KR20130110658A

KR20130110658A - Method for preparing ginsenoside rg3 using recombinant beta-glucosidase gene and hsccc (high speed counter current chromatography) after mplc (medium pressure chromatography) pre-treatment

Info

Publication number: KR20130110658A
Application number: KR1020120032792A
Authority: KR
Inventors: 손성호
Original assignee: 주식회사 비트로시스
Priority date: 2012-03-30
Filing date: 2012-03-30
Publication date: 2013-10-10

Abstract

PURPOSE: A method for preparing ginsenoside Rg3 using recombinant beta-glucosidase gene-derived enzyme and high speed counter current chromatography (HSCCC) after medium pressure liquid chromatography (MPLC) pretreatment is provided to enhance the concentration and the purity of Rg3 in 1 ml of a red ginseng concentrate with a sugar content of 65 brix to approximately 280 mg and 75%, respectively. CONSTITUTION: A method for effectively preparing a large amount of Rg3 from a red ginseng concentrate comprises the steps of: fermenting a red ginseng concentrate using enzyme produced from a recombinant beta-glucosidase gene; treating the red ginseng concentrate by microwaves for bioconversion of general ginsenoside into ginsenoside Rg3; and isolating and purifying the bioconverted Rg3 by MPLC and HSCCC. A strain which produces beta-glucosidase includes Bacillus subtilis, Paenibacillus polymyxa, Thermotoga neapolitana, Escherichia coli, Agrobacterium tumefaciens, Salmonella typhimurium, and Thermus caldophilus. [Reference numerals] (AA) Sample: ginsenoside fraction from red ginseng extract (250mg)

Description

Method for preparing ginsenoside Rg3 using recombinant beta-glucosidase gene and HSCCC (High Speed Counter Current Chromatography) after MPLC (Medium Pressure Chromatography) ) pre-treatment}

The present invention relates to a method for efficiently preparing a large amount of high content of Rg3 from red ginseng, more specifically, an enzyme obtained by inserting a beta-glucosidase gene isolated from various microorganisms into E. coli. The present invention relates to a method for biotransformation of ginsenosides present in red ginseng into ginsenosides Rg3, followed by separation and purification of Rg3 using MPLC and HSCCC.

Ginseng has been cultivated in Korea since ancient times and mainly uses medicinal roots grown for 4-6 years. The main ingredient of ginseng is ginsenosides known as ginseng saponin, and other polysaccharides and phenolic ingredients are included. Ginsenosides, ginseng saponins, are mostly polymer sugars that are not absorbed well in the body, and must be decomposed by intestinal bacteria after ingestion to properly perform pharmacological action. Ginseng-specific ginsenosides are present in red ginseng dried by steaming ginseng, which are ginsenosides Rg3, Rk1, Rg5, Ginsenosides Rb1, Rb2, Rc, and Rd. It is produced by converting to Rh1, Rh2 and the like. However, Rg3 is thus converted to about 20mg per 1g dry weight is not a large amount. In order to overcome this problem, a technique for culturing microorganisms having a beta-glucosidase gene and converting Rg3 to the enzyme has been developed, but the production of Rg3 is still low.

Even if it is converted to Rg3 by heat treatment or enzyme, separation and purification of Rg3 from other impurities is also an important process. There is a method using preparative HPLC to efficiently purify Rg3. However, in this case, since the production amount of Rg3 is only a small amount, it is necessary to develop a large amount of purified bioconverted Rg3.

Therefore, in the present invention, after directly separating the gene from various microorganisms having a beta-glucosidase gene, a strain having a high Rg3 biotransformation rate is developed by recombinant technology, and the recombinant origin The technical task of the present invention is to establish the industrial production technology of Rg3 by completing a method of bioconversion of a large amount of ginsenoside Rg3 using the enzyme of and a combination of MPLC and HSCCC for efficient separation and purification of Rg3. I am doing it.

In order to achieve the above object, the present invention isolates the beta-glucosidase gene from a variety of microorganisms having a beta-glucosidase gene and insert it into E. coli Preparations were made, and among the enzymes produced through the expression of this recombinant gene, the strain with the highest Rg3 bioconversion rate was selected.

The enzyme produced from the selected strain is applied to the actual red ginseng concentrate to convert the Rg3, and to investigate how much more efficient the production method according to the present invention than the conventional heat treatment or the general enzyme method, to solve the above problems.

In addition, the present invention is to solve the problem that only a small amount of production through HPLC by applying HSCCC after MPLC pretreatment in order to obtain an industrial-grade high-purity Rg3 fraction.

According to the present invention, it is possible to obtain Rg3 of more than about 280 mg from 1 g of red ginseng concentrate, which can be expected to replace Rg3 imported from China in the future, and also to export to overseas using high purity. Is considered. In addition, Rg3 is expected to contribute significantly to related industries, as its number is expected to increase rapidly as a raw material for health functional foods or pharmaceuticals in Korea.

1 is a flow chart of a large amount of ginsenoside Rg3 production using beca-glucosidase.
2 is a beta-glucosidase gene isolated from each strain.
Figure 3 is the expression of recombinant genes inserted into E. coli.
Figure 4 is an illustration of HPLC (High Performance Liquid Chromatography) analysis to increase the Rg3 after treating the enzyme produced from the present invention to the red ginseng concentrate.
5 is an exemplary diagram of HPLC (High Performance Liquid Chromatography) of red ginseng concentrate separated using MPLC.
6 shows an example of separating Rg3 using HSCCC

The present invention relates to a method for efficiently preparing a large amount of high content of Rg3 from red ginseng, by separating and recombining beta-glucosidase gene from various microorganisms, and using the produced enzyme Biotransformation of other ginsenosides in red ginseng into Rg3, and a method for producing ginsenoside Rg3 on an industrial scale, comprising separating and purifying Rg3 using a resin and HSCCC. .

The various microorganisms in the above are Bacillus subtilis , Paenibacillus polymyxa , Thermotoga neapolitana , Escherichia coli , Agrobacterium tumefaciens , Salmonella typhimurium , and Thermus It is characterized by caldophilus and the like, and provides a method for producing a large amount of beta-glucosidase enzyme by separating and beta-glucosidase synthesis gene from them, and a high content from fermented mature red ginseng concentrate using these enzymes A method of producing Rg3 and a method of mass producing pure Rg3 using resin, MPLC, and HSCCC are provided.

The present invention relates to ginsenoside Rg3 bioconversion from red ginseng concentrate using beta-glucosidase having saponin-degrading ability, and a gene capable of synthesizing beta-glucosidase. If you have microorganisms with no limits, but Bacillus subtilis , Paenibacillus polymyxa , Thermotoga neapolitana , Escherichia coli , Agrobacterium tumefaciens , Salmonella typhimurium , and Thermus It may be selected from the group consisting of caldophilus .

Hereinafter, the present invention will be described in detail. The present invention is a microorganism having high conversion rate to ginsenoside Rg3 by separating beta-glucosidase, an enzyme having saponin-degrading ability, from microorganisms, treating it with red ginseng concentrate, and fermenting and ripening at a specific temperature and method. Can be selected. Even the same beta-glucosidase enzyme may show a difference in activity, and in the present invention, an enzyme having higher activity than the existing enzyme could be obtained.

The prior art discloses that ginsenoside (beta-glucosidase) bioconversion may be caused by inoculating ginseng or red ginseng and incubating with lactic acid bacteria. However, the bioconversion of ginsenosides by these lactic acid bacteria is likely to be small, and in particular, the conversion to ginsenoside Rg3 is considered to be insignificant. Among the ginsenosides, the present invention aims to bioconvert to Rg3.

Bioconversion to ginsenoside Rg3 in the present invention is the majority of conversion by the beta-glucosidase enzyme isolated in the present invention. However, when the fermented red ginseng concentrate was analyzed by HPLC, the relative Rg3 content was low, which was thought to be due to the glucose not completely attached to the chemical back bone of ginseng saponin. Therefore, in the present invention, in order to further increase the conversion to ginsenoside Rg3, microwaves were used to convert ginsenosides, which may remain unconverted, to Rg3.

The prior art uses lactic acid bacteria to induce the conversion of ginsenosides, but the heat treatment is also performed in parallel. In general, the heat treatment temperature is said to be processed in the range of 90 ~ 150. In this case, however, the heat treatment procedure may be more complicated than the case of using a microwave because it further includes a step such as pressurization. The microwave method used in the present invention has the advantage that the heat treatment for ginsenoside conversion under atmospheric pressure can be carried out more conveniently.

According to the present invention, the microwave heat treatment may be performed in a range of 10 to 40 minutes, more preferably 15 to 25 minutes, most preferably 20 minutes. According to the present invention, the Rg3 contained in about 10 mg in 1 ml of the existing 65 brix red ginseng concentrate can be increased to about 280 mg, and the purity can be increased to 75% or more when using HSCCC.

According to the present invention, any method is not particularly limited as long as it can analyze and purify the bioconverted Rg3. Among them, the crude total saponin can be obtained using the resin. As the resin to be used, there is no limitation as long as it can collect ginsenosides. Among them, HP-20 resin is preferably used. The solvent used to recover the saponins adsorbed on the resin will not be limited as long as the saponins can be collected. Among them, ethanol anhydride is most preferred.

Any method can be used if only Rg3 can be recovered from the total saponin collected using the anhydrous ethanol, but in the present invention, a large amount was collected on a commercial scale using MPLC and HSCCC. In the present invention, total saponin recovered from the HP-20 resin column was first collected using MPLC to fractionate Rg3. The fraction collection time is between 30 minutes and 70 minutes, preferably between 40 and 60 minutes, and most preferably between 45 and 55 minutes.

The collected fraction includes Rg3 as a main component, and any method may be used to purely recover high-purity Rg3, but in the present invention, which aims to produce a large amount of pure Rg3 at a commercial level, it is preferable to use HSCCC. desirable. It was possible to analyze Rg3 from the time-phased fractions that passed, and to collect high purity Rg3 therefrom.

In obtaining the Rg3 S type fraction using HSCCC, it was confirmed that the separation efficiency of each saponin was very low when MPLC was not used as the pretreatment. It appeared to be essential.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments of the present invention. However, the scope of the present invention is not determined accordingly, but is presented by way of example only.

Isolation and Cloning of the Beta-Glucosidase Gene

Microorganisms with beta-glucosidase genes were selected through genomic database search. Based on this, Bacillus subtilis , Paenibacillus polymyxa , Thermotoga neapolitana , Escherichia coli , Agrobacterium tumefaciens , Salmonella typhimurium , and Thermus caldophilus were selected, and the beta-glucosidase synthetic genes were isolated from these using restriction enzymes, and the isolated beta-glucosidase synthetic genes were isolated from the TA cloning kit (Invitrogen, USA). After cloning (manual) according to the manual using and inserted into E. coli.

[Table 1] (Exemplary diagram showing the size of the selected strain, beta-glucosidase synthetic gene and produced protein through genomic database search) Entry Name Gene Name Protein Name Strain Protein (kDa) BGL1_BACSU bglH beta-glucosidase Bacillus subtilis 53.289 BGL2_BACSU yckE probable beta-glucosidase Bacillus subtilis 55.140 BGLA_PAEPO bglA beta-glusosidase A Paenibacillus polymyxa 51.649 BGLB_PAEPO bglB beta-glucosidase B Paenibacillus polymyxa 51.573 BGLX_ECOLI bglX periplasmic beta-glucosidase Escherichia coli 83.460 BGLA_THENE bglA beta-glucosidase A Thematoga neapolitana 51.493 BGLS_AGRTU bglA beta-glucosidase Agrobacterium tumefaciens 88.290 BGLX_SALTY bglX periplasmic beta-glucosidase Salmonella typhimurium 83.392 Q8GHE5_THECA none beta-glusocidase Thermus caldophilus 48.661

Isolation of Beta-glucosidase Enzymes

E. coli containing the recombinant vector was incubated for 5 days in a 5L bioreactor containing a liquid culture medium. The microbial cells were recovered by centrifugation and washed twice with sterile water. Water was removed from the cells using filter paper and finely powdered using liquid nitrogen. About 3 times the volume of the powdered cells was added to sodium phosphage (pH 7.0) buffer and sonicated to destroy the cells. After standing in ice for about 15 minutes, centrifuged for 15 minutes at 4, 42000 rpm.

Red ginseng concentrate preparation

After washing the ginseng, steamed red ginseng at 121 for 1 hour, dried it with hot air, and dried red ginseng. The dried red ginseng was extracted four times with 70% ethanol solution, filtered and concentrated again to about 65 brix. Red ginseng concentrate was prepared in the state.

Rg3 Bioconversion Using Beta-glucosidase Enzyme

The beta-glucosidase enzyme was added to the red ginseng concentrate and fermented for 3 days in a constant temperature room maintained at 25 to 28. When the Rg3 bioconversion rate in the fermented red ginseng concentrate was insufficient, it was further aged for one day. After fermentation, the red ginseng concentrate was heated for about 20 minutes using microwave. The red ginseng concentrate thus prepared was diluted 20 times with purified water, and then centrifuged at about 15,000 rpm to remove the residue. Bioconverted Rg3 was confirmed using TLC and HPLC. HPLC analysis was performed using Agilent 1200 series (Agilent Technologies, USA), the column was ZORBAX Eclipse XDB-C18 (Agilent Technologies, USA), the oven temperature of the column was 30, the loading amount of the sample was 10. The mobile phase was obtained by concentration gradient for 60 minutes with 100% water and 100% acetonitrile. The flow rate was 1 ml per minute and was detected at 203 nm.

Bioconverted Rg3 Isolation and Analysis

The dilution solution from which the scum was removed was filled in a HP-20 (Mitsubishi Chemical Corporation, Japan) resin column swelled with water so that ginseng saponin adhered to HP-20, and then washed three times with purified water, followed by adsorbing saponin. Were removed. Ginseng saponin attached to the resin was collected again using anhydrous ethanol and then received the ginseng saponin fraction by time zone using MPLC. MPLC analysis was performed using the SP System (Biotage, Sweden) and the column was KP-C18-HS (Biotage, Sweden). The mobile phase was obtained by concentration gradient in 70% with 100% water and 100% acetonitrile. The flow rate was 20 ml per minute and was detected at 203 nm.

Mass production of Rg3 via HSCCC

When the fraction containing Rg3 obtained above was concentrated to about 35brix, it was injected into the mobile phase to HSCCC. The fractions containing Rg3 were collected separately by running the HSCCC and flowing the fractions. When the fraction containing the Rg3 is collected, the purified water is added enough to adjust the brix of the fraction to about 5 ~ 10, and then placed for 3 days at a low temperature of about 10 degrees. After 3 days, a white precipitate was found to sink to the bottom, which was collected, washed with purified water, and centrifuged and dried to obtain a high concentration of R type Rg3 white powder. The fraction from which the precipitate was removed was concentrated to about 65 brix to obtain Rg3 S type in the same manner.

None

Claims

Fermenting red ginseng concentrate using an enzyme produced from a recombinant beta-glucosidase gene to bioconvert general ginsenosides to ginsenoside Rg3;

Fermenting red ginseng concentrate using an enzyme produced from a recombinant beta-glucosidase gene in the bioconversion method, followed by microwave treatment;

The bio-converted Rg3 is separated and purified using MPLC and HSCCC to efficiently prepare a large amount of Rg3 from red ginseng concentrate.

The method of claim 1,
The beta-glucosidase enzyme producing strain is Bacillus subtilis , Paenibacillus polymyxa , Thermotoga neapolitana , Escherichia coli , Agrobacterium tumefaciens , Salmonella typhimurium , and Thermus How to use caldophilus .

The method of claim 1,
After fermentation of red ginseng concentrate using the enzyme and heating for 20 minutes with a microwave or a related technology

The method of claim 1,
Method of using MPLC and HSCCC or related technology to isolate and purify the bioconverted Rg3 in mass production

The method of claim 1,
When separating and purifying the bioconverted Rg3, MPLC pretreatment step before HSCCC.

The method of claim 1,
Purification of Rg3 and other saponins using the MPLC and HSCCC and related technologies.

The method of claim 1,
A technique for extracting and purifying saponin from red ginseng using the technique shown in the drawings of the present invention or a similar technique.