KR101156204B1 - Beta-glycosidase and Production of aglycon protopanaxadiol and compound K by the thermostable beta-glycosidase - Google Patents

Abstract

The present invention relates to a method for preparing a compound g and aglycon protopanaxadiol, which are rare ginsenosides, using beta glycosidase (β-glycosidase), and more particularly, pyro pyro Pyrococcus furiosus ) a recombinant expression vector containing beta-glycosidase, a method for producing a large amount of beta-glycosidase using E. coli transformed therein, and the high temperature beta-glucosidase as a substrate Cenoside Rd or ginseng extract is reacted with a reaction solvent to provide a method for preparing ginsenoside compound K and aglycone protoparanaxadiol.

Description

Beta-glycosidase and Production of aglycon protopanaxadiol and compound K by the thermostable beta-glycosidase}

The present invention utilizes a thermophilic beta-glycosidase enzyme, but is present in ginsenoside compound K belonging to the rare ginsenoside which is not present in ginseng, but present in trace amounts in red ginseng, and ginseng and red ginseng. The present invention relates to a method for preparing aglycon protopanaxadiol, which is not present, and more specifically, to pyrooccus pyriocus ( Pyrococcus). furiosus ) using a high-temperature beta-glycosidase enzyme derived from the strain to selectively hydrolyze only the glucose of Ginsenosides at high temperature to finally synthesize the ginsenoside compound K and the aglycone propanacanadiol. It relates to a manufacturing method.

Ginsenoside compound k (formula 1) and aglycone protoparanaxadiol (formula 2) are known to have various excellent effects such as immune enhancement, tumor angiogenesis inhibition, cancer cell invasion inhibition and cancer cell proliferation inhibition.

Conventional techniques for the production of compound k to date, beta-galactosidase enzyme (Korean Patent Publication No. 2003-94757), cellulase or Aspergillus genus isolated from the genus microorganism of the genus Penicillium By treating beta-galactosidase (Korean Patent No. 377546), P. nagininase isolated in Penicillium, or Pectinase (Korean Patent No. 418604) isolated in Aspergillus. Methods of making compound k are known.

In addition, a single molecule of glucose in the compound K may be hydrolyzed to produce aglycone propananaxadiol, which is a method of alkali hydrolysis using sodium (Na) metal (Korean Journal of Pharmaceutical Sciences, Vol. 38, 425-429 (1994)). However, there are difficult reaction conditions, long reaction times and low yields. On the other hand, the method for preparing aglycone propanacanadiol using the acid hydrolysis ability of perchloric acid (HClO ₄ ) is 92.65%, but the chemical method of epimerization, hydration, and hydrosilylation ( There is a problem that an addition reaction such as hydroxylation may occur. This problem can be overcome by culturing with microorganisms and biological conversion methods using enzymes of microorganisms. In particular, the biological conversion method does not produce by-products and thus is environmentally friendly and has a merit of producing a desired substance in high yield because of substrate specificity for a specific sugar. Although there have been reports of conversion of aglycone protopanaxadiol through incubation of intestinal bacteria and ginsenoside algi 3 (Rg3), it requires a culture time of more than 40 hours and a low yield (Bae-Eun Ah et al. 2002 metabolism of 20 ( S )-and 20 ( R ) -ginsenoside Rg3 by human lntestinal bacteria and its relation to in vitro b iological activities. Biol. Pharm. Bull. 25: 1 58-63), the production of aglycone propanacanadiol by enzyme has not been reported.

The production of the compound k and aglycone protoparnaxadiol of the present invention increases the solubility of the substrate ginsenoside Rd or Ginseng root extract due to the high temperature reaction temperature by using a high temperature beta-glycosidase. The present invention was successfully completed by providing a high production method of compound K and aglycone protoparanaxadiol by investigating the optimum concentration of ginsenoside Aldi (Rd) and ginseng extract as an enzyme unit / ml and a substrate as a substrate. It was.

The present invention has been made to solve the above problems and the object of the present invention is to provide an enzyme capable of mass production of compound k, and aglycon protopanaxadiol (Ginsenoside compound K and aglycon protopanaxadiol) .

It is another object of the present invention to provide a method for mass production of compound K and aglycone propanacanadiol by reacting a high temperature beta-glycosidase enzyme with a ginsenoside aldi or ginseng extract as a substrate. will be.

Another object of the present invention is to provide a compound K, and a composition for the production of aglycone protoparnaxadiol.

In order to achieve the above object, the present invention provides a beta-glycosidase used in the production of compound k and aglycone protoparanaxadiol.

In one embodiment of the present invention, the thermophilic beta-glycosidase is an ultra-high temperature microorganism Pyrococcus furiosus ) is preferably derived from a strain, but may also be obtained by a method such as polymerase chain reaction (PCR) or chemical synthesis.

In an embodiment of the present invention, the beta-glycosidase is within the range that the beta-glycosidase activity indicated by the amino acid sequence described in SEQ ID NO: 1 (Fig. 8) and proteins having these amino acid sequences is not impaired, Variant beta-glycosidase introduced with deletion, substitution and addition of at least one amino acid of at least one amino acid.

As an example, the gene sequence encoding the beta-glycosidase of this invention is represented by SEQ ID NO: 2. In addition, the beta-glycosidase gene encoding the above-mentioned mutant beta-glycosidase obtained by mutating the nucleotide sequences of SEQ ID NO: 2 is also included in the beta-glycosidase gene according to the present invention.

In another aspect, the present invention provides a composition for producing ginsenoside compound k containing the enzyme of the present invention.

In another aspect, the present invention provides a composition for preparing aglycone propananaxadiol comprising the enzyme of the present invention.

In another aspect, the present invention provides a process for producing compound K and aglycone protoparanaxadiol by treating ginsenoside aldi or ginseng extract with the enzyme of the present invention.

In addition, the present invention includes a recombinant vector containing the beta-glycosidase gene, transformants transformed by the recombinant vector. The present invention also includes a method for producing beta-glycosidase, wherein the transformant is cultured to separate beta-glycosidase from the culture obtained.

Hereinafter, the present invention will be described.

The beta-glycosidase gene of the present invention is isolated from Pyrococcus puriosus. First, chromosomal DNA is obtained from Pyrococcus puriosus with beta-glycosidase gene. Next, the designed oligonucleotide is used as a primer, and the polymerase chain reaction (PCR) is performed using chromosomal DNA of Pyrococcus puriosus as a template to partially amplify the beta-glycosidase gene. The PCR amplified fragment thus obtained was a fragment having 100% homology with the beta-glycosidase gene of Pyrococcus puriosus, and a high S / N ratio was expected as a probe for colony hybridization. At the same time, it facilitates stringency control of hybridization. The PCR amplification fragments are labeled with appropriate reagents, and colony hybridization is performed on the chromosomal DNA library to select beta-glycosidase enzyme genes (Current Protocols in Molecular Biology, Vol. 1, p. 603, 1994). year).

DNA fragments containing the beta-glycosidase gene can be obtained by recovering the plasmid from the E. coli selected by the above method using the alkaline method (Current Protocols in Molecular Biology, Vol. 1, p. 161, 1994). . After determining the nucleotide sequence by the above method, it is possible to obtain the entire gene of the present invention by hybridizing the DNA fragment prepared by digestion by restriction enzymes of the DNA fragment having the nucleotide sequence as a probe.

SEQ ID NO: 2 shows the nucleotide sequence of the beta-glycosidase gene of the present invention, SEQ ID NO: 1 shows the amino acid sequence encoded by the gene.

The transformed microorganism of the present invention is obtained by introducing the recombinant vector of the present invention into a host suitable for the expression vector used when producing the recombinant vector. For example, when a bacterium such as Escherichia coli is used as a host, the recombinant vector according to the present invention is capable of autonomous replication in the host and at the same time, contains a promoter, a DNA containing a beta-glycosidase gene, a transcription sequence, and the like. It is desirable to have a configuration necessary for the expression of. Although pET24a was used as the expression vector used in the present invention, any expression vector satisfying the above requirements can be used.

Production of beta-glycosidase according to the present invention is carried out by culturing a transformant obtained by transforming a host by a recombinant vector having a gene encoding the same, and beta- which is a gene product in a culture (culture cell or culture supernatant). It is performed by generating and accumulating glycosidase and obtaining an enzyme from the culture.

Acquisition and purification of beta-glycosidase can be carried out by centrifuging the cells or supernatants from the obtained culture, and by cell alone, or by combining cell disruption, affinity chromatography, cation or anion exchange chromatography, or the like. .

The present invention clones a gene encoding beta-glycosidase from a gene of Pyrococcus puriosus to produce industrially useful beta-glycosidase, and analyzes the nucleotide sequence of the electric gene and the amino acid sequence inferred therefrom. do.

Beta-glycosidase of the present invention refers to an enzyme having the ability to convert ginsenoside aldi or ginseng extract to the compound K and aglycone propananaxadiol.

In addition, the present invention provides a production method for obtaining a compound K and aglycone protopanaxadiol in large quantities by investigating the optimum concentration of beta-glycosidase and ginsenoside Aldi and ginseng root extract as substrates. .

Hereinafter, the present invention will be described in detail.

The present invention provides a method for preparing ginsenoside compound K and aglycone propanacanadiol by reacting ginsenoside aldi or ginseng root extract as a substrate with a beta-glycosidase in a reaction solvent.

The present invention also provides a beta-glycosidase enzyme for use in the production of the rare ginsenosides of the compound K and aglycone protopanaxadiol. Specifically, the present invention provides beta-glycosidase derived from Pyrococcus puriosus strain capable of producing compound k and aglycone propanacanadiol using ginsenoside aldi or ginseng root extract as a substrate. .

The beta-glycosidase is present in a variety of animals and plants, microorganisms, etc., as a enzyme that generates sugar and aglycone by hydrolyzing glycosidic bonds of glucose or small sugars, glucosides or cyanogenics attached to aryl, amino, and alkyl groups. It is responsible for hydrolyzing galactosides and oligosaccharides or disaccharides. It is an enzyme that plays a fundamental role in carbohydrate production and disruption in the regulation of metabolism (Park. Et al. (2007) J. Microbiol, Biotechnol 17 (3), 454-460).

In addition, the thermophilic beta-glycosidase of the present invention is a microorganism belonging to the genus Pyrococcus , which is a thermophilic microorganism, preferably Pyrococcus furiosus ) strain. The Pyrococcus puriosus is a native bacterium (Archaea); Euryarchaeota Moon; Thermococci steels; Thermo kokal less (Thermococcales) neck; Thermo Coca Kane (Thermococcaceae) with: a Pyro to strain belonging to the genus Rhodococcus (Pyrococcus), pie Lactococcus furiosus (Pyrococcus furiosus) strain is known to exhibit optimal growth at 100 ℃ and pH 7.

Beta-glycosidase in the production of compound K, aglycone protopanaxadiol using the pyrococcus puriosus derived beta-glycosidase, characterized in that the optimum activity temperature is 70 ~ 95 ℃ do.

In addition, obtaining the thermophilic beta-glycosidase of the present invention from a Pyrococcus puriosus strain can be achieved by 1) isolation and purification directly from the strain, or 2) by cloning the beta-glycosidase gene from the strain. It can be obtained by expression and purification. This process for obtaining enzymes from microorganisms is by conventional methods in the art (Sambrook, J. and Russell, D. W. Molecular Cloning 3rd Ed. Cold Spring Harbor Laboratory, 2001).

Thus, the composition for preparing the ginsenoside compound K and the aglycone protopanaxadiol containing the high temperature beta-glycosidase of the present invention is a mixture of ginsenoside aldi or ginseng root extract and a buffer, an aqueous solvent or an aqueous solvent and an organic solvent. When reacting in a mixed solution, the reaction is carried out at a high temperature of 70 ~ 95 ℃ to increase the solubility of the substrate exhibits the effect of producing a high yield of ginsenoside compound K and aglycone Protoparanaxadiol in a short time.

In a preferred embodiment of the present invention, a) DNA fragments containing the pyrogenic beta-glucosidase gene are amplified by PCR using genomic DNA of Pyrococcus puriosus as a template and primers with forward and reverse directions. Next, the amplified DNA fragment was cloned into the expression vector pET24a after treatment with restriction enzymes BamH I and Sal I to prepare a recombinant expression vector pET24a / beta-glycosidase. C) transform it to E. coli ER2566 by transformation; d) incubate the transformed E. coli and induce overexpression to produce high-temperature beta-glycosidase and e) express the expressed beta-glycosidase enzyme. Isolate and purify.

The beta-glycosidase enzyme cultures E. coli transformed with the recombinant expression vector comprising the beta-glycosidase enzyme gene, induces the expression of beta-glycosidase enzyme, isolates and expresses the expressed enzyme protein. It is preferable to use beta-glycosidase as the purification process.

And e) separating the beta-glycosidase enzyme expressed in the process a) disrupting the microorganism and b) centrifuging the cell debris to obtain a supernatant; (c) heat treatment at 90 ° C. for 10 minutes to further centrifuge; (d) filtering the supernatant obtained therefrom; This can be done by separating the enzyme liquid into the process.

It is preferable that the reaction temperature of the said enzyme is made in the range of 70 degreeC thru | or 95 degreeC, and it is more preferable to react in about 95 degreeC temperature range. In addition, the reaction solvent of the enzyme may be a sodium citrate buffer, a buffer solution such as potassium ginseng buffer, an aqueous solvent or a mixture of an aqueous solvent and an organic solvent, and the reaction between the high temperature beta-glycosidase enzyme and the substrate is pH 4.5 to It is preferable to make it in the range of pH 6.5, and it is more preferable to react in the range of pH 5.5.

The present invention also provides a production method for obtaining a large amount of compound K and aglycone protoparanadiol by investigating the optimum concentration of beta-glycosidase and Rd and ginseng extract as substrates.

In the case where ginsenoside aldi purely separated as a substrate in the reaction, the beta-glycosidase enzyme unit is preferably 8 to 50 units / ml. Specifically, it is more preferable to use 8 units / ml for compound k production, and more preferably 48 units / ml for aglycone propananaxadiol production.

In the case of using ginseng extract as a substrate in the reaction, the beta-glycosidase enzyme unit is preferably 8 to 100 units / ml. Specifically, it is more preferable to use 16 units / ml for compound k production, and more preferably 96 units / ml for aglycone propananaxadiol production.

Ginsenosides when 8 units / ml or 48 units / ml of beta-glycosidase are used in the production of compound K and aglycone propanacanadiol in the case of using ginsenoside aldi purely separated as a substrate in the reaction. It is preferred that the aldi is in the range of 1 mM to 8 mM, more preferably in 4 mM ginsenoside aldi.

In case of using ginseng root extract as a substrate in the reaction, ginseng root extract 5% to 20 when 16 units / ml or 96 units / ml of beta-glycosidase is used in the production of compound K and aglycone protopanaxadiol. It is preferably made in the range of%, more preferably made of 10% ginseng root extract. The ginseng root extract can be used as a ginseng saponin mixture or extract obtained by extracting and separating from the root or ground portion of Korean ginseng, Samchil ginseng, American ginseng, bamboo shoot ginseng, Himalayan ginseng or Vietnamese ginseng.

According to the method for preparing rare ginsenosides using the high-temperature beta-glycosidase enzyme of the present invention, when a pure ginsenoside ginsenoside aldi or ginseng root extract is used using one enzyme that is stable at high temperature, each rare ginsenoside is used. With regard to the production of sidein ginsenoside compound k and aglycone protoparnaxadiol, the compound k and aglycone protoparnasediol can be selectively produced by optimizing the substrate and enzyme concentration, and thus industrially useful.

Method for preparing compound K, and aglycone protoanaxadiol by reacting the high temperature beta-glycosidase enzyme of the present invention with a ginsenoside aldi or ginseng root extract as a substrate It is about.

Pyrococcus furiosus ) Thermophilic beta-glycosidase derived from the strain exhibits a stable activity even at high temperatures, and increases the reaction rate by increasing the solubility of the substrate by reaction at high temperatures. In addition, due to the low substrate specificity for the sugars other than glucose, only the main ginsenoside (Rb1, Rb2, Rc, Rd) glucose present in the ginseng root extract specifically hydrolyzed to produce a compound k, and the compound k One molecule of glucose is hydrolyzed to produce aglycone propanacandiol.

The present invention can be efficiently produced using enzymes containing compound K and aglycone protoparanadiol, which have various excellent effects such as immune enhancement, tumor angiogenesis inhibition, cancer cell invasion inhibition and cancer cell proliferation inhibition. The compound k production method shows the highest productivity among previously reported compound k productivity, and the production method of aglycone protoparanaxadiol is environmentally friendly and the first enzymatic method compared to the conventional chemical production method. .

In addition, by optimizing the substrate and enzyme concentration, compound k and aglycone protoparnaxadiol can be selectively produced in high yield, which can be used industrially.

1 shows two substances converted as a result of the reaction with ginsenoside aldi. Ginsenoside Aldi is consumed, resulting in Compound K and Uglycon Protoparnaxadiol.
Figure 2 shows by examining the enzyme activity according to the temperature and pH of the beta- glycosidase. (a) is temperature, (b) is irradiation of pH.
Figure 3 shows the temperature stability at each temperature of beta-glycosidase.
Figure 4 shows the optimum unit / ml of beta-glycosidase in the production of compound K and aglycone protoparnaxadiol, (a) is 4 mM Rd, (b) using 10% ginseng extract It was investigated.
FIG. 5 shows the optimum ginsenoside aldi and ginseng substrate concentrations at the optimum unit / ml of FIG. 3 in the production of compound K and aglycone protoparanaxadiol. (a) and (b) are the optimal concentrations of Rd using beta-glycosidase 8 or 48 units / ml, and (c) and (d) ginseng using beta-glycosidase 16 or 96 units / ml The optimum concentration of the extract was investigated.
FIG. 6 shows production over time with the optimal enzyme units / ml and substrate concentrations of FIGS. 4 and 5 in compound k and aglycone protoparanaxadiol production. (a) and (b) show production of compound k and aglycone propanoparanadiol using 4 mM ginsenoside aldi and beta-glycosidase 8 or 48 units / ml, and (c) and (d ) Shows the production of compound k and aglycone propananaxadiol using 10% ginseng extract and 16 or 96 units / ml of beta-glycosidase.
Figure 7 shows the nucleotide sequence of pyrococcus puriosus derived beta-glycosidase.
8 shows the amino acid sequence of Pyrococcus puriosus derived beta-glycosidase.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example 1 Preparation of Recombinant Expression Vectors and Transgenic Microorganisms Comprising Thermophilic Beta-Glycosidase

In the present invention, the beta-glycosidase gene derived from the pyrococcus puriosus strain was first isolated in order to prepare a high temperature beta-glycosidase. Specifically, pyrococcus furiosus, which has a gene sequence and an amino acid sequence already specified Genomic DNA of the DSM 3638 strain was purchased (Microbank, Daejeon, Korea) (FIG. 7).

Published known DNA sequences of beta-glycosidases derived therefrom (see Sequence Listing; A forward primer (5'-GGATCCATGAAGTTCTCCAAAAAAC-3 '; SEQ ID NO: 3) and a reverse primer (5'-GTCGACCTTTCTTGTAACAAATT-3'; SEQ ID NO: 4) were prepared based on Genebank Accession No. AAC25555 (Genebank Accession No. AAC25555) It was. Next, a polymerase chain reaction (PCR) was performed using the genomic DNA and primers to amplify the gene. The expression vector pET24a / beta- used in this study was inserted into the plasmid vector pET24a (Novagen, Darmstadt, Germany) using the beta-glycosidase gene fragment amplified by the above procedure using restriction enzymes BamH I and Sal I. Glycosidase was produced. In addition, the recombinant expression vector thus obtained was transformed into E. coli ER2566 strain (New England Biolabs, Hertfordshire, UK) by a conventional transformation method.

The transformed E. coli was stored frozen before the incubation with the addition of 20% glycerin (glycerine) solution. The recombinant E. coli was named E. coli ER2566 pET24a / beta-glycosidase strain.

Example  2. Thermophilic Beta- Glycosidase  Expression and Purification

In the present invention, a tube containing 3 ml of LB medium containing 20 mg / l kanamycin of E. coli ER2566 pET24a / beta-glycosidase strain, which is stored frozen for mass production of beta-glytosidase (Test tube) was inoculated and the seed culture was carried out by shaking incubator at 37 ℃ until absorbance at 2.0 at 600 nm. The cultured spawn was then added to a 2 l Erlenmeyer flask containing 500 ml of LB medium containing 20 mg / l kanamycin and incubated until the absorbance at 600 nm was 0.8 and the stirring speed was 200 rpm. , The culture temperature was adjusted to 37 ℃. In addition, after the absorbance at 600 nm became 0.8, 0.1 mM ITPG (isopropyl-beta-thiogalactoside) was added thereto to induce mass expression of beta-glycosidase. After addition of IPTG, the stirring rate was adjusted to 150 rpm and the incubation temperature was adjusted to 16 ° C. for 12 hours.

In addition, the beta-glycosidase produced by overexpression as described above was centrifuged at 6,000 x g for 30 minutes at 6,000 × g, washed twice with 0.85% sodium chloride (NaCl) and then 50 mM Sodium citrate buffer (50 mM, Sodium citrate buffer, pH 5.5) was added to disrupt the cell solution with a sonicator. The cell lysate was again centrifuged at 13,000 × g for 20 minutes at 4 ° C. and heat treated at 90 ° C. for 10 minutes at high temperature, and then the heat treatment obtained therefrom was centrifuged again at 4 ° C. for 20 minutes at 13,000 × g. After that, only the supernatant was obtained and separated as an enzyme solution used for the production of compound K and aglycone propanacanadiol.

Example  3. Thermophilic Beta- Glycosidase  Effect on enzyme activity pH  And temperature effects investigation

In the present invention, in order to investigate the activity of the beta-glycosidase enzyme according to the pH and temperature changes, the enzyme and the substrate Rd were reacted under various pH and temperature conditions and the enzyme activity was compared.

end. Thermophilic Beta- Glycosidase  Enzyme Activity Measurement

Purely isolated ginsenoside aldi was used to measure beta-glycosidase activity provided in the composition for the production of the rare ginsenosides presented herein. Specifically, the reaction was diluted with a final concentration of 1 mM ginsenoside aldi, 50 mM pH 5.5 sodium citrate buffer and enzyme to a total volume of 1 ml, and then the reaction was performed at 95 ° C. for 20 minutes. The reaction was also stopped by addition of 1 ml n -butanol. At this time, the true amount of the compound K generated due to ginsenosides Aldi and enzyme reaction used was calculated by performing a VWD detector and a YMC-PackTM Pro C ₁₈ TM a column equipped with a high pressure liquid chromatography of Photography, YMC-PackTM Pro C ₁₈ TM The column was allowed to pass acetonitrile from 0% to 80% at 37 ° C. at 1 ml / min rate. At this time, the unit of the enzyme (Unit) was defined as the amount of enzyme to produce 1 micromole (μmole) compound k. In addition, the amount of the enzyme protein was calculated using the Bradford method based on bovine serum albumin.

I. Thermophilic Beta- Glycosidase  Effect on enzyme activity pH  And temperature effects investigation

First, to investigate the effect of temperature on the activity of beta-glycosidase enzyme, the enzymatic reaction was carried out using a 50 mM pH 5.5 sodium citrate buffer solution containing 0.01 unit / ml enzyme and 1 mM ginsenoside aldi. The reaction was carried out using 70 ~ 95 ℃. The reaction was then stopped by the addition of 1 ml n- butanol and the activity of the enzyme was measured by high pressure liquid chromatography. As a result, it was confirmed that the optimum temperature in the temperature range we tested was 95 ℃ (Fig. 2a).

In addition, to investigate the effect of beta-glycosidase on pH on the enzyme activity, the enzyme reaction was performed using 50 mM pH 5.5 sodium citrate buffer containing 0.01 unit / ml enzyme and 1 mM ginsenoside aldi. pH was carried out from 4.5 to 6.0, and was performed from pH 6.0 to 6.5 using a 50 mM pH 5.5 potassium ginseng buffer solution containing 0.01 unit / ml enzyme and 1 mM ginsenoside aldi. The reaction was then stopped by the addition of 1 ml n- butanol and the activity of the enzyme was measured by high pressure liquid chromatography. As a result, it was confirmed that the optimum pH was pH 5.5 (FIG. 2B). In the activity of the beta-glycosidase enzyme according to the pH in Figure 2b is ● sodium citrate buffer solution, ○ represents a phosphate buffer solution.

3 shows the temperature stability of the thermophilic beta-glycosidase enzyme enzyme activity. The enzyme activity half-life is shown as 69 hours at 85 ° C, 118 hours at 90 ° C, and 69 hours at 95 ° C (FIG. 3). . In the activity of the beta-glycosidase enzyme according to the temperature stability in Figure 3 ● is 75 ℃, ○ is 80 ℃, ▼ is 85 ℃, △ is 90 ℃, ■ is 95 ℃.

Example  4. Thermophilic Beta- Glycosidase  Used Compound Kwa Uglycon  Investigation of Optimal Enzyme Unit / mL and Optimal Substrate Concentration in the Production of Protoparanaxadiol

When the beta-glycosidase obtained through the above process was used as ginsenoside aldi or ginseng root extract, the optimum enzyme unit and substrate concentration were investigated in the production of compound K and aglycone propanacanadiol.

end. beta- Glycosidase  Used Compound Kwa Uglycon  In the production of protoparnacindiol Gin Senocide  Beta- Using Aldi or Ginseng Root Extract as Substrate Glycosidase  Optimum unit / ml count

Reaction with ginseng extract was carried out to investigate the effect observed in units / ml of the enzyme when reacted with ginsenoside aldi or ginseng root extract. To prepare ginseng extract, add 100 g of dried ginseng powder to 1 l of MeOH / water mixture (4: 1 v / v), extract it for 1 hour at 80 ℃, filter it, concentrate it, and dissolve it in 1 l of distilled water. (Kim SK, Kwak YS, Kim SW, Hwang YS, Ko ys, Yoo CM: Improved method for the preparation of crude ginseng saponin. J. Ginseng Res 1988; 22; 155-160)

In the enzyme reaction, 4 mM ginsenoside aldi or ginseng root extract was used as a substrate. When ginsenoside Aldi was used as a substrate, it reacted with beta-glycosidase for 5 hours. As a result, the optimal unit / ml of beta-glycosidase in compound K production was 8 units / ml, and aglycone propanacanadiol was used. The optimum unit of beta-glycosidase / ml in production is 48 units / ml (FIG. 4A). In the beta-glycosidase optimal enzyme unit / ml irradiation in Figure 4a, ● is ginsenoside aldi, 는 is compound K, ▼ is aglycone propanacanadiol.

When ginseng root extract was used, it was reacted with beta-glycosidase for 6 hours. As a result, the optimum unit / ml of beta-glycosidase in compound K production was 16 units / ml, and beta in aglycone propananaxadiol production. -Glycosidase optimal unit / ml is 96 units / ml (FIG. 4B). In the beta-glycosidase optimal enzyme unit / ㎖ irradiation in Figure 4b is ● ginsenoside alb 1 (Rb1), ○ is ginsenoside AL (Rc), ▼ is ginsenoside alb 2 (Rb2), △ is Ginsenoside Aldi, ■ is compound K, and □ is aglycone Protoparnaxadiol.

I. beta- Glycosidase  Used Compound Kwa Uglycon  In the production of protoparnacindiol Gin Senocide  Optimal Substrate Concentration when Using Aldi or Ginseng Root Extract as Substrate

When Rd was used to investigate the optimal concentration of Rd and ginseng extract as a substrate in the use of the optimal unit / ml of beta-glycosidase, 8 units / ml of beta-glycosidase was used in the production of the compound K ( Figure 5a), productivity and yield using 48 units / ml of beta-glycosidase in the production of aglycone propanacanadiol, the optimum substrate concentration is 4 mM (Figure 5b).

In order to investigate the optimal concentration of ginseng extract, 16 units / ml of beta-glycosidase was used in the production of compound K (FIG. 5c), and 96 units / ml of beta-glycosidase in production of aglycone propanacanadiol. As a result of using the productivity and yields, the optimum substrate concentration is 10% ginseng root extract (FIG. 5D).

In the investigation of the optimum concentration of ginsenoside aldi or ginseng root extract in Figure 5 ● indicates productivity, ○ indicates the conversion rate.

Example  4. Optimal Enzyme Units / mL Beta- With glycosidase  optimal Substrate concentration Rd  Or over time of the ginseng extract composition Compound K , Uglycon  Production of Protoparnacindiol

Optimum pH (pH 5.5) of pyrogenic beta-glycosidase to develop a ginsenoside compound K, aglycone propanacanadiol production method over time using the pyrogenic beta-glycosidase in the present invention and The reaction was carried out at a temperature (95 ° C.) considering the time at which the enzyme activity was reduced by half.

When the reaction was performed at 8 units / ml, which is the optimum enzyme unit / ml of compound k production in the case of using the 4 mM ginsenoside aldi obtained by the above experiment, the yield of the compound k was 2.06 mg / ml after 5 hours. It shows the productivity of 413 mg / l / h, and after 32 hours all of the compound k is hydrolyzed to produce 1.79 mg / ml of agglycon propanpanaxadiol (56 mg / l / h) (Fig. 6a) .

Aglycone Propanacanadiol when using 4 mM ginsenoside aldi obtained in the above experiments was measured at 48 units / ml, which is the optimum enzyme unit / ml. A yield of 1.78 mg / ml yields a productivity of 351 mg / l / h (FIG. 6B). In the compound k and aglycone protoparnaxadiol production in FIGS. 6a and 6b, x represents ginsenoside aldi, ○ represents a compound k, and a represents aglycone protoparanaxadiol.

Compound K production in case of using 10% ginseng root extract obtained in the above experiment When the reaction was performed at 16 units / ml, which is the optimum enzyme unit / ml, the yield of hourly production of compound k produced 1.81 mg / ml after 6 hours. It shows the productivity of mg / l / h, and after 32 hours all of the compound k is hydrolyzed to produce 1.69 mg / ml of agglycone propananaxadiol (52.8 mg / l / h) (Fig. 6c).

Aglycone Protoparnaxadiol production using the 10% ginseng root extract obtained in the above experiments measured the hourly production when reacted at 96 units / ml, which is the optimum enzyme unit / ml. It yields 1.75 mg / ml producing 292 mg / l / h (FIG. 6D). In the production of compound k and aglycone protoparnaxadiol in FIGS. 6C and 6D, 는 is ginsenoside albi 1, 는 is ginsenoside AL, ▼ is ginsenoside albi 2, △ is ginsenoside Aldi, and Is the compound K, □ represents the aglycone Protoparanadiol.

To date, the enzymes capable of producing ginsenoside compound k include commercial enzymes Novozyme Co. Pectinex 100 and Sulfolobus solfataricus beta-glycosidase. . When reacting at a concentration of 10% of ginseng extract, Pectinex produced 1.3 mg / ml of compound K at 24 hours and showed 54 mg / l / h of productivity (Bo-Hye Kim et al. 2006, Biotransformation of Korean Panax ginseng by Pectinex.Biol.Pharm.Bull. 29, 2472-2487), Sulfolobus Ashdo caldarius acidocaldarius ) beta-glycosidase produces 1.2 mg / ml of compound k at 24 hours, yielding 50 mg / l / h of productivity (Kyeong-Hwan Noh et al. 2009 Production of the rare ginsenosides compound K, compound Y , and compound Mc by a thermostable beta-glycosidase from Sulfolobus acidocaldarius . Biol Pharm Bull 32 1830-1835), Sulfolobus beta-glycosidase of solfataricus has been reported to have a productivity of about 136 mg / l / h, about 2.5 times higher (Kyeong-Hwan Noh et al. 2009 Ginsenoside compound K production from ginseng root extract by a thermostable betagalaactosidase from Sulfolobus solfataricus.Biosci.Biotechnol.Biochem 73 316-321).

When the enzyme is reacted under optimal reaction conditions using ginsenoside aldi or ginseng root extract, it finally produces compound K and aglycone propananaxadiol by 1.7 mg / ml, respectively, and 300 to 420 mg / l /. The productivity of h, the compound k productivity is more than twice the productivity reported previously, the agglycone propanpanasediol is produced in a high yield using environmentally friendly and substrate specificity rather than production by conventional chemical methods It is the first to be produced by the enzymatic method that can be expected.

Furthermore, no results have been reported to date for the production of ginsenoside compound k and aglycone propanacanadiol produced using the same enzymes. This has the advantage of converting ginseng extract and purely isolated ginsenoside aldi into ginsenoside compound K and aglycone protoparanaxadiol with one enzyme.

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Claims (10)

delete delete delete delete delete delete delete delete A method for producing aglycone propananaxadiol by reacting beta-glycosidase having the amino acid sequence of SEQ ID NO: 1 with ginsenoside aldi or ginseng extract in a reaction solvent. 10. The method of claim 9, wherein the ginseng extract is obtained by extracting and extracting from the root or ground portion of Korean ginseng, Samchil ginseng, American ginseng, bamboo shoot ginseng, Himalayan ginseng, or Vietnamese ginseng. .

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