Rb2, Rc, and Rd, which are PPD-derived ginsenosides, are metabolized and absorbed by 20-O- β-D-glucopyranosyl-20 (S) -protopanaxadiol (CK) in the intestine. PPT ginsenosides Re, Rf, and Rg1 are metabolized in the intestine to Rh1, F1, and Protopanaxatriol. The PPD family ginsenosides Rb1, Rb2, Rc and Rd are converted into 20 (R) - / 20 (S) -ginsenoside Rg3 by intestinal microorganisms and Rg3 is converted into Rh2 by intestinal microorganisms.

In recent years, the effective ingredient of ginseng is easily absorbed into the human body, and a method of fermenting ginseng using intestinal microorganism or lactic acid bacteria or treating an enzyme with a trace amount of ingredients in ginseng is used. Korean Patent Publication No. 10-1017378 discloses a method for producing fermented red ginseng and fermented red ginseng and a Bacillus subtilis strain derived from chungkukjang, which is suitable for the fermented ginseng and fermented red ginseng, using a microorganism belonging to the genus Bacillus isolated from chungkukjang. And a technique of fermenting red ginseng to increase the content of ginsenoside Rd.

Korean Patent Registration No. 10-1240192 discloses a fermented ginseng extract for improving insulin secretion using Lactobacillus acidophilus strain K-59 and strain K-59 and a method for producing the same. Rg1, Rb1, Rg1 or Re, which is a low bioavailability component of ginseng, is converted into ginsenoside Rh1, Rg3, Rh2 or CK ( Lactobacillus acidophilus ) by using Lactobacillus acidophilus strain K-59 (KFCC11467P) A method for converting the above-mentioned compound is disclosed.

On the other hand, Gynostemma pentaphyllum (Thunb.) Makino is also a vineous perennial plant belonging to the genus Cucurbitaceae, also known as the seventh leaf. The stem is rolled up on another plant or object with a vine beard. Leaves are alternate, new plantar-like or compound leaf-like compound leaf. There are 3 ~ 7 small leaves, ovate lanceolate, sharp edges, sharp beneath the leaves, coarse teeth and short hairs. Flowers bloom in August ~ September with greenish green blooms, and the leaves are rounded from the axilla to the end, and the fruit is berry, spherical, ripe in black green and 6 ~ 8mm in diameter. Native species other than stones are Korea, Japan, China, Sri Lanka, Bangladesh, Nepal and India. They grow mainly in high humidity places such as coasts and rivers.

In addition to the stones, there are various Dammarane type saponins, which are called Gynosaponin or Gypenoside, depending on the country and region where they grow. Extra saponin is known to exhibit the effects of lipid metabolism improving action, cardiovascular disease protective action, hypoglycemic action, central nervous system action, anticancer action, platelet aggregation inhibition action, and tonic action, (Ginsenoside), an active ingredient of the &lt; RTI ID = 0.0 &gt; Meyer &lt; / RTI &gt; This fact is recognized because both plants contain a common saponin of fumaran.

In recent years, research is being conducted on a technique for converting such Dhammarine type saponin, Gypenoside, into Ginsenoside, which is an easily absorbable form in the body. Korean Patent Registration No. 10-1427882 discloses a method for producing ginsenosides Rd and F2 using a novel ginsenoside glycosidase derived from Flavobacterium Johnson. The novel ginsenoside glycoside A transformant into which a vector containing a gene encoding a protein coding for the Ginsenoside glycosidase protein or a gene encoding the protein is transformed or a ginseng saponin of PPD type is transformed using the transformant culture to obtain ginsenoside Rd or F2 Discloses a technique relating to a method for producing the same.

The present inventors have made intensive efforts to develop a method for bioconversion of ginsenoside from Gypenoside to a high yield. As a result, Lactobacillus casei GFC 1 strain (accession number: (Ginsenoside) protein consisting of the amino acid sequence shown in SEQ ID NO: 1 derived from Gentamicin (KCTC18333P) is used to treat extrinsic Gypenoside XVll in a form that is easily absorbed in the body (Ginsenoside) F2 or Ginsenoside Compound K (CK), and completed the present invention.

Korean Patent Publication No. 10-1017378 Korean Patent Publication No. 10-1240192 Korean Registered Patent No. 10-1427882

It is an object of the present invention to provide a ginsenosidase protein capable of bioconversion of Gypenoside into Ginsenoside in a high yield, a nucleic acid encoding the Ginsenoside protein, and a recombinant vector containing the nucleic acid .

Another object of the present invention is to provide a method for bioconversion of Gypenoside XVll to Ginsenoside in high yield.

Another object of the present invention is to provide a cosmetic composition comprising Ginsenoside F2 or Ginsenoside Compound K (CK).

In order to accomplish the above object, the present invention provides a ginsenosidase protein comprising the amino acid sequence shown in SEQ ID NO: 1.

In the present invention, the ginsenoside protein is characterized in that it is derived from a strain of Lactobacillus casei GFC 1 (accession number: KCTC18333P).

In addition, the present invention provides a nucleic acid encoding the ginsenoside protein.

In the present invention, the nucleic acid is characterized by having the nucleotide sequence of SEQ ID NO: 2.

The present invention also provides a recombinant vector comprising the nucleic acid.

The present invention also provides a method for producing ginsenoside by treating the ginsenoside protein with ginsenoside XVll.

In the present invention, the treatment is carried out at a pH of from 6 to 9 at a temperature of from 20 to 40 캜.

In the present invention, the treatment is characterized by adding at least one metal ion selected from the group consisting of Co ²⁺ , Fe ³⁺ , Cu ²⁺ , Zn ²⁺ , NH ⁴⁺ and Mg ²⁺ .

In the present invention, the treatment is characterized by adding NH ⁴⁺ or Mg ²⁺ metal ions.

In the present invention, the ginsenoside is Ginsenoside F2 or Ginsenoside Compound K (C-K).

The present invention also provides a cosmetic composition comprising ginsenoside prepared by the above method.

In the present invention, the ginsenoside is characterized by having antioxidant, anti-inflammatory and anti-wrinkle effects.

The novel ginsenoside protein of the present invention is a compound of the formula (I) wherein Gypenoside XVll is converted into Ginsenoside F2 or Ginsenoside Compound K (CK) There is an effect that can be switched. It is possible to produce Ginsenoside F2 or Ginsenoside Compound K (CK) in a minor amount, which is produced only in a trace amount from a natural state, and has a limited use. It can be used as an antioxidant, anti- It can be used as a cosmetic composition having an improving effect.

Fig. 1 shows the results of SDS-PAGE analysis of purified ginsenosidase. (M: Molecular weight standard, 1: Lactobacillus casei GFC 1 DNA, 2: Recombinant microorganism lysate, 3: Purified ginsenoside)
FIG. 2 is a graph showing the stability and activity of purified ginsenoside according to pH (A) and temperature (B) of the present invention.
Figure 3 is a TLC analysis result of biological conversion using ginsenoside according to the present invention. (S; Standard; C; Gypenoside XV11, 1; Examples 5-6 and 2; Examples 5-8 (12hr treatment) 12 hr treatment))
Figure 4 shows the HPLC (High Performance Liquid Chromatography) analysis of Gypenoside XV11 converted to Ginsenoside F2 or Ginsenoside Compound K (CK) after treatment with ginsenoside. (A: before treatment, B: after 12 hours treatment)

The terms used in the present invention are defined as follows.

As used herein, the term "nucleic acid" refers to any DNA or RNA, for example, chromosomes, mitochondria, viruses and / or bacterial nucleic acids present in a tissue sample. Includes one or both strands of a double-stranded nucleic acid molecule and includes any fragment or portion of the intact nucleic acid molecule.

As used herein, the term "gene " refers to any nucleic acid sequence or portion thereof that has a functional role at the time of protein coding or transcription, or in the control of other gene expression. The gene may consist of only a portion of the nucleic acid encoding or expressing any nucleic acid or protein that encodes the functional protein. The nucleic acid sequence may comprise an exon, an intron, an initiation or termination region, a promoter sequence, another regulatory sequence, or a gene abnormality within a particular sequence adjacent to the gene.

As used herein, the term "primer" refers to a nucleic acid that hybridizes to a complementary RNA or DNA target polynucleotide, and is a starting point for the stepwise synthesis of a polynucleotide from a mononucleotide, for example, by the action of a nucleotidyltransferase that occurs in a polymerase chain reaction Functional oligonucleotide &lt; / RTI &gt; sequence.

As used herein, the term "recombinant vector" means an expression vector capable of expressing a desired protein in a suitable host cell, and a nucleic acid preparation containing an essential regulatory element operably linked to the expression of the nucleic acid insert.

In the present invention, "homology" means the percentage of identity between two polynucleotides or polypeptide mimetics. The homology between sequences from one moiety to another can be determined by known techniques. For example, homology between two motifs can be determined by aligning the sequence information and directly aligning the sequence information between two polynucleotide molecules or two polypeptide molecules using a computer program that is readily available. In addition, homology between polynucleotides can be determined by hybridization of the polynucleotide under conditions that result in a stable double strand between homologous regions, followed by degradation to single-strand-specific nucleases to determine the size of the resolved fragment.

The term " Gypenoside XVll "in the present invention refers to a compound of the triterpene saponin series which is isolated from the outside of the stones." Gynosaponin S; Gypenoside-XVII; 3 硫 - (硫 -D-Glucopyranosyloxy) beta] -D-glucopyranosyl- [beta] -D-glucopyranoside; (3beta, 12beta) -3- (beta-D-Glucopyranosyloxy) -12-hydroxydamma- 20-yl 6-O-beta-D-glucopyranosyl-beta-D-glucopyranoside "

Hereinafter, the present invention will be described in detail.

In order to achieve the above object, the present invention provides a novel ginsenosidase protein consisting of the amino acid sequence shown in SEQ ID NO: 1.

The ginsenoside protein refers to a glycoprotein hydrolyzing enzyme which converts ginsenoside saponin into extracellular saponin, preferably a protein consisting of the amino acid sequence shown in SEQ ID NO: 1. The ginsenosidease protein is an active ingredient of ginsenoside that bioconverts Gypenoside XVll to Ginsenoside F2 or Ginsenoside Compound K (CK) . In addition, as the sequence having such homology, if it is an amino acid sequence having substantially the same or corresponding biological activity as the Ginsenosidase protein, a protein variant having an amino acid sequence in which a part of the sequence is deleted, modified, substituted or added And are included within the scope of the present invention.

The homology refers to a degree of similarity to an amino acid sequence of a wild type protein or a nucleic acid sequence encoding the wild type protein, and includes the sequence having the same sequence as the amino acid sequence or the nucleic acid sequence of the present invention, do. This comparison of homology can be performed using a visual program or a comparison program that is easy to purchase.

Herein, the ginsenosidease protein according to the present invention is derived from a Lactobacillus casei GFC 1 strain (accession number: KCTC18333P) (hereinafter abbreviated as 'GFC 1').

In one embodiment of the present invention, a Ginsenosidase protein (SEQ ID NO: 1) derived from Lactobacillus casei GFC 1 strain (accession number: KCTC18333P) was isolated and purified. The enzyme is an enzyme belonging to the Glycoside hydrolase family 3 and the amino acid sequence has 487 residues.

The present invention also relates to a nucleic acid encoding the ginsenoside protein.

Herein, the nucleic acid may preferably be a nucleic acid having a nucleotide sequence represented by SEQ ID NO: 2, but is not limited thereto. The nucleic acid encoding the ginsenoside protein is not limited to a nucleic acid encoding a protein having the activity of ginsenosidease but may be a nucleic acid encoding a protein having substantially the activity of ginsenosidease And a sequence encoding a protein. The homology is as described above.

The present invention also relates to a recombinant vector comprising said nucleic acid.

In one embodiment of the invention, the recombinant vector may be a pMAL-c5X vector.

The present invention also relates to a method for producing ginsenoside by treating Ginsenosidase protein consisting of the amino acid sequence shown in SEQ ID NO: 1 with Ginsenoside XVll.

The ginsenoside may be Ginsenoside F2 or Ginsenoside Compound K (CK).

In one embodiment of the present invention, the Ginsenosidase protein was treated with Gypenoside XVll and analyzed by TLC. As a result, it was found that the Ginsenosidase protein bound Gypenoside XVll It was confirmed that the biosynthesis of Ginsenoside F2 or Ginsenoside Compound K (CK) was confirmed.

Specifically, the ginsenosidase protein consisting of the amino acid sequence shown in SEQ ID NO: 1 converts Gypenoside XV11 to Ginsenoside F2 and, over time, converts ginsenoside ( Ginsenoside F2 is biotransformed into Ginsenoside Compound K (CK).

When ginsenoside protein consisting of the amino acid sequence represented by SEQ ID NO: 1 is treated with Gypenoside XVll to prepare ginsenoside, the treatment is carried out at pH 6 to 9 To 20 &lt; 0 &gt; C to 40 &lt; 0 &gt; C.

If the pH and temperature conditions are out of the above range, the activity and stability of ginsenoside protein are lowered, which is undesirable. That is, the hydrolytic activity of sugar is inhibited, and the saponin is not converted to saponin from extracellular saponin, so that the function of ginsenoside protein is lost.

Further, when treating the ginsenoside Let the (Ginsenosidase) protein consisting of the amino acid sequence represented by the SEQ ID NO: 1 to jipe furnace side (Gypenoside) XVll the production of ginsenoside (Ginsenoside), the processing is Co ^{2+, Fe 3+, Cu 2+, Zn 2+} , NH 4+ , and it is preferable to add at least one metal ion selected from the group consisting of Mg ^2+, more preferably NH ^{4 +} or Mg ^{2 +} metal ions Is preferred because the conversion rate to Ginsenoside F2 or Ginsenoside Compound K (CK) is the highest.

Although it has proved its pharmacological effect, it can be used to produce a minor form of Ginsenoside F2 or Ginsenoside Compound K (CK), which was produced only from natural to trace amounts and was restricted in use. have.

Further, the present invention relates to a cosmetic composition characterized by containing ginsenoside prepared by the above method.

The obtained ginsenoside may be one having antioxidant, anti-inflammatory and anti-wrinkle effects.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not to be construed as limiting the scope of the present invention. It will be self-evident.

Example 1: Acquisition of microorganisms

A strain capable of converting Gypenoside into Ginsenoside was isolated and identified from flowers of ginseng through screening. One of them was identified as Lactobacillus casei GFC 1 (accession number: : KCTC18333P). It was confirmed that the strain could hydrolyze Gypenoside XVll into Ginsenoside F2 and finally hydrolyze it with Ginsenoside Compound K (CK).

Example 2: Preparation of Ginsenosidase

The DNA sequence of the GFC 1 strain was analyzed by National Center for Biotechnology Information ' s ORF FINDER. For the expected ORF, BLAST was used to find an ORF presumed to be ginsenoside, and the template DNA was amplified by PCR using the oligonucleotide primer shown in Table 1 below. The PCR conditions were 95 ° C 5 minutes, 30 seconds [Denaturation] at 95 ° C, 30 seconds [Annealing] at 60 ° C, and 30 seconds [Extension] at 72 ° C. This enzyme belongs to the Glycoside hydrolase family 3 and the amino acid sequence has 487 residues.

primer The sequence (5'-3 ') SEQ ID NO: Forward primer
(Gin primer F) CGG ATC CAT GTA CAG TAA AGA ATC AAT CAC AG 3 Reverse primer
(Gin primer R) CGA ATC CTT CTA ATG TTT TAC CGT TACT TTCA 4

Example 3 Molecular Cloning, Expression and Purification of Recombinant Ginsenosidase

The fragment amplified in Example 2 was cloned into a pMAL-c5X expression vector (available from New England Biolabs), and the recombinant pMAL-c5X was introduced into E. coli BL21 (DE3) cells to prepare a transformed strain Respectively. In order to mass-produce ginsenosidase, the transformed strain was inoculated into an Erlenmeyer flask containing LB medium supplemented with Ampicillin and incubated at 27 ° C until the absorbance at 600 nm reached 0.6 The seed culture was carried out in a shaking incubator at 100 rpm. The expression of ginsenosidase was induced by treatment with 50 mM IPTG (Isopropyl-beta-D-thiogalactoside). When the strain was in a stationary phase, the culture broth of the strain was centrifuged at 5000 rpm for 30 minutes, suspended in 20 mM sodium phosphate buffer (pH 7.0), and the cell solution was applied to a sonicator ). The cell lysate was again centrifuged at 9000 rpm for 30 minutes, and finally ginsenoside was obtained from the supernatant which can be used for production. Ginsenosidase obtained by separation and purification using Glutathione Resin and protease cleavage was analyzed by SDS-PAGE together with Lactobacillus casei GFC 1 DNA and recombinant microorganism debris. As a result, the ginsenosidase protein showed a molecular weight of about 71.1 kDa (Fig. 1).

In order to confirm the amino acid sequence of the ginsenosidase protein, the ratio of the 16S rRNA gene was analyzed and sequencing was commissioned to Solgent (Daejeon, Korea). The 16S rRNA gene full-length sequence was collected using SeqMan software (DNASTAR) and blasted in the NCBI database to obtain a taxonomically related 16S rRNA gene sequence from GenBank. The amino acid sequence was confirmed after the transfection using the obtained nucleotide sequence.

As a result, the number of amino acids of the ginsenoside protein was 487, and the amino acid sequence was represented by SEQ ID NO: 1.

Example 4: Determination of the stability of recombinant ginsenoside according to pH and temperature

The effect of pH on the ginsenosidase protein enzyme activity of Example 3 was measured. The substrate was pNPG (4-Nitrophenyl? -D-glucuronide; Sigma) and the pH was adjusted to 2 to 10 using a buffer as described below.

HCl (pH 8), glycine-HCl (pH 3), sodium acetate (pH 4 and pH 5), sodium phosphate (pH 6 and pH 7), Tris-

To determine the effect of pH on the stability of the enzyme, the enzyme was incubated in the above-mentioned buffer for 24 hours at a temperature of 30 ° C, and analyzed for pNPG in 50 mM potassium buffer. Respectively. Residual activity was analyzed by standard analytical methods and the activity probabilities obtained at optimal pH are shown in Figure 2-A.

As a result, as shown in FIG. 2-A, the stability and activity of the recombinant ginsenoside were excellent in the range of pH 6 to 9, more preferably in the range of pH 7.0.

In addition, the effect of the temperature on the enzyme activity of the ginsenoside protein of Example 3 was measured. Similar to the pH stability measurement, pNPG (4-Nitrophenyl β-D-glucuronide; Sigma) was used as a substrate and the pH was adjusted to pH 7.0. The enzyme equivalent in 50 mM potassium phosphate buffer for 30 minutes at 20 to 80 ° C. The temperature stability analysis was performed by culturing. The temperature stability analysis was performed by confirming the residual activity measured by the standard analysis method, and the result is shown in FIG. 2-B.

As a result, as shown in FIG. 2-B, the activity and thermal stability of the recombinant ginsenoside were excellent in the range of 20 to 40 占 폚, more preferably in the range of 28 to 35 占 폚.

Example 5 Evaluation of Bioconversion Ability from Gypenoside to Ginsenosidase by Metal Treatment

5 Kg of the dried off-shore outpost was extracted by refluxing with 50% ethanol in an organic solvent for 24 hours. The filtrate was concentrated under reduced pressure in a water bath at 40 ° C or lower and repeated twice. The concentrated concentrate was homogeneously suspended in distilled water 10 times the total amount of the concentrate and then fractionated. The fractions were separated primarily from non-glycosides (Aglycon) using ethyl acetate (Ethyl Acetate). A fraction containing high purity gypenoside was obtained by using n-butanol in the remaining water layer and then lyophilized to prepare a high-fraction powder containing gypenoside in a final powder state.

When the strain was placed in a stationary phase before obtaining purified ginsenoside by the method of Example 3, the culture broth of the strain was centrifuged at 5000 rpm for 30 minutes, and diluted with 20 mM sodium phosphate buffer, pH 7.0). The cell solution was disrupted with a crusher, and centrifuged at 9000 rpm for 30 minutes to obtain a supernatant, that is, ginsenoside. One ml of the supernatant was taken, Treated with 10 mg / ml of high-fraction powder containing gypenoside, and the component changes are shown in Table 2.

Converted ginsenosides  (mg / ml) Gypenoside XVll Ginsenoside  F2 Ginsenoside  C-K Total Ginsenosidase
Before processing 10 - - 10 Ginsenosidase
After processing 8.80 0.74 - 9.54

Further, in order to improve the biosynthesis from the zinfenide to the ginsenoside, Co ²⁺ , Fe ³⁺ , Cu ²⁺ , Zn ²⁺ , K ⁺ , Na ⁺ , NH ^{4 +} and Mg ^{2 +} were each treated to a final concentration of 10 uM. The results of TLC analysis after 12 hours of treatment are shown in Table 3 below.

Metal ions Production of Ginsenoside F2 Production of Ginsenoside C-K Example 5-1 (Co ²⁺ ) (+) - Example 5-2 (Fe ³⁺ ) (+) - Example 5-3 (Cu ^{+ 2)} (+) - Example 5-4 (Zn ²⁺ ) (+) - Example 5-5 (K ⁺ ) - - Example 5-6 (Na ⁺ ) - - Examples 5-7 (NH ⁴⁺ ) ++ + Examples 5-8 (Mg ²⁺ ) + ++

From Table 3, ginsenoside let compared to the first one only treatment, the metal ion treatment when examined the biotransformation capacity of the furnace side by jipe ginsenoside result, except for K ⁺ and Na ⁺ In Examples 5-1 to 5-4 and 5-7 to 5-8 in which Co ²⁺ , Fe ³⁺ , Cu ²⁺ , Zn ²⁺ , NH ⁴⁺ and Mg ²⁺ metal ions were treated, (Gypenoside) XVll was converted to ginsenoside. In particular, in Examples 5-7, it was confirmed that the conversion of ginsenoside F2 was greatly increased and the conversion to ginsenoside Compound K (CK) was also increased. In addition, in Examples 5-8, the conversion from Gypenoside XVll to Ginsenoside F2 was increased and the conversion of Ginsenoside Compound K (CK) was greatly increased I could.

As a reference, "++" indicates that the conversion rate from zifenoid to ginsenoside is 30% to 50% is "+" and "+" indicates that the conversion rate from ginsenoside is 5% to 30% A ~ 5% increase was marked as "-".

Example 6: Analysis of Gypenoside XVll transformation by Chromatography

5 g of water-saturated butanol was added to 1 g of each sample prepared from the products of Examples 5-6 to 5-8 and the high fractionated powder containing gypenoside, followed by mixing. The supernatant of the butanol mixture was taken and concentrated under reduced pressure Respectively. At this time, TLC analysis was performed on the butanol mixture before concentration (FIG. 3). The resulting concentrate was dissolved in methanol (HPLC grade), filtered through a 0.2 μm membrane filter, and used as a sample for HPLC analysis. HPLC analysis was carried out on a C18 column (250 x 4.6 mm, ID 5 m) at UV 203 nm. The mobile phase was graded using Acetonitrile (Solvent A) and Water (Solvent B) and separated at a flow rate of 1.6 ml / min. Table 4 shows the gradient conditions of the mobile phase.

Time (min) Moblie  phase Acetonitrile (solvent A) Water (solvent B) 0-5 15 85 5-25 21 79 25-70 58 42 70-82 90 10 82-100 15 85

The results of the changes of the components of the gypenoside XVll of the products of Examples 5-7 to 5-8 and the high fractionated powder containing gypenoside are shown in Table 5 and FIG.

Converted ginsenosides  (mg / ml) Gypenoside XVll Ginsenoside  F2 Ginsenoside  C-K Total Zifenoid
High-fraction powder 1000 - - 1000 Examples 5-7
product 132 542 51 725 Examples 5-8
product 0 690 114 804

As a result, as shown in Table 5 and FIG. 4, the products of Examples 5-7 were obtained by converting most of Gypenoside XV11 into Ginsenoside F2 and only a part of Ginsenoside Compound K (CK), respectively. In addition, in the case of the products of Examples 5-8, all of the Gypenoside XV11 was converted to Ginsenoside F2, and the Ginsenoside F2 was converted into a product of the product of Examples 5-7 (Ginsenoside) Compound K (CK).

Example 7: Antioxidant efficacy test: DPPH radical scavenging activity

The antioxidant efficacy of DPPH (2,2-Diphenyl-1-picrylhydrazyl) scavenging activity of the products of Examples 5-7 to 5-8 and the high fractionated powder containing gypenoside was tested.

Free radicals generally act as a pathologic factor that causes the skin to dry out by destroying the skin moisturizing layer and reacting to external stimuli, which is sensitive to external stimuli. The antioxidant activity of the products of Examples 5-7 to 5-8 and the fractionated powder containing gypenoside according to the present invention can be confirmed by free radical scavenging ability through DPPH experiment.

First, the samples of the products of Examples 5-7 to 5-8 and the high fractionated powder containing ziphenocide were prepared, and the samples were treated so as to have a final concentration of 50 ppm to prepare 100 μM DPPH (1,1-diphenyl-1-picrylhydrazyl ) Solution. After reacting at 4 ° C for 30 minutes, the amount of DPPH was measured at 520 nm in a 96-well plate. As a control, the free radical scavenging activity (%) of the sample was calculated by the following equation (1). At this time, 20 uM (final concentration) of ascorbic acid known to have antioxidant effect was set as Positve control. The degree of antioxidation by DPPH free radical scavenging assay was expressed by Equation (1) and the results of antioxidative efficacy were shown in Table 6.

[Equation 1]

Free radical scavenging ability (%) = 100- (absorbance of the group treated with the sample / absorbance of the group not treated with the sample X 100)]

sample   DPPH radical scavenging (%)   Positve control   76.74 + 0.34   Zifenoid-containing high-fraction powder   52.1 ± 0.68   The products of Examples 5-7   85.3 ± 0.13   The products of Examples 5-8   69.1 ± 0.67

As a result, as shown in Table 6, the products of Examples 5-7 to 5-8 after treatment with ginsenoside in comparison with the high-fraction powder containing Gypenoside before the treatment with ginsenoside (Ginsenoside) Showed high antioxidant activity.

Example 8: Anti-inflammatory efficacy test: Nitric oxide (NO) production inhibitory activity

In order to measure the anti-inflammatory activity of the products of Examples 5-7 to 5-8 and the high fractionated powder containing gypenoside, an experiment for measuring the concentration of nitric oxide (NO) produced by the inflammation induction reaction Respectively. When stimulated with LPS (Lipopolysaccharide), an inflammation inducing substance, NOS (NO synthase) generates NO through the activated pathway. NO was present in the cell culture in the form of NO ^{2 -} and the content was measured using NO kit (Nitric oxide detection kit, iNtRON 21021, Korea).

First, the products of Examples 5-7 to 5-8 and the high fractionated powder containing giphenoid were respectively dissolved in water (DW) and diluted. Mouse macrophage Raw 264.7 was dispensed into a 96-well plate at a concentration of ⁵ × 10 ⁵ cells / ml and cultured in a 5% CO ₂ incubator for 24 hours. Then, the cell RAW 264.7 was treated with 1.5 μg / mL of LPS (sigma), and the products of Examples 5-7 to 5-8 and the fractionated powder containing gpenoside were treated to a final concentration of 50 ppm Lt; / RTI &gt; for 24 hours.

Next, the supernatant of the culture was reacted with the same amount of griess reagent (Sigma), and the absorbance at 540 nm was measured with an ELISA reader, and the nitric oxide production rate was converted into a percentage. NO ^{2 -} was measured using nitrite as a standard solution. After setting up the anti-inflammatory effect of Dexamethasone 20uM (final concentration) known to a Positve control (positive control), and is treated in the same amount as sample water treated with a Negative control (negative control) comparing the generated NO concentration ^2- And the results are shown in Table 7 below.

sample   NO Production (μM)   Positve control   17 ± 0.15   Negative control   62 ± 0.21  Zifenoid-containing high-fraction powder   39 ± 0.24   The products of Examples 5-7   11 ± 0.36   The products of Examples 5-8   8 ± 1.01

As a result, as shown in Table 7, the products of Examples 5-7 to 5-8 produced a small amount of NO products as compared with the high-fraction powder containing gypenoside, Could know.

Example 9: Wrinkle-improving efficacy test: MMP-1 inhibitory activity

The MMP-1 (Matrix Metallo peptidase-1) inhibitory activity of the products of Examples 5-7 to 5-8 and the high-fraction powder containing gypenoside was measured.

Human fibroblasts (HDF) were inoculated into each well of a 6-well plate to be 18 × 10 ⁵ cells, and then cultured in a 5% CO ₂ environment at 37 ° C. for 24 hours. Then, the cells were washed three times with phosphate buffered saline (PBS), and ultraviolet light was irradiated to induce skin aging. At this time, ultraviolet rays were irradiated with a total energy of 50 mJ / cm 2 using an ultraviolet A lamp (Sankyo Denki FL8BL lamp, Sankyo Denki Co., Japan) at an energy amount of 27 mW / cm 2 per second. Immediately after irradiation with ultraviolet light, the cells were washed with phosphate buffer again, and the products of Examples 5-7 to 5-8 and the high-fractionated powder containing Gypenoside were added to a DMEM medium not containing fetal bovine serum to a final concentration of 50 ppm And further cultured for 24 hours. The MMP-1 ELISA kit (MMP-1 human, ELISA system) was used to determine the expression level of MMP-1, a type I procollagen degrading enzyme, Biotrak Assay, Amersham Biosciences, USA) according to the manufacturer's protocol. The concentration of MMP-1 was expressed as mg of protein per 1 ml of total cell culture medium, and the results are shown in Table 8. EGCG (Epigallocatechin gallate) was used as a positive control (positive control) and water was used as a negative control (negative control).

sample   MMP-1 inhibition (%)  Positive control   76.0 + - 0.66  Negative control   27.0 + - 0.57 Zifenoid-containing high-fraction powder   29.0 + - 0.84  Examples 5-7   71.0 ± 1.75  Examples 5-8   80.5 ± 0.59

As a result, as shown in Table 8, in Examples 5-7 to 5-8 relative to the high-fraction powder containing gypenoside in the case of irradiating ultraviolet light to human fibroblasts (ultraviolet light alone treatment group), MMP-1 Expression was inhibited. In particular, when Example 5-8 was treated, the expression of MMP-1 was remarkably decreased, and it was confirmed that the wrinkles of the skin were improved.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> GFC CO., LTD. <120> NOVEL GINSENOSIDASE PROTEIN AND USE THEREOF <130> P17012 <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 487 <212> PRT <213> Artificial Sequence <220> <223> Ginsenosidase <400> 1 Met Tyr Ser Lys Glu Ser Ile Thr Gly Phe Pro Lys Asp Phe Leu Trp   1 5 10 15 Gly Gly Ala Thr Ala Ala Asn Gln Ile Glu Gly Ala Trp Gln Val Asp              20 25 30 Gly Lys Gly Leu Thr Thr Ala Glu Val Val Arg Lys Ala Glu Asp Arg          35 40 45 Arg His Gly Ser Met Gly Asp Val Asn Ala Asp Thr Leu Gln Glu Ala      50 55 60 Ile Ala Asp His Ser Asp Val His Tyr Pro Lys Arg Arg Gly Val Asp  65 70 75 80 Phe Tyr His His Tyr Lys Glu Asp Ile Ala Leu Phe Ala Glu Met Gly                  85 90 95 Phe Lys Val Phe Arg Leu Ser Ile Ala Trp Ser Arg Ile Phe Pro Thr             100 105 110 Gly Leu Glu Glu Asn Pro Asn Glu Ala Gly Leu Ala Phe Tyr Asp Arg         115 120 125 Val Phe Asp Glu Leu Asp Arg Tyr His Ile Glu Pro Leu Val Thr Leu     130 135 140 Ser His Tyr Glu Met Pro Ile Glu Leu Thr Lys Arg Tyr Asn Gly Trp 145 150 155 160 Leu Asp Arg Glu Val Ile Lys Ala Phe Asn His Tyr Thr Glu Thr Val                 165 170 175 Phe Lys Arg Tyr Lys Asp Arg Val Lys Tyr Trp Leu Thr Phe Asn Glu             180 185 190 Ile Asn Thr Ser Ala Trp Gly Phe His Glu Thr Gly Ala Met Asp Gly         195 200 205 Asp Leu Ser Thr Gln Glu Gln Leu Gln Val Arg Tyr Gln Ala Val His     210 215 220 His Gln Phe Val Ala Ser Ala Ala Thr Lys Gln Leu Lys Glu Ile 225 230 235 240 Ala Pro Asn Ala Lys Ile Gly Ser Met Leu Ala Arg Met Gln Thr Tyr                 245 250 255 Pro Ala Thr Ala Asn Pro Val Asp Val Gln Ala Ala Gln Val Glu Asp             260 265 270 Gln Lys Asn Leu Phe Tyr Thr Asp Val Gln Ala Arg Gly Glu Tyr Pro         275 280 285 Glu Tyr Met Asn Arg Tyr Phe Asn Glu Asn Gly Ile Val Ile Asn Met     290 295 300 Ser Ala Asp Asp Glu Lys Ile Leu Lys Gln Tyr Pro Val Asp Phe Ile 305 310 315 320 Ser Phe Ser Tyr Met Thr Asn Val Thr Gly Val Asp Gly Val Glu                 325 330 335 Asp Gly Ile Gly Asn Met Ser Leu Gly Gly Arg Asn Pro Tyr Leu Lys             340 345 350 Glu Ser Glu Trp Gly Trp Gln Ile Asp Pro Ile Gly Leu Arg Val Ser         355 360 365 Leu Asn Val Leu Trp Asp Arg Tyr Arg Lys Pro Leu Phe Ile Val Glu     370 375 380 Asn Gly Leu Gly Ala Val Asp Glu Leu Thr Ala Asp Tyr Lys Val His 385 390 395 400 Asp Asp Tyr Arg Ile Asp Tyr Leu Arg Leu His Ile Glu Gln Met Lys                 405 410 415 Glu Ala Val Ile Asp Gly Val Asp Leu Met Gly Tyr Thr Met Trp Ala             420 425 430 Pro Leu Asp Ile Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Arg Ser         435 440 445 Gly Leu Ile Tyr Val Asp Gln Asp Asp Ala Gly Asn Gly Thr Leu Lys     450 455 460 Arg Tyr Lys Lys Asp Ser Phe Phe Trp Tyr Gln Lys Val Ile Glu Ser 465 470 475 480 Asn Gly Lys Thr Leu Glu ***                 485 <210> 2 <211> 1461 <212> DNA <213> Artificial Sequence <220> <223> Ginsenosidase <400> 2 atgatacagta aagaatcaat cacaggcttt ccaaaggact ttttatgggg tggcgcaact 60 gctgctaatc aaattgaagg ggcgtggcaa gtagatggga agggcttaac aacagctgaa 120 gtcgtacgta aagctgaaga ccgacgtcac ggttcaatgg gagatgttaa cgcagatacc 180 ttacaagaag ctatcgccga tcattcggat gtacattatc caaaacgtcg tggagttgat 240 ttttatcatc actacaaaga agatattgca ctatttgcag aaatgggttt taaagtattt 300 cgactttcaa tcgcttggtc acgtattttt ccaactggtt tagaagaaaa tccaaatgag 360 gctgggttag ctttttatga ccgagtattt gatgaactag atagatacca tattgaacca 420 ctcgtgactc tttcacatta tgagatgcca attgaactaa cgaagcgtta caatggttgg 480 ttggaccgtg aggtcatcaa ggcattcaat cactatacaga aaactgtttt taaaagatac 540 aaggaccgag ttaagtactg gttgacattc aatgaaatta atactagtgc ttggggattt 600 catgaaactg gcgctatgga tggtgattta agtacgcaag aacagttgca agtgcgctat 660 caggcagttc atcatcaatt tgttgccagc gctattgcga caaagcaatt aaaggaaatt 720 gcgcccaacg ccaaaattgg ttcaatgtta gctcgcatgc aaacgtatcc agcaacagca 780 aacccggttg atgtgcaagc agcacaagtt gaagatcaga aaaatttgtt ttatacggat 840 gtccaagcac gaggtgaata tccagaatac atgaatcgat atttcaatga aaatggtatc 900 gttattaata tgtctgctga tgatgaaaaa attttgaaac agtatccggt tgattttatt 960 agtttcagtt attatatgac caacgtgaca ggagtagatg gtgttgaaga cgggattggt 1020 aatatgagtt tgggtggtcg taatccctat ttgaaggaat cagaatgggg atggcaaatc 1080 gacccaatcg gcttgcgagt ttctctgaat gtcctgtggg atcgttaccg taaaccatta 1140 tttattgttg aaaatggctt aggagcagtt gacgaattaa cagcagatta caaagtacac 1200 gatgattatc gtattgacta ccttcgtttg cacattgagc aaatgaaaga ggctgtgatt 1260 gatggcgttg acttaatggg ctacacaatg tgggcaccac tagatattat tagttttagt 1320 acttctgaga tgagtaagcg ttatggcttg atttacgtag atcaagatga cgcaggaaat 1380 ggcacattaa agcgttacaa aaaggattcg ttcttctggt atcaaaaagt tattgaaagt 1440 aacggtaaaa cattagaata a 1461 <210> 3 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Gin primer F <400> 3 cggatccatg tacagtaaag aatcaatcac ag 32 <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Gin primer R <400> 4 cgaatccttc taatgtttta ccgttacttt ca 32

Claims (12)

A ginsenosidase protein consisting of the amino acid sequence shown in SEQ ID NO: 1.
The ginsenosidease protein according to claim 1, wherein the ginsenosidease protein is derived from a strain of Lactobacillus casei GFC 1 (accession number: KCTC18333P).
9. A nucleic acid encoding the ginsenosidase protein of claim 1.
4. The nucleic acid according to claim 3, wherein the nucleic acid has the nucleotide sequence of SEQ ID NO: 2.
A recombinant vector comprising the nucleic acid of claim 3.
A method for producing ginsenoside by treating Ginsenosidase protein of claim 1 with Ginsenoside XVll.
7. The method according to claim 6, wherein the treatment is carried out at a pH of from 6 to 9 at a temperature of from 20 to 40 占 폚.
7. The method of claim 6, wherein the treatment further comprises adding at least one metal ion selected from the group consisting of Co2 ⁺ , Fe3 ⁺ , Cu2 ⁺ , Zn2 ⁺ , NH4 ^+, and Mg2 ⁺ A method for producing ginsenoside.
9. The method of claim 8, wherein the treatment adds NH4 ⁺ or Mg2 ⁺ metal ions.
The method of claim 6, wherein the ginsenoside is Ginsenoside F2 or Ginsenoside Compound K (CK). 7. The method of claim 6, wherein the ginsenoside is Ginsenoside F2 or Ginsenoside Compound K (CK).
11. A cosmetic composition comprising ginsenoside prepared by the method of any one of claims 6 to 10.
12. The cosmetic composition according to claim 11, wherein the ginsenoside has antioxidant, anti-inflammatory and anti-wrinkle effects.

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