KR102011853B1 - Manufacturing method of ginsenoside C-K - Google Patents

Abstract

The present invention relates to a method for preparing ginsenoside CK, specifically, a) a kitty having an amino acid sequence of SEQ ID NO: 1 in a ginsenoside containing one or more ginsenosides selected from ginsenosides Rb1, Rb2, Rc and Rd Reacting the old woman by adding an enzyme derived from Chitinophaga pinensis ; And b) adding the cellulase complex enzyme Cellic CTec2 to the reaction product obtained in step a) to react with the ginsenoside CK.
According to the present invention, ginsenoside CK, which is a rare ginsenoside in the minor form present in trace amounts in ginseng or processed products of ginseng, can be efficiently produced using a safe enzymatic method. In particular, the method of the present invention has the advantage that the process is very simple and the production efficiency is very excellent, allowing mass production for industrial use.

Description

Manufacturing method of ginsenoside C-K

The present invention relates to a method for preparing ginsenoside CK, specifically, a) a kitty having an amino acid sequence of SEQ ID NO: 1 in a ginsenoside containing one or more ginsenosides selected from ginsenosides Rb1, Rb2, Rc and Rd Reacting the old woman by adding an enzyme derived from Chitinophaga pinensis ; And b) adding the cellulase complex enzyme Cellic CTec2 to the reaction product obtained in step a) to react with the ginsenoside CK.

Saponin means a substance composed of several cyclic compounds in the glycosides widely present in the plant system, but not sugar. Triterpene saponin, a saponin component contained in ginseng or red ginseng as a main bioactive ingredient, has a chemical structure different from that found in other plants, so ginseng (Ginseng) is distinguished from other plant-based saponins. ) Glycoside is called Ginsenoside.

Ginsenosides are protopanaxadiol-type (PPD type) ginsenosides and Protopanaxatriol-type (PPT type) ginsenosides and oleanolinic acids according to the structure of aglycone. (oleanolic acid type) can be classified into three types of ginsenosides. These three groups are again classified according to the location and number of sugar moieties that are attached by glycosicid bonds to the 3rd carbon, 6th carbon and 20th carbon positions of the ring in the compound structure. . The basic skeleton of the oleanolinic acid ginsenoside is cyclic, and there is only ginsenoside Ro, and the aglycone is oleanolic acid.

Currently, over 180 ginsenosides have been isolated, most of which are PPD type ginsenosides. Ginsenosides of the PPD type include Rb1, Rb2, Rb3, Rc, Rd, Gypenoside XVII, Compound O, Compound Mc1, F2, Compound Y, Compound Y, Compound Mc (Compound Mc), Rg3, Rh2, CK. Ginsenosides of the PPT type include Re, Rg1, Rf, Rg2, Rh1 and the like.

In addition, it occupies 90% or more of ginsenosides in dried ginseng, but it is very low in vivo due to its large size near 1,000 daltons. Therefore, in order to increase the efficacy of ginsenosides, it is necessary to convert major ginsenosides into minor ginsenosides which are relatively well absorbed and have better efficacy. That is, major ginsenosides require a conversion process to remove glucose, arabinose, rhamnose, xylose, and the like which constitute sugar in order to effectively exhibit physiological activity in vivo. Major ginsenosides include Rg1, Re, Rb1, Rb2, Rc and Rd, and minor ginsenosides (rare ginsenosides) present in trace amounts include F2, Rg3, Rk1, Rg5, Rh1, Rh2, Rk2, Rh3, gypenoside (Gyp) XVII, zipenoside LXXV and compounds K, CK, Mc, Mc1 (compound K, CK, Mc, Mc1) and the like.

Among minor ginsenosides, CK and the like are present in a very small amount in ginseng and contain a very small amount of ginsenosides during the process of red ginseng, and the therapeutic effect of sepsis caused by Gram-negative bacteria (Korea Patent No. 10-0913213) ), Inhibiting or treating obesity (Korean Patent No. 10-0847252), prevention, delay and treatment of macular degeneration disease (Korean Patent No. 10-1539573), as well as anti-cancer effect, there are various pharmacological effects Is the substance of interest.

As described above, the minor ginsenosides such as C-K may be used for various useful purposes, but since the present amount is small in ginseng, it is necessary to develop a technology for mass production thereof.

As a method for producing minor ginsenosides present in trace amounts, a chemical decomposition method, an enzymatic method, and a method using glycoside synthesis have been proposed, but such methods have to be performed through several steps in the manufacturing process; In the manufacturing process, the desired component is lost, 3) the use of an unsuitable catalyst for food, or 4) the low yield still limited production.

Particularly, among the enzymatic methods, enzymes that can be used include β-glucosidase, α-L-arabinopyranosidase, and α-L-arabinofuranosidase. Although several enzymes belonging to (α-L-arabinofuranosidase) and α-L-rhamnosidase are known, there are problems in that they are not effective or costly as mass production methods.

Although it belongs to the above enzyme classification, there is a big difference in the substrate and the final product according to the origin of the enzyme, and in fact, the enzyme having the activity capable of converting major ginsenoside to minor ginsenoside is very rare. Much effort is needed to identify new enzymes that can efficiently produce minor ginsenosides through biotransformation of cenosides.

In this regard, the present inventors have endeavored to discover an enzyme useful for producing useful minor ginsenosides through the bioconversion of major ginsenosides as described above. As a result, chitinophaga having the amino acid sequence of SEQ ID NO. pinensis ) -derived enzymes can be used to efficiently convert ginsenosides Rb1, Rb2, Rc and Rd to ginsenoside F2, and finally the ginsenoside CK using the commercialization enzyme Cellic CTec2. It was confirmed that can be efficiently produced and the present invention was completed.

Republic of Korea Patent No. 10-0913213 Republic of Korea Patent No. 10-0847252 Republic of Korea Patent No. 10-1539573

Therefore, the main object of the present invention is to efficiently prepare ginsenoside CK, a minor ginsenoside in the minor form present in ginseng or processed products of ginseng from major ginsenosides by enzymatic method. To provide a way.

According to one aspect of the invention, the invention is a) chitinopa having a amino acid sequence of SEQ ID NO: 1 in ginsenosides containing one or more ginsenosides selected from ginsenosides Rb1, Rb2, Rc and Rd Reacting by adding an enzyme derived from Chitinophaga pinensis ; And b) adding the cellulase complex enzyme Cellic CTec2 to the reaction product obtained in step a) to react with the reaction product obtained in step a).

In the ginsenoside C-K production method of the present invention, the irradiated ginsenoside composition is preferably obtained by extracting Korean ginseng with an alcoholic solution having a alcohol content of 50 to 90%.

In the ginsenoside C-K production method of the present invention, the reaction of step a) is preferably performed at pH 6.5 to 7.5 and 30 to 37 ℃.

In the ginsenoside C-K production method of the present invention, it is preferable that the reaction of step b) is performed at pH 5 to 5.5 and 45 to 50 ℃.

According to the present invention, ginsenoside C-K, which is a rare ginsenoside in the minor form (minor form) present in ginseng or processed products of ginseng, can be efficiently produced using a safe enzymatic method. In particular, the method of the present invention has the advantage that the process is very simple and the production efficiency is very excellent, allowing mass production for industrial use.

1 is a block diagram showing a ginsenoside CK manufacturing process according to an embodiment of the present invention.
2 is a result of HPLC analysis of the irradiated phonyponin used in one embodiment of the present invention.
FIG. 3 is a HPLC analysis result of an enzyme reaction product in which an irreversible reaction reaction of chiponinin with a chitinophaga pinensis- derived enzyme according to an embodiment of the present invention.
4 is an HPLC analysis result of an enzyme reaction product in which chitinopa reacts with Chitinphaga pinensis- derived enzyme product with Cellic CTec2.

A method for preparing ginsenoside CK of the present invention is a) chitinopa having a amino acid sequence of SEQ ID NO: 1 in a ginsenoside containing one or more ginsenosides selected from ginsenosides Rb1, Rb2, Rc, and Rd. Reacting by adding an enzyme derived from Chitinophaga pinensis ); And b) adding the cellulase complex enzyme Cellic CTec2 to the reaction product obtained in step a).

Step a) may be referred to a step of obtaining ginsenoside F2 by treating the ginseng checkonin containing one or more ginsenosides selected from ginsenosides Rb1, Rb2, Rc and Rd with an enzyme derived from chitinopaga pinensis. have. According to the present invention, the chitinopaga pinensis-derived enzyme can convert ginsenosides Rb1, Rb2, Rc and Rd into ginsenoside F2 very efficiently. At this time, the enzyme reaction is preferably made at pH 6.5 to 7.5 and 30 to 37 ℃ in order to increase the conversion efficiency to ginsenoside F2.

The chitinofa pinenesis-derived enzyme used in step a) is originally composed of the amino acid sequence of SEQ ID NO: 1 and is registered as ACU59334.1 in GenBank. It is known to have the activity of beta-galactosidase, but the ginsenosides Rb1, Rb2, Rc and Rd can be used as substrates or ginsenosides based on the ginsenosides. It is not known at all whether it can be converted to F2, and it is first revealed through the present invention.

The enzyme is produced by Chitinophaga pinensis DSM 2588, and the strains of chitinopaga Dens 2588 include DSM, KACC 12763, ATCC 43595, IFO (now NBRC) 15968, LMG 13176, Deposited as UQM 2034. The enzyme of the present invention can be obtained through a purification method for culturing the strain according to the culture method suggested by the deposit institution and for purifying a conventional beta-galactosidase or a purification method using the amino acid sequence. In addition, the gene encoding the enzyme is composed of the nucleotide sequence of SEQ ID NO: 2, it can also be obtained by heterologous expression in other organisms such as E. coli using this sequence. Accordingly, the enzyme of the present invention may also add various amino acid sequences to the N-terminus or C-terminus of the amino acid sequence of SEQ ID NO: 1. There is no particular limitation on the sequence to be added, but it would be desirable to add targeting sequences, tags, labeled residues, half-life or amino acid sequences designed for specific purposes to increase the stability of the peptide. For example, a plurality of histidines may be added to the N-terminus or the C-terminus for ease of purification.

Ginseng research saponin (crude saponin) refers to the crude saponin before it is divided into components. Generally, the ginseng sample is extracted with a solvent such as ethanol or alcohol and adsorbed using a porous resin such as HP-20 or AB-8 resin. After it can be obtained by adjusting the concentration of ethanol again. In the present invention, any of these ginseng irradiated saponinins may be used as long as they contain Rb1, Rb2, Rc or Rd. Such investigationonin can be obtained by extracting alcohol from the leaves and roots of other ginseng, including Korean ginseng, hwagi, and bamboo shoots, and more preferably, ginseno of the sugar and protopanaxatriol family using HP-20 resin. It is preferable to use a probeponin with a higher content of Rb1, Rb2, Rc or Rd by removing the side and the like. It is effective to use alcohol having 50 to 90% (v / v) alcohol content.

In the present invention, step b) may be referred to a step of converting ginsenoside F2 generated in step a) to ginsenoside C-K using the cellulase complex enzyme Celic CTec2. Celic CTec2 is a cellulase complexase sold by Novozymes as a commercial enzyme, and is mainly used for hydrolyzing lignocellulosic materials. According to the present invention, the celtic CTec2 can convert ginsenoside F2 to ginsenoside C-K very efficiently. At this time, the enzyme reaction is preferably performed at pH 5 to 5.5 and 45 to 50 ℃ in order to increase the conversion efficiency to ginsenoside C-K.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

EXAMPLE

1. Survey Phonine Extract

The leaves and roots of Korean ginseng were extracted two or more times (1-2 hours at room temperature) with alcohol containing 80% (v / v) of 20 times the volume of alcohol and dried to obtain an extract. The extract was dissolved again in water, adsorbed onto HP-20 resin, washed with water to remove sugars, and washed first with alcohol containing 40% (v / v) alcohol to remove ginsenosides Re and Rg1, which are protopanaxatriol. After the removal, the alcohol content of 80% (v / v) was washed with alcohol and ginsenosides Rb1, Rb2, Rc, Rd of the protopanaxadiol-based eluting was obtained and dried. As a result, analysis of the irradiated phononine by HPLC is shown in FIG. 2.

2. Manufacture of ginsenoside F2

After dissolving the probeponin prepared in 1 above in 0.1M phosphate buffer (pH 7.0), the enzyme derived from Chitinophaga pinensis having the amino acid sequence of SEQ ID NO: 1 was 1/100 to 1/10 of the total volume. The reaction was carried out by addition. At this time, the enzyme reaction was carried out at 37 ℃ for 24 hours.

The chitinofa pinensis-derived enzyme amplifies an enzyme gene from the genomic DNA of chitinpaga pinensis KACC 12763 using primers of SEQ ID NOs: 3 and 4, and uses pGEX4T- using a BamHI and XhoI restriction enzyme site. After cloning into one vector to prepare a recombinant vector, the recombinant vector was transformed with E. coli BL21, overexpressed through IPTG induction while culturing, and cultured cells were obtained by crushing and centrifuging (supernatant).

As a result of analyzing the enzyme reaction obtained by the enzyme reaction by HPLC, almost all ginsenosides Rb1, Rb2, Rc, and Rd were converted to ginsenoside F2 as shown in FIG.

This means that the chitinopa is a pinene-derived enzyme capable of converting ginsenosides Rb1, Rb2, Rc and Rd into ginsenoside F2 very efficiently.

3. Preparation of Ginsenoside C-K

Adjust the enzyme reaction obtained in step 2 to pH 5 using acetate buffer (0.1M, pH 5.0), and then add Noviczyme's cellulase complex enzyme Cellic CTec2 to 1/2 to 1/10 of the total volume. The reaction was carried out. At this time, the enzyme reaction was carried out at 50 ℃ for 24 hours.

As a result of analyzing the enzyme reaction obtained by the Cellic CTec2 enzymatic reaction by HPLC, ginsenoside F2 was converted to ginsenoside C-K as shown in FIG. 4.

<110> BACK, So-young          Shine & Shine Co., Ltd. <120> Manufacturing method of ginsenoside C-K <130> PA-D17286 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 451 <212> PRT <213> Unknown <220> <223> Chitinophaga pinensis <400> 1 Met Glu Gln Phe Ser Ala Pro Phe Ser Arg His Asp Phe Gly Glu Asn   1 5 10 15 Phe Leu Trp Gly Val Thr Ile Ser Ala Phe Gln Asn Glu Gly Ala Cys              20 25 30 Asp Ala Asp Gly Lys Gly Lys Ser Ile Trp Asp Glu Phe Thr Thr Lys          35 40 45 Lys Gly Lys Ile Lys Asp Gly Thr Asn Ala Leu Val Thr Val Asp Phe      50 55 60 Tyr Asn Arg Tyr Arg Glu Asp Ile Gln Leu Val Lys Gln Met Gly Phe  65 70 75 80 Asp Val Phe Arg Phe Ser Ile Ser Trp Pro Arg Ile Leu Pro Glu Gly                  85 90 95 Thr Gly Lys Val Asn Gln Ala Gly Ile Asp Tyr Tyr His Arg Val Ile             100 105 110 Asp Ala Cys Leu Glu Ala Gly Leu Ile Pro Tyr Val Thr Leu Tyr His         115 120 125 Trp Asp Leu Pro His Ala Ile Glu His Lys Gly Gly Trp Cys His Arg     130 135 140 Gly Val Ile Phe Ala Phe Glu Glu Tyr Val Arg Ile Cys Val Gln Ala 145 150 155 160 Phe Gly Asp Lys Val Lys Asn Trp Ile Val Met Asn Glu Pro Phe Gly                 165 170 175 Phe Thr Ser Leu Gly Tyr Met Leu Gly Val His Ala Pro Gly Lys Phe             180 185 190 Gly Val Ser Tyr Phe Leu Pro Ala Val His His Val Leu Leu Ala Gln         195 200 205 Ala Val Gly Ala Lys Val Ile Arg Glu Thr Val Lys Glu Ala Asn Ile     210 215 220 Gly Thr Cys Leu Ser Cys Ser Tyr Ile Tyr Pro Tyr Thr Gln Gln Glu 225 230 235 240 Gly Asp Ile Gln Ala Ala Lys Lys Ala Asp Ala Leu Phe Asn Arg Leu                 245 250 255 Phe Leu Glu Pro Val Leu Gly Met Gly Tyr Pro Val Asp Asp Phe Pro             260 265 270 Leu Leu Arg Arg Ile Glu Arg Arg Tyr Ala Leu Trp Arg Asp Trp Asp         275 280 285 Gln Leu Ser Phe Asp Phe Asp Phe Ile Gly Val Gln Asn Tyr Phe Pro     290 295 300 Leu Val Val Arg Arg Asn Ala Phe Met Pro Val Val Gly Ile Ser Glu 305 310 315 320 Val Lys Pro Lys Ser Arg Arg Val Pro Met Thr Ala Leu Gly Trp Glu                 325 330 335 Ile Ser Gly Glu Gly Met Tyr Ala Ile Leu Lys Gln Phe Gly Ala Tyr             340 345 350 Lys Gly Ile Lys Arg Leu Met Val Thr Glu Ser Gly Ala Ala Phe Ala         355 360 365 Asp Ile Met Ser Gly Gly Ala Ile Asp Asp Gln Asp Arg Ile Gly Tyr     370 375 380 Phe Lys Glu Tyr Leu Ala Gly Val Leu Lys Ala Lys Arg Glu Gly Ile 385 390 395 400 Pro Ile Asp Gly Tyr Phe Ala Trp Thr Leu Thr Asp Asn Phe Glu Trp                 405 410 415 Ala Glu Gly Tyr Lys Ala Arg Phe Gly Leu Val His Val Asp Arg Asp             420 425 430 Thr Gln Val Arg Thr Met Lys Ser Ser Gly Gln Trp Phe Ser Glu Leu         435 440 445 Leu Lys Lys     450 <210> 2 <211> 1356 <212> DNA <213> Unknown <220> <223> Chitinophaga pinensis <400> 2 atggaacaat tcagcgcacc tttcagccgt catgattttg gagagaattt cctttgggga 60 gtgacgattt ccgcatttca gaatgaaggg gcctgcgatg ccgatggtaa agggaagtct 120 atctgggacg agtttaccac caagaaagga aagattaaag atggtaccaa tgcacttgtg 180 acagtcgatt tctataaccg ttaccgggaa gatattcagc tggtcaaaca aatgggtttt 240 gatgtgttcc gtttttctat ttcctggccg cgtattctgc cggaaggtac cggcaaggtg 300 aatcaggcgg gcatcgatta ttatcaccgg gtgatagacg cctgtctgga agccggactg 360 ataccatacg ttactttata tcactgggat ttaccgcatg caattgaaca caagggagga 420 tggtgccacc gtggcgttat tttcgctttt gaagaatatg tgcgtatatg tgtacaggcg 480 tttggagata aggtgaagaa ctggattgtg atgaacgagc cttttggttt tacatcatta 540 ggctacatgt tgggcgtcca tgcacccggt aaatttggcg tatcctattt tcttccggcg 600 gtacatcatg tgctgcttgc acaggcagta ggtgcgaaag tcatccggga gaccgtcaaa 660 gaagccaata tagggacttg tctgtcctgt tcctatattt acccttacac acagcaagag 720 ggcgatatac aggctgcaaa gaaggcggat gcgctattca atcgcctgtt cctggagccg 780 gtactgggca tggggtatcc ggtggatgat ttccccttat tacgccggat agaaagacgc 840 tatgccttgt ggcgggattg ggaccagctg tcattcgatt ttgattttat cggcgtccag 900 aactatttcc cgttggtggt acgcaggaat gcgtttatgc cggtggtcgg catatccgaa 960 gtaaaaccga agtcccgccg ggtgccaatg acggcgttag gctgggagat cagcggggaa 1020 gggatgtatg ccatcctgaa acagtttggt gcttataaag gcattaaacg gctgatggta 1080 acggaaagcg gggctgcctt tgctgatatt atgagtgggg gtgctatcga tgatcaggac 1140 cgtattggtt actttaagga atatttggct ggtgtactga aggcgaagag ggaggggata 1200 ccgatagacg ggtattttgc ctggacgctg acagataatt tcgaatgggc ggaaggatat 1260 aaggcccgct ttgggttagt gcatgtagat cgtgatacgc aggtgcggac gatgaaaagc 1320 tccggacagt ggttcagcga gttgttaaaa aaataa 1356 <210> 3 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for beta-galactosidase of Chitinophaga pinensis          KACC 12763 <400> 3 cgggatccga acaattcagc gcacctttca gccgt 35 <210> 4 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for beta-galactosidase of Chitinophaga pinensis          KACC 12763 <400> 4 cccctcgagt tattttttta acaactcgct gaacca 36

Claims (4)

a) reacting a ginsenoside containing one or more ginsenosides selected from ginsenosides Rb1, Rb2, Rc and Rd by adding an enzyme derived from Chitinophaga pinensis having an amino acid sequence of SEQ ID NO: 1 step; And
b) a ginsenoside CK manufacturing method comprising the step of reacting the reaction product obtained in step a) by adding the cellulase complexase Cellic CTec2. The method of claim 1,
The method of producing ginsenoside CK, characterized in that the irradiated ginseng obtained by extracting Korean ginseng with alcohol solution of 50 to 90% (v / v) alcohol. The method of claim 1,
Ginsenoside CK manufacturing method characterized in that the reaction of step a) is made at pH 6.5 to 7.5 and 30 to 37 ℃. The method of claim 1,
Ginsenoside CK production method characterized in that the reaction of step b) is made at pH 5 to 5.5 and 45 to 50 ℃.

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