A method for preparing ginsenoside Rh2 by biological enzyme conversion

Abstract

This patent application discloses a method for preparing ginsenoside Rh2 by bioenzymatic conversion. The method includes using a glycosidase composition derived from Aminobacter sp. Gsoil204 bacteria (GenBank: AGA60126.1) and a β-glycosidase derived from Sphingopyxis alaskensis RB2256 bacteria (GenBank: ABF52736.1) to catalyze the reaction of ginsenoside Rb1, and convert it into ginsenoside Rh2 in a reaction system. After the reaction, the content of ginsenoside Rh2 reaches 36% and the conversion rate reaches more than 60%. The generated enzyme-catalyzed product has a relatively simple composition, is simple to operate, is easy to scale up production, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a method for preparing ginsenoside Rh2 by using ginsenoside Rb1 as a raw material and adopting a biological enzyme conversion method. Background Art

[0002] Ginsenosides are the primary components of ginseng and other herbs that exert various pharmacological effects. Protopanaxadiol saponins are composed of dammarane-type triterpenoid saponins, primarily including common ginsenosides (such as Rb1, Rb2, Rb3, and Rd) and rare ginsenosides (Rg3, Rh2, and CK). They exhibit specific pharmacological activities, including anti-tumor, antioxidant, anti-immune, rapid apoptosis-inducing, and anti-aging properties. Rare saponins share the same sapogenin structure as their high-content common ginsenoside counterparts, differing only in the number of sugar groups. Therefore, rare saponins can be prepared by selectively hydrolyzing the sugar groups of high-content saponins.

[0003] Ginsenoside Rh2, a protopanaxadiol-type ginsenoside, is the actual entity of ginsenoside Rb1 that is absorbed and exerts its effects in the body. Existing studies have shown that Rh2 can inhibit the proliferation and growth of various tumor cells by inducing differentiation or apoptosis, and exhibits low toxicity, making it an important tumor suppressor.

[0004] In recent years, the research on the preparation of ginsenoside Rh2 has gradually transitioned from acid hydrolysis, alkaline hydrolysis, and synthesis to biotransformation. Among them, the glycosidase biotransformation method does not use a large amount of acid and alkali in the production process, avoiding the disadvantages of acid and alkali treatment after conversion. The reaction conditions are mild, the equipment requirements are not high, and it is easy to scale up. Therefore, the enzymatic preparation of ginsenoside Rh2 is an ideal preparation method.

[0005] However, technicians have not yet developed a method to directly convert ginsenoside Rb1 into ginsenoside Rh2 in one reaction system. Summary of the Invention

[0006] In view of the above technical difficulties in the prior art, the object of the present invention is to provide a glycosidase composition and a method for directly converting ginsenoside Rb1 into ginsenoside Rh2 in a reaction system using the glycosidase composition.

[0007] To solve the above technical problems, the inventors conducted in-depth research and found that by combining the glycosidase from Aminobacter sp. Gsoil 204 (GenBank: AGA60126.1) and the β-glycosidase from Sphingopyxis alaskensis RB2256 (GenBank: ABF52736.1), ginsenoside Rb1 can be used as a raw material to carry out an enzyme-catalyzed reaction in a reaction system to produce ginsenoside Rh2, with a high conversion rate (more than 60%), thus completing the present invention.

[0008] That is, the present invention includes:

[0009] 1. A glycosidase composition comprising glycosidase 1 and glycosidase 2, wherein the glycosidase 1 is a glycosidase derived from Aminobacter sp. Gsoil204 (GenBank: AGA60126.1), and the glycosidase 2 is a β-glycosidase derived from Sphingopyxis alaskensis RB2256 (GenBank: ABF52736.1).

[0010] 2. The glycosidase composition according to item 1, wherein the weight ratio of the glycosidase 1 to the glycosidase 2 is 1:10 to 10:1.

[0011] 3. Use of the glycosidase composition according to item 1 or 2 as a catalyst for preparing ginsenoside Rh2 by enzymatic reaction in a reaction system using ginsenoside Rb1 as raw material.

[0012] 4. The use according to item 3, wherein the enzymatic reaction is carried out in one reaction system.

[0013] 5. A method for preparing ginsenoside Rh2, wherein ginsenoside Rb1 is used as a raw material and the glycosidase composition according to item 1 is used as a catalyst to prepare ginsenoside Rh2 through an enzymatic reaction.

[0014] 6. The method according to item 5, wherein the enzymatic reaction is carried out in one reaction system.

[0015] 7. The method according to item 6, wherein the reaction system comprises acetate buffer, phosphate buffer or citrate buffer.

[0016] 8. The method according to item 6, wherein the pH of the reaction system is 4 to 5.5, preferably 5.

[0017] 9. The method according to item 5, wherein the temperature of the enzymatic reaction is 35 to 50°C, preferably 40°C.

[0018] The beneficial effects of the present invention are: 1) the present invention realizes the direct conversion of ginsenoside Rb1 into rare ginsenoside Rh2 in the same system by combining two enzymes from different sources; 2) the combined dosage of the two enzymes can make the ginsenoside Rh2 content reach more than 36% and the conversion rate reach more than 60%; 3) the preparation method provided by the present invention is simple to operate, the generated enzyme-catalyzed product has a relatively simple composition, is easy to scale up production, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the HPLC detection spectrum of Example Sample 1 after reaction.

[0020] Figure 2 This is the HPLC detection spectrum of Example Sample 2 after reaction.

[0021] Figure 3 This is the HPLC detection spectrum of ginsenoside Rh2 standard.

[0022] Figure 4 This is the HPLC detection spectrum of Example Sample 3 after reaction.

[0023] Figure 5 This is the HPLC detection spectrum of Example Sample 4 after reaction. DETAILED DESCRIPTION

[0024] The present invention will be described in detail below through specific examples. It should be noted that these descriptions are merely exemplary and do not constitute a limitation on the scope of the present invention.

[0025] The glycosidase from Aminobacter sp. Gsoil204 (GenBank: AGA60126.1, whose amino acid sequence is shown in SEQ ID No: 1) and the β-glycosidase from Sphingopyxis alaskensis RB2256 (GenBank: ABF52736.1, whose amino acid sequence is shown in SEQ ID No: 2) used in this example were both homemade in the laboratory.

[0026] The preparation method is as follows:

[0027] 1. Construction of expression strain

[0028] The amino acid sequences described in SEQ ID NO: 1 and SEQ ID NO: 2 were translated into gene sequences and codon-optimized for expression in E. coli. The optimized gene sequences were sent to Xi'an Qingke Gene Co., Ltd. for full gene synthesis and ligated into the pET28a plasmid using the NcoI and XbaI restriction sites. The constructed plasmids were then transformed into the E. coli BL21DE3 host.

[0029] 2. Enzyme Solution Preparation

[0030] The constructed strain was inoculated into LB medium and cultured until OD 600 To between 0.8-1, add 0.1mM IPTG, cool to 25℃ for induction, and collect the bacteria after 8 hours of induction. Prepare the collected bacteria into a 200g / L bacterial suspension and perform ultrasonic cell wall disruption at a power of 600W. Centrifuge and take the supernatant as the enzyme solution. Example 1: Catalytic effect of glycosidase of Aminobacter sp. Gsoil204 (GenBank: AGA60126.1) on ginsenoside Rb1

[0031] Weigh two appropriate amounts of ginsenoside Rb1 raw material. One portion was added directly to a sodium acetate buffer (sample 1). The other portion was mixed with a glycosidase enzyme solution from Aminobacter sp. Gsoil204 at a 1:1 weight ratio and added to the sodium acetate buffer (sample 2). Both samples were subjected to the enzymatic reaction at pH 5 and 40°C. After 6 hours, the reaction was terminated, centrifuged, and the precipitate dried. Liquid chromatography was performed to determine the formation of ginsenoside Rh2, and the generated Rh2 content was calculated using an external standard method.

[0032] Table 1 Catalytic effect of glycosidase from Aminobacter sp. Gsoil204 on ginsenoside Rb1

[0033]

[0034] The HPLC detection spectrum of sample 1 after reaction is as follows Figure 1 As shown, the HPLC detection spectrum of sample 2 after reaction is as follows Figure 2 As shown. As a reference, the HPLC detection spectrum of ginsenoside Rh2 standard is as follows Figure 3 As shown in the figure, the retention time of the ginsenoside Rh2 standard is 12.7 min. This shows that the glycosidase from Aminobacter sp. Gsoil204 alone is basically unable to convert the ginsenoside Rb1 raw material into ginsenoside Rh2.

[0035] Example 2 Catalytic reaction of β-glucosidase from Sphingopyxis alaskensis RB2256 (GenBank: ABF52736.1) on Rb1

[0036] Appropriate amounts of ginsenoside Rb1 and β-glucosidase enzyme solution from Sphingopyxis alaskensis RB2256 were weighed and added to sodium acetate buffer at a weight ratio of 1:1 (referred to as Sample 3). The enzymatic reaction was carried out at pH 5 and 40°C. After 6 hours, the reaction was terminated, centrifuged, and the precipitate dried. Liquid chromatography was performed to determine the formation of ginsenoside Rh2, and the Rh2 content was calculated using the external standard method.

[0037] Table 2 Catalytic effect of β-glucosidase on ginsenoside Rb1

[0038]

[0039] The HPLC detection spectrum of sample 3 after reaction is as follows Figure 4 As shown in the figure, it can be seen that the β-glucosidase derived from Sphingopyxis alaskensis RB2256 alone is basically unable to convert the raw material ginsenoside Rb1 into ginsenoside Rh2.

[0040] Example 3 Glycosidase composition catalyzes the production of Rh2

[0041] Appropriate amounts of glycosidase solution from Aminobacter sp. Gsoil204 and β-glycosidase solution from Sphingopyxis alaskensis RB2256 were combined at weight ratios of 1:1, 10:1, and 1:10, respectively. These were referred to as combination enzyme 1, combination enzyme 2, and combination enzyme 3, respectively.

[0042] Three appropriate amounts of ginsenoside Rb1 raw material were weighed and added to sodium acetate buffer along with the enzyme solution of the above-mentioned combined enzyme at a weight ratio of 1:1 (samples 4, 5, and 6, respectively). The enzymatic reaction for each of these three samples was performed at pH 5 and 40°C. The reaction was terminated after 6 hours, followed by centrifugation, drying of the precipitate, and liquid chromatography analysis to determine the formation of ginsenoside Rh2. The generated Rh2 content was then calculated using the external standard method.

[0043] Table 3 Catalytic effect of combined enzymes on ginsenoside Rb1

[0044]

[0045] The HPLC detection spectrum of sample 4 after reaction is as follows Figure 5It can be seen that the above-mentioned combined enzymes can convert ginsenoside Rb1 raw material into ginsenoside Rh2 in one reaction system, and the conversion rate reaches more than 60%, and the content of ginsenoside Rh2 reaches more than 36%.

[0046] Sequence information

[0047] SEQ ID NO: 1

[0048] 1mtqtifpddf lwgaataayq iegspladga gpsiwqrfsh dprlmaakgdtgdiacdhyn61rmpadvalmk elglqayrfs vnwgrilpeg tgrvnepgld fyerlvdellrhgieplltl121hhwdlpaald drggwlnrds agwfadyaai myrrldgrvk kwvtlnepwvitdggylhga

[0049] 181lapghrnmfe apiashnlmr ahgaavqayr segaheiglv vniepkypas dgiedvaaaa

[0050] 241rahaymnrqy ldpalkgsyp aelaeifgea wpqwpaedmr sicqpvdfig inyytrnvvq

[0051] 301adpnqwplra spvrqnatht ttdwevcppa ltdmliwfrd tygnipvyit engaafydpp

[0052] 361tagpagiddp lrcdylrthi saigdairqg vdvrgymaws lldnlewslg yskrfgivhv

[0053] 421dyetqvrtpk rsarfyssvi rsnggnlg SEQ ID NO: 2

[0054] 1mtqtsfpddf lwgaataayq iegspladga gasiwqrfsh dprlmankgdtgdiacdhyn61rmesdvalmk alglkayrfs vnwgrvlpeg vgrvneaglg fyerlvdellkhdieplltl121hhwdlpvald drggwlnrds adwfaeygsv lyrrldgrvk kwvtlnepwvitdggylhga

[0055] 181lapghrnlfe tpiashnlmr ahgaavrayr segaheiglv vniepkypas dspedvaata

[0056] 241rahaymnrqy lepallghyp aelaeifgea wpdwpecdlk dicqpldfvg inyytrnvvr

[0057] 301adpnqwplga spvrqtatht ttdwevyppa ltdmlvwfrd tfgnipvyit engaafydpp

[0058] 361qagpdgiddp lrcdylrthi aaigdairqg vdvrgymvws lldnlewslg fskrfgivhv

[0059] 421dyetqvrtpk rsarfysnii atnggnva

Claims

1. A glycosidase composition comprising glycosidase 1 and glycosidase 2, wherein the glycosidase 1 is derived from Aminobacter The amino acid sequence of the glycosidase from sp. Gsoil204 is shown in SEQ ID No: 1; the glycosidase 2 is derived from Sphingopyxis alaskensis β-glucosidase from bacteria RB2256, the amino acid sequence of which is shown in SEQ ID No: 2; in, The weight ratio of the glycosidase 1 to the glycosidase 2 is 1:10 to 10:

1.

2. Use of the glycosidase composition according to claim 1 as a catalyst for preparing ginsenoside Rh2 by enzymatic reaction in a reaction system using ginsenoside Rb1 as raw material.

3. A method for preparing ginsenoside Rh2, wherein: Ginsenoside Rh2 is prepared by enzymatic reaction using ginsenoside Rb1 as raw material and the glycosidase composition according to claim 1 as catalyst.

4. The method according to claim 3, wherein: The enzymatic reaction is carried out in a reaction system.

5. The method according to claim 4, wherein The reaction system includes acetate buffer, phosphate buffer or citrate buffer.

6. The method according to claim 4, wherein: The pH of the reaction system is 4-5.

5.

7. The method according to claim 4, wherein: The pH of the reaction system is 5.

8. The method according to claim 3, wherein: The temperature of the enzymatic reaction is 35-50°C.

9. The method according to claim 3, wherein: The temperature of the enzymatic reaction was 40°C.

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