# US 10,870,857 B2 · Method of producing ginsenosides 20(S)-Rg3 and 20(S)-Rh2 using ginsenoside glycosidases

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- Number: US 10,870,857 B2
- Document ID: US10870857B2
- Status: Granted
- Kind: B2
- Application number: 16/213,729
- Filed: 2018-12-07
- Published: 2019-06-27
- Granted: 2020-12-22
- Earliest claimed: 2017-10-20
- Assignees: Modnbio, Inc.
- Applicants: Modnbio, Inc.; Eun Kyung Do
- Inventors: Jong Cheon Hahm; Eun Kyung Do
- Web page: https://patentgrep.com/patent/US10870857B2

## CPC

- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12N 9/2408: {acting on alpha -1,4-glucosidic bonds}
- C12N 9/2445: {Beta-glucosidase (3.2.1.21)}
- C12N 15/74: Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12P 33/00: Preparation of steroids
- C12P 33/20: containing heterocyclic rings
- C12Y 302/01: Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y 302/01021: Beta-glucosidase (3.2.1.21)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Y: ENZYMES

## Abstract

The present invention relates to a method of producing ginsenoside 20(S)—Rg3 or 20(S)—Rh2 using a novel ginsenoside glycosidase in order to obtain ginsenoside 20(S)—Rg3 or 20(S)—Rh2 with high efficiency and high purity. According to the production method of the present invention, a large amount of 20(S)—Rg3 or 20(S)—Rh2, which is a minor form of rare ginsenoside present in very small amounts in ginseng or processed ginseng products, may be safely and efficiently produced. In particular, the method according to the present invention has an advantage in that it may produce a large amount of 20(S)—Rg3 or 20(S)—Rh2 for industrial applications, since the process is very simple and the production efficiency is very high.

## Claims

### Claim 1

A method of producing ginsenoside 20(S)—Rg3 comprising: reacting ginseng crude saponin with
a ginsenoside glycosidase having an amino acid sequence of at least 95% sequence similarity to the sequence of SEQ ID NO: 3.

### Claim 2 (depends on 1)

The method of claim 1, wherein the amino acid sequence has at least 98% similarity to the sequence of SEQ ID NO: 3.

### Claim 3 (depends on 1)

The method of claim 1, wherein the amino acid sequence is identical to the sequence of SEQ ID NO: 3.

### Claim 4 (depends on 1)

The method of claim 1, wherein the ginsenoside glycosidase is expressed by a transformant introduced with a vector comprising a nucleic acid encoding the ginsenoside glycosidase having an amino acid sequence of at least 95% sequence similarity to the sequence of SEQ ID NO: 3.

### Claim 5 (depends on 4)

The method of claim 4, wherein the ginseng crude saponin is reacted with a culture of the transformant containing the ginsenoside glycosidase.

### Claim 6 (depends on 1)

The method of claim 1, wherein the reacting step is at a pH of 6.0 to 8.0 and a temperature of 28 to 40° C. for an incubation time of 12 to 72 hours.

### Claim 7

A method of producing ginsenoside 20(S)—Rh2 comprising:
(a) reacting ginseng crude saponin with
a ginsenoside glycosidase having an amino acid sequence of at least 95% sequence similarity to the sequence of SEQ ID NO: 3 to prepare a reaction solution comprising ginsenoside 20(S)—Rg3; and
(b) reacting the reaction solution prepared in the step (a) with
a ginsenoside glycosidase having an amino acid sequence of at least 95% sequence similarity to the sequence of SEQ ID NO: 4 to produce ginsenoside 20(S)—Rh2.

### Claim 8 (depends on 7)

The method of claim 7, wherein the ginsenoside glycosidase in the step (a) comprises an amino acid sequence that has at least 98% similarity to the sequence of SEQ ID NO: 3, and the ginsenoside glycosidase in the step (b) comprises an amino acid sequence that has at least 98% similarity to the sequence of SEQ ID NO: 4.

### Claim 9 (depends on 7)

The method of claim 7, wherein the ginsenoside glycosidase in the step (a) comprises an amino acid sequence of SEQ ID NO: 3, and the ginsenoside glycosidase in the step (b) comprises an amino acid sequence of SEQ ID NO: 4.

### Claim 10 (depends on 7)

The method of claim 7, wherein the ginsenoside glycosidase is expressed by a transformant introduced with a vector comprising a nucleic acid encoding the ginsenoside glycosidase having an amino acid sequence of at least 95% sequence similarity to the sequence of SEQ ID NO: 3, and the reaction solution prepared in the step (a) is reacted with ginsenoside glycosidase expressed by a transformant introduced with a vector comprising a nucleic acid encoding the ginsenoside glycosidase having an amino acid sequence of at least 95% sequence similarity to the sequence of SEQ ID NO: 4.

### Claim 11 (depends on 10)

The method of claim 10, wherein the ginseng crude saponin is reacted with a culture of the transformant containing the ginsenoside glycosidase having an amino acid sequence of at least 95% sequence similarity to the sequence of SEQ ID NO: 3, and wherein the reaction solution prepared in the step (a) is reacted with a culture of the transformant containing the ginsenoside glycosidase having an amino acid sequence of at least 95% sequence similarity to the sequence of SEQ ID NO: 4.

### Claim 12 (depends on 7)

The method of claim 7, wherein step (a) comprises reacting the crude ginseng crude saponin with the ginsenoside glycosidase having an amino acid sequence of at least 95% sequence similarity to the sequence of SEQ ID NO: 3 at a pH of 6.0 to 8.0 and a temperature of 28 to 40° C. for an incubation time of 12 to 72 hours, and wherein the treatment step (b) comprises reacting the crude ginseng crude saponin with the ginsenoside glycosidase having an amino acid sequence of at least 95% sequence similarity to the sequence of SEQ ID NO: 4 at a pH of 6.0 to 8.0 and a temperature of 28 to 40° C. for an incubation time of 12 to 72 hours.

### Claim 13 (depends on 12)

The method of claim 12, further comprising, before the step (b), a step of performing deactivation of the ginsenoside glycosidase having an amino acid sequence of at least 95% sequence similarity to the sequence of SEQ ID NO: 3 by heating at 70° C. to 90° C. for at least 4 hours.