Application of alpha-glucosidase in synthesis of alpha-salidroside

Abstract

The invention relates to application of alpha-glucosidase in synthesis of salidroside, and belongs to the technical field of biosynthesis. According to the technical scheme, the application of the alpha-glucosidase in the aspect of synthesizing the alpha-salidroside is characterized in that the alpha-glucosidase is derived from Ensifer adhaerens, the amino acid sequence of the alpha-glucosidase is shown in SEQ ID No: 1, and the Ensifer adhaerens is CCTCC AB 2019220. The invention provides an alpha-salidroside preparation technology which is simple and convenient in preparation process, high in green safety and low in production cost, and industrial large-scale production is facilitated.

Description

Technical Field

[0001] The invention relates to application of alpha-glucosidase in synthesizing salidroside, and belongs to the technical field of biosynthesis. Background Art

[0002] α-Glucosidase is an indispensable enzyme in the sugar metabolism pathway of organisms. It can directly participate in the metabolism of starch and glycogen and regulate the sugar metabolism of the human body.

[0003] Rhodiola rosea usually grows in pollution-free areas at high altitudes and high altitudes. It has been used in traditional medical treatment for thousands of years. It can also be eaten as a health product. It also has a strong cosmetic effect and can be used as an effective ingredient in skin care products. Salidroside (p-hydroxyphenylethyl-β-D-glucoside, CAS: 10338-51-9) is an active substance isolated from Rhodiola rosea. It has been confirmed to be an effective medicinal ingredient in the traditional Chinese medicine Rhodiola rosea. It has multiple physiological effects and functions such as anti-hypoxia, anti-fatigue, anti-radiation, anti-aging, cardiovascular and cerebrovascular protection, anti-inflammation, anti-tumor, immune regulation, whitening and freckle removal, etc., and it is highly water-soluble and easy to absorb. Therefore, the current market demand for it is increasing day by day, and it has great development prospects.

[0004] Due to the scarcity of wild Rhodiola rosea resources and the difficulty of artificial cultivation, it has been listed as a national rare plant and is prohibited from being mined. In order to make up for the shortage of wild resources, researchers have begun to try to artificially synthesize salidroside, generally through chemical or biological methods. The chemical synthesis of salidroside requires harsh reaction conditions and expensive catalysts, and the process is complicated and easy to generate by-products that are difficult to remove. Chinese patents 202211708333.0, 202211682663.7, etc. use genetic recombination technology to transform microorganisms such as yeast or Escherichia coli, and synthesize salidroside through biological fermentation. The biological method has low production cost, can be industrialized, is environmentally friendly, and has higher product quality and safety, which is more conducive to the application of salidroside in food, medicine, health products and cosmetics.

[0005] The natural salidroside extracted from plants is β-salidroside, and its glycosidic bond is β-type. Chinese patent 202211359878.5 discloses an α-salidroside, whose chemical name is: 2-(4-hydroxyphenyl)ethyl-α-D-glucoside. Compared with the known β-salidroside, it has better free radical scavenging activity. The patent also discloses that α-salidroside can be prepared by catalytic reactions of common commercial enzyme preparations such as glycosyltransferase, amylase, aromatase, cellulase, cycloglucan transferase and glycoside transferase. When the glycosyltransferase is derived from Aspergillus sp., the conversion rate of tyrosol to α-salidroside is 2%; when the glycosyltransferase is derived from rthrobacter sp., the conversion rate of tyrosol to α-salidroside is 20%.

[0006] Due to the need for iteration of product production processes, it is urgent to develop a green, simple, scalable, high-efficiency, high-conversion, and low-cost salidroside production technology. Summary of the invention

[0007] Purpose of the invention: To provide an application of α-glucosidase in the synthesis of α-salidroside.

[0008] Technical solution: The applicant successfully synthesized α-salidroside using α-glucosidase derived from Ensifer adhaerens CCTCC AB 2019220 and improved the tyrosol conversion rate.

[0009] The technical scheme of the present invention is: application of alpha-glucosidase in synthesizing alpha-salidroside.

[0010] The α-glucosidase is derived from Ensifer adhaerens, and preferably, its amino acid sequence is shown in SEQ ID No:1.

[0011] The Ensifer adhaerens is preferably CCTCC AB 2019220.

[0012] The expression method of α-glucosidase, the Ensifer adhaerens includes a wild strain of the genus Ensifer separated from nature, or a mutant strain after physical and chemical mutagenesis, or an engineered bacterium after genetic engineering.

[0013] The α-glucosidase may be used in the form of a crude enzyme solution, but preferably a purified enzyme solution is used.

[0014] The α-salidroside compound has a chemical name of 2-(4-hydroxyphenyl)ethyl-α-D-glucoside and a chemical formula of Formula I.

[0015]

[0016] In the application, the glycosyl donor of the reaction system is maltose, the substrate is tyrosol, and α-salidroside is synthesized by catalysis. When the glycosyl donor is trehalose or maltose, the conversion rate of tyrosol is low.

[0017] In the application, preferably, the reaction solvent is an aqueous solvent or a solution containing solids.

[0018] In the application, the reaction temperature is preferably 20-47°C; further preferably 30-45°C, and more preferably 35-45°C.

[0019] In the application, the preferred enzyme addition amount is 0.05-1 mg / mL; more preferably, it is about 0.10-0.30 mg / mL. When the enzyme addition amount exceeds 1 mg / mL, the conversion rate of tyrosol is not greatly affected.

[0020] In the application, preferably, the pH of the reaction system is in the range of 6-9; more preferably, pH 6.5-8, and further preferably, pH 7.

[0021] In the application, preferably, the molar ratio of the substrate tyrosol to the glycosyl donor maltose is in the range of 1:4-7, preferably 1:4.5-6. When the molar ratio of tyrosol to maltose is lower than 1:4, the conversion rate of tyrosol is low, and when it is higher than 1:7, the conversion rate of tyrosol does not increase significantly.

[0022] Beneficial effects:

[0023] The present invention utilizes α-glucosidase derived from Ensifer adhaerens CCTCC AB 2019220 to catalyze and synthesize α-salidroside compounds through enzymatic reactions, and the tyrosol conversion rate can reach about 50%, which is significantly improved compared with the prior art. The present invention provides a preparation technology for α-salidroside with simple process, high green safety and low production cost, which is conducive to industrial large-scale production and has positive significance for the industrialization and application of salidroside and its derivatives in the fields of food, medicine or cosmetics. DETAILED DESCRIPTION

[0024] The experimental methods used in the following examples are conventional methods unless otherwise specified; all materials, reagents, etc., are commercially available unless otherwise specified.

[0025] The present invention is further described in detail below through examples. It should be understood that the specific implementation methods described herein are only used to explain the present invention and are not used to limit the present invention. Modifications or replacements to the details and forms of the technical solution without departing from the structural concept and scope of use of the present invention all fall within the protection scope of the present invention.

[0026] Unless otherwise defined, all technical terms and scientific terms used in this specification have the same meanings as those commonly understood by those skilled in the art. In general, the nomenclature used in this specification and the experimental methods described below are well known and commonly used in the art.

[0027] It should be noted that the terms used here are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are to describe specific embodiments rather than to limit the scope of protection of the present invention. In the following specific embodiments, if the experimental methods of specific conditions are not indicated, they are generally based on conventional methods and conditions of molecular biology within the technology of the art, and such techniques and conditions are fully explained in the literature, or according to the conditions recommended by the manufacturer.

[0028] In the following examples and test examples, the product obtained by the enzyme-catalyzed reaction refers to the application number of the applicant 202211359878.5, and the name is "An α-salidroside and its preparation method and application". The TLC, HPLC, LC-MS, and NMR methods described in Example 10 are used for analysis and identification, and the concentration of the enzyme-catalyzed reaction product is determined by high performance liquid chromatography (HPLC) analysis method. The entire content of patent 202211359878.5 is incorporated by reference into the present invention and constitutes a part of the present invention.

[0029] Example 1. Preparation of α-glucosidase from Ensifer adhaerens CCTCC AB 2019220.

[0030] (1) Cultivation of Ensifer adhaerens CCTCC AB 2019220.

[0031] Ensifer adhaerens CCTCC AB 2019220 was cultured in a medium containing 5.0 g / L maltose, 0.1 g / L yeast extract, 2.0 g / L ammonium sulfate, 1.0 g / L potassium dihydrogen phosphate, 1.0 g / L potassium hydrogen phosphate, and 0.2 g / L magnesium sulfate heptahydrate at 25°C for 48 h, after which the culture broth was centrifuged at 12,000 × g for 10 min at 4°C to collect the cells.

[0032] (2) Preparation of α-glucosidase from Ensifer adhaerens CCTCC AB 2019220.

[0033] The cells obtained above were resuspended in 25 mM potassium phosphate buffer (pH 7.0), and disrupted on ice using an ultrasonicator (Ohtake, Tokyo, Japan), and centrifuged at 14000×g for 30 min at 4°C to remove cell debris. The obtained supernatant was the crude enzyme solution of Ensifer adhaerens CCTCC AB 2019220.

[0034] Enzyme purification: The crude enzyme solution was precipitated with ammonium sulfate [40%-60% (w / v) saturation] and dialyzed, and then purified using an AKTA purification system (GE Healthcare, Uppsala, Sweden). The dialysate was loaded into a GE MonoQ anion exchange column (5 / 50 GL; GE Healthcare), connected to an AKTA, equilibrated with 10 mM sodium phosphate buffer (pH 7.5), and the protein was eluted with a 0-1.0 M sodium chloride gradient. The active fractions were collected and desalted by repeated dilution. The sample was loaded into a GE MonoQ anion exchange column again. The active fractions were collected and concentrated by ultracentrifugation using an Amicon Ultra-2 centrifugal filter device (nominal molecular weight limit, 10,000; Merck). The concentrated eluate was loaded into a Superdex 200 column (10 / 300 GL; GE Healthcare), the active fractions were collected and dialyzed, and the dialysate was used as the purified enzyme.

[0035] Example 2. Study on the substrate specificity of α-glucosidase from Ensifer adhaerens CCTCC AB 2019220.

[0036] 55mM tyrosol was used as the glycosyl acceptor, 280mM isomaltose, cellobiose, lactose, amylose, sucrose, trehalose or maltose were used as the glycosyl donors, and an appropriate amount of α-glucosidase (phosphate buffer pH 7.0) was added and incubated at 25°C for 4h. The substrate specificity of the enzyme was determined by measuring the level of α-salidroside production in the reaction solution by HPLC. The production rate of α-salidroside was defined as the ratio of the peak area of ​​α-salidroside to the total area, and the data were normalized relative to the results of maltose.

[0037] The results showed that Ensifer adhaerens CCTCC AB 2019220 α-glucosidase was active on maltose, sucrose and trehalose (see Table 1 for details), and the production of α-salidroside was not observed in isomaltose, cellobiose, lactose and amylose.

[0038] Table 1. Study on substrate specificity of α-glucosidase from Ensifer adhaerens CCTCC AB 2019220

[0039] Substrate Relative activity (%) maltose 100 sucrose 11.1 Trehalose 2.5 .

[0040] Example 3. Synthesis of α-salidroside by enzyme-catalyzed reaction.

[0041] The purified enzyme solution obtained in Example 1 was used to carry out enzyme catalysis reaction.

[0042] The system is: 20mM potassium phosphate buffer (pH 6.0), 55mM tyrosol, 280mM maltose, 0.10mg / mL purified enzyme solution, incubated at 45°C for 16h. The α-salidroside content determined by HPLC is 4.5mg / mL, and the tyrosol conversion rate is 27.3%.

[0043] Example 4. Synthesis of α-salidroside by enzyme-catalyzed reaction.

[0044] The purified enzyme solution obtained in Example 1 was used to carry out enzyme catalysis reaction.

[0045] The system is: 20mM potassium phosphate buffer (pH 6.5), 55mM tyrosol, 280mM maltose, 0.15mg / mL purified enzyme solution, incubated at 45°C for 16h. The α-salidroside content determined by HPLC is 7.5mg / mL, and the tyrosol conversion rate is 45.5%.

[0046] Example 5. Synthesis of α-salidroside by enzyme-catalyzed reaction.

[0047] The purified enzyme solution obtained in Example 1 was used to carry out enzyme catalysis reaction.

[0048] The system was: 20 mM Britton-Robinson buffer (pH 8.5), 55 mM tyrosol, 280 mM maltose, 1.0 mg / mL purified enzyme solution, incubated at 40°C for 24 h. The yield of α-salidroside in the reaction solution was 7.0 mg / mL by HPLC, and the tyrosol conversion rate was 42.4%.

[0049] Example 6. Synthesis of α-salidroside by enzyme-catalyzed reaction.

[0050] The purified enzyme solution obtained in Example 1 was used to carry out enzyme catalysis reaction.

[0051] The system is: 20mM potassium phosphate buffer (pH7.0), 55mM tyrosol, 280mM maltose, 0.30mg / mL purified enzyme solution, incubated at 30°C for 24h. The yield of α-salidroside in the reaction solution was 8.1mg / mL by HPLC, and the tyrosol conversion rate was 49.1%.

[0052] Example 7. The purified enzyme solution obtained in Example 1 was used to carry out enzyme catalytic reaction.

[0053] The purified enzyme solution obtained in Example 1 was used to carry out enzyme catalysis reaction.

[0054] The system is: 20mM potassium phosphate buffer (pH 7.0), 55mM tyrosol, 220mM maltose, 0.15mg / mL purified enzyme solution, incubated at 47℃ for 16h. The α-salidroside content determined by HPLC is 5.3mg / mL, and the tyrosol conversion rate is 32.1%

[0055] Example 8. The purified enzyme solution obtained in Example 1 was used to carry out enzyme catalytic reaction.

[0056] The purified enzyme solution obtained in Example 1 was used to carry out enzyme catalysis reaction.

[0057] The system is: 20mM potassium phosphate buffer (pH 7.0), 55mM tyrosol, 330mM maltose, 0.15mg / mL purified enzyme solution, incubated at 45°C for 16h. The α-salidroside content determined by HPLC is 8.2mg / mL, and the tyrosol conversion rate is 49.7%.

[0058] Example 9. The purified enzyme solution obtained in Example 1 was used to carry out enzyme catalytic reaction.

[0059] The purified enzyme solution obtained in Example 1 was used to carry out enzyme catalysis reaction.

[0060] The system is: 20mM potassium phosphate buffer (pH 7.0), 55mM tyrosol, 385mM maltose, 0.15mg / mL purified enzyme solution, incubated at 45°C for 16h. The α-salidroside content determined by HPLC is 8.5mg / mL, and the tyrosol conversion rate is 51.5%.

[0061] Example 10. The purified enzyme solution obtained in Example 1 was used to carry out enzyme catalytic reaction.

[0062] The purified enzyme solution obtained in Example 1 was used to carry out enzyme catalysis reaction.

[0063] The system is: 20mM potassium phosphate buffer (pH 7.0), 55mM tyrosol, 330mM maltose, 0.15mg / mL purified enzyme solution, incubated at 20°C for 16h. The α-salidroside content determined by HPLC is 4.9mg / mL, and the tyrosol conversion rate is 29.7%.

[0064] Example 11. Synthesis of α-salidroside by enzyme-catalyzed reaction.

[0065] The purified enzyme solution obtained in Example 1 was used to carry out enzyme catalysis reaction.

[0066] The system is: 20mM potassium phosphate buffer (pH 9.0), 55mM tyrosol, 280mM maltose, 0.15mg / mL purified enzyme solution, incubated at 45°C for 16h. The α-salidroside content determined by HPLC is 4.7mg / mL, and the tyrosol conversion rate is 28.5%.

[0067] Control Example 1. Synthesis of α-salidroside by enzyme-catalyzed reaction.

[0068] The purified enzyme solution obtained in Example 1 was used to carry out enzyme catalysis reaction.

[0069] The system was: 20 mM Britton–Robinson buffer (pH 4.0), 55 mM tyrosol, 280 mM maltose and 0.15 mg / mL purified enzyme solution, incubated at 35°C for 0.5 h. The HPLC method determined the α-salidroside content to be 0.68 mg / mL, and the tyrosol conversion rate to be 4.1%.

[0070] The above shows that when maltose is selected as the glycosyl donor at pH 4.0, the tyrosol conversion rate is very low and has no industrial application value.

[0071] Control Example 2. Synthesis of α-salidroside by enzyme-catalyzed reaction.

[0072] The purified enzyme solution obtained in Example 1 was used to carry out enzyme catalysis reaction.

[0073] The system was: 20mM Britton-Robinson buffer (pH 10.0), 55mM tyrosol, 280mM maltose and 1.0mg / mL purified enzyme solution, incubated at 40°C for 24h. The yield of α-salidroside in the reaction solution was 0.78mg / mL by HPLC, and the tyrosol conversion rate was 4.7%.

[0074] Control Example 3. Synthesis of α-salidroside by enzyme-catalyzed reaction.

[0075] The purified enzyme solution obtained in Example 1 was used to carry out enzyme catalysis reaction.

[0076] The system is: 20mM potassium phosphate buffer (pH7.0), 55mM tyrosol, 280mM maltose and 0.15mg / mL purified enzyme solution, incubated at 60°C for 24h. The yield of α-salidroside in the reaction solution is 1.2mg / mL and the tyrosol conversion rate is 7.3% by HPLC.

[0077] Control Example 4. Synthesis of α-salidroside by enzyme-catalyzed reaction.

[0078] The purified enzyme solution obtained in Example 1 was used to carry out enzyme catalysis reaction.

[0079] The system is: 20mM potassium phosphate buffer (pH 7.0), 55mM tyrosol, 280mM sucrose and 0.15mg / mL purified enzyme solution, incubated at 15°C for 20h. The α-salidroside content determined by HPLC is 0.35mg / mL, and the tyrosol conversion rate is 2.1%.

[0080] Control Example 5. Synthesis of α-salidroside by enzyme-catalyzed reaction.

[0081] The purified enzyme solution obtained in Example 1 was used to carry out enzyme catalysis reaction.

[0082] The system was: 20 mM Britton-Robinson buffer (pH 6.0), 55 mM tyrosol, 280 mM maltose, 0.05 mg / mL purified enzyme solution, incubated at 35°C for 0.5 h. The HPLC method determined that the α-salidroside content was 0.1 mg / mL and the tyrosol conversion rate was 0.6%.

[0083] Control Example 6. Synthesis of α-salidroside by enzyme-catalyzed reaction.

[0084] The purified enzyme solution obtained in Example 1 was used to carry out enzyme catalysis reaction.

[0085] The system is: 20mM potassium phosphate buffer (pH10.0), 55mM tyrosol, 280mM maltose, 0.15mg / mL purified enzyme solution, incubated at 45°C for 16h. The α-salidroside content determined by HPLC is 0.4mg / mL, and the tyrosol conversion rate is 2.4%.

[0086] The above description is only an embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention, as well as the research and selection of process parameters such as the temperature of the catalytic reaction, the amount of enzyme added, the reaction time, and the pH value, shall fall within the scope of the present invention.

[0087] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. Application of α-glucosidase in the synthesis of α-salidroside.

2. The use according to claim 1, characterized in that: The α-glucosidase is derived from Ensiferadhaerens.

3. The use according to claim 1, characterized in that: The amino acid sequence of the α-glucosidase is shown in SEQ ID No:

1.

4. The use according to claim 1, characterized in that: The α-glucosidase source bacteria is Ensiferadhaerens CCTCC AB 2019220.

5. The use according to claim 1, characterized in that: The α-glucosidase is a crude enzyme solution or a purified enzyme solution.

6. The use according to claim 1, characterized in that: The applied glycosyl donor is maltose and the substrate is tyrosol.

7. The use according to claim 6, characterized in that In the application, the reaction temperature is 20-47°C; preferably 30-45°C, more preferably 35-45°C. In the application, preferably, the enzyme is added in an amount of 0.05-1 mg / mL; preferably 0.1-0.3 mg / mL. In the application, preferably, the pH of the reaction system is in the range of 6-9; further preferably, pH 6.5-8.5, and further preferably, pH 7. In the application, preferably, the molar ratio of the substrate tyrosol to the glycosyl donor maltose is in the range of 1:4-7, preferably 1:4.5-6.

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