Use of glycosyltransferase bs-yji c in bacillus subtilis 168

Abstract

The application discloses application of a glycosyltransferase Bs-YjiC in Bacillus subtilis 168 and comprises the following steps: catalyzing reaction of ginsenoside Rg2, the glycosyltransferase Bs-YjiC and uridine diphosphate glucose, so as to glycosylate C 20 hydroxyl group of the ginsenoside Rg2 to generate ginsenoside Re; or catalyzing reaction of notoginsenoside R2, the glycosyltransferase Bs-YjiC and uridine diphosphate glucose, so as to glycosylate C 20 hydroxyl group of the notoginsenoside R2 to generate notoginsenoside R1; the application utilizes the glycosyltransferase Bs-YjiC of microbial origin to catalyze C 20 hydroxyl group glycosylation of the ginsenoside Rg2 or the notoginsenoside R2, and since the ginsenoside Re and the notoginsenoside R1 have important pharmacological effects, the glycosyltransferase Bs-YjiC of microbial origin can be utilized to catalyze synthesis of the ginsenoside Re and the notoginsenoside R1 beneficial to human health.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology and botany, and relates to application of a glycosyltransferase Bs-YjiC in Bacillus subtilis 168. BACKGROUND

[0002] Triterpenoid saponins are a class of naturally occurring triterpenoid glycosides with diverse structures and rich pharmacological activities, including Ginsenoside, Notoginsenoside, and Saikosaponin. Among them, Ginsenoside and Notoginsenoside are more widely used.

[0003] Ginsenoside is the main active substance of ginseng and is a glycoside compound composed of aglycone and sugar group. At present, more than 60 kinds of ginsenoside monomers including ginsenoside Rh2, Rg2 and Re have been isolated and identified from different ginseng varieties. Among them, the relatively more studied saponins include ginsenoside Re. Ginsenoside Re has the effects of antiarrhythmia, anti-heart failure and central nervous system inhibition. The content of ginsenoside in ginseng root is only about 4%. Therefore, the biosynthesis of ginsenoside Re plays an important role in the systematic study of ginsenoside Re.

[0004] Notoginsenoside is the main active substance of Panax notoginseng. At present, a variety of saponin components have been isolated from Panax notoginseng, including notoginsenoside R2 and R1. With the in-depth study of the pharmacological effects of saponin components, it is found that many saponins have excellent pharmacological activities. Among them, notoginsenoside R1 is one of the unique saponin components in Panax notoginseng, which has the effects of regulating lipid metabolism, mediating oxidative stress, inhibiting inflammatory response and improving atherosclerosis. In the 2020 edition of Chinese Pharmacopoeia, the content of R1 is one of the main identification indexes for judging the quality of Panax notoginseng. Therefore, the biosynthesis of notoginsenoside R1 can effectively promote the systematic study of the saponin component.

[0005] At present, a number of plant-derived glycosyltransferases have been discovered from ginseng and Panax notoginseng. Plant-derived glycosyltransferases can catalyze the synthesis of a variety of ginsenosides or notoginsenosides. Compared with plant-derived glycosyltransferases, microbial-derived glycosyltransferases have the characteristics of high heterologous expression level, wide substrate spectrum and high catalytic activity. Studies have shown that microbial-derived glycosyltransferase Bs-YjiC can catalyze the synthesis of C3, C6 and C 12 hydroxyl groups of ginsenosides or notoginsenosides, while its C 20 hydroxyl catalytic function has not been reported. SUMMARY

[0006] The application aims to overcome the deficiencies of the prior art and provide the application of glycosyltransferase Bs-YjiC in Bacillus subtilis 168.

[0007] The technical scheme of the application is summarized as follows:

[0008] The application of glycosyltransferase Bs-YjiC in Bacillus subtilis 168 comprises the following steps:

[0009] The ginsenoside Rg2 is subjected to catalytic reaction with the glycosyltransferase Bs-YjiC and uridine diphosphate glucose, so that the C 20 hydroxyl group of the ginsenoside Rg2 is glucosylated to generate the ginsenoside Re;

[0010] Or the notoginsenoside R2 is subjected to catalytic reaction with the glycosyltransferase Bs-YjiC and uridine diphosphate glucose, so that the C 20 hydroxyl group of the notoginsenoside R2 is glucosylated to generate the notoginsenoside R1;

[0011] The amino acid sequence of the glycosyltransferase Bs-YjiC is shown in SEQ ID NO. 1.

[0012] Advantages of the application:

[0013] The application uses the glycosyltransferase Bs-YjiC from a microbial source as a catalyst, uses the ginsenoside Rg2 as a substrate, catalyzes the C 20 hydroxyl group of the ginsenoside Rg2 to be glucosylated, and synthesizes the ginsenoside Re. Or uses the notoginsenoside R2 as a substrate, catalyzes the C 20 hydroxyl group of the notoginsenoside R2 to be glucosylated, and synthesizes the notoginsenoside R1. Since the ginsenoside Re and the notoginsenoside R1 have important pharmacological effects, the glycosyltransferase Bs-YjiC mined by the application can be used to catalyze the synthesis of the ginsenoside Re and the notoginsenoside R1 which are beneficial to human health. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 A is a reaction formula of the ginsenoside Rg2 generating the ginsenoside Re under the catalysis of the glycosyltransferase Bs-YjiC. Figure 1 B is a reaction formula of the notoginsenoside R2 generating the notoginsenoside R1 under the catalysis of the glycosyltransferase Bs-YjiC.

[0015] Figure 2 A is a high performance liquid chromatography analysis glycosylation product diagram of the ginsenoside Rg2 catalyzed by the glycosyltransferase Bs-YjiC; Figure 2 B is a high performance liquid chromatography-mass spectrometry analysis glycosylation product diagram of the ginsenoside Rg2 catalyzed by the glycosyltransferase Bs-YjiC.

[0016] Figure 3A is the high performance liquid chromatography analysis of glycosyltransferase Bs-YjiC catalyzed panax saponins R2 glycosylation product map; Figure 3 B is the high performance liquid chromatography-mass spectrometry analysis of glycosyltransferase Bs-YjiC catalyzed panax saponins R2 glycosylation product map.

[0017] Figure 4 It is the SDS-PAGE protein gel map of glycosyltransferase Bs-YjiC after expression and purification. DETAILED DESCRIPTION

[0018] The glycosyltransferase Bs-YjiC of the application is from Bacillus subtilis 168, and the glycosyltransferase can be expressed and purified by constructing an expression vector and transforming into a host strain.

[0019] The glycosyltransferase of the application can in vitro prepare ginsenoside Re and panax saponins R1, and the amino acid sequence is as shown in SEQ ID NO. 1.

[0020] The structural formula of ginsenoside Re is shown in Figure 1 A; the structural formula of panax saponins R1 is shown in Figure 1 B.

[0021] The application will be further described in detail below in combination with specific embodiments.

[0022] In order to further illustrate the technical means adopted by the application and its effects, the application will be further described below in combination with examples. It can be understood that the specific embodiments described herein are only used to explain the application, and not to limit the application.

[0023] If the specific technology or condition is not specified in the examples, the technology or condition described in the literature in the art or according to the product manual is used. If the reagent or instrument is not specified by the manufacturer, it is a conventional product that can be commercially available through a regular channel.

[0024] The application will be further described in detail below in combination with examples.

[0025] The examples are implemented on the premise of the technical solutions of the application, and detailed implementation modes and specific operation processes are given. The examples will help to understand the application, but the protection scope of the application is not limited to the examples below.

[0026] The enzyme activity determination method in the application is as follows:

[0027] Glycosyltransferase enzyme activity assay: in 100 μL reaction system containing ginsenoside Rg2 or notoginsenoside R2 (0.5 mM), uridine diphosphate glucose (5 mM), 100 mM phosphate buffer (pH 7.5), 1 (v / v) % Tween-20, 10 ug glycosyltransferase Bs-YjiC, 37 °C water bath reaction for 12 h, 100 μL methanol was added to terminate the reaction, and the consumption of ginsenoside Rg2 and notoginsenoside R2 was detected by high performance liquid chromatography.

[0028] Example 1, synthesis, expression and purification of glycosyltransferase Bs-YjiC

[0029] The GenBank accession number of glycosyltransferase Bs-YjiC is NP_389104. Bs-YjiC gene was synthesized by Jiangsu Jinyuizhi Biotechnology Co., Ltd. (The amino acid sequence of the glycosyltransferase Bs-YjiC is shown as SEQ ID NO. 1.)

[0030] The glycosyltransferase Bs-YjiC gene was inserted into the pET-28a(+) expression vector treated with the same restriction endonuclease by using DNA ligase to construct the recombinant vector pET28-Bs-YjiC. Then the recombinant plasmid pET28-Bs-YjiC was transformed into Escherichia coli BL21(DE3) competent cells and the expression of the target protein was induced; the induction conditions of glycosyltransferase Bs-YjiC were as follows: in LB liquid medium (10 g / L NaCl, 10 g / L peptone and 5 g / L yeast powder) containing 50 mg / L kanamycin, 37 °C culture for about 4 hours to OD 600 of the bacterial liquid was 0.6-0.8, then IPTG was added to a final concentration of 0.5 mM, and the culture was carried out at 18 °C, 180 r / min for 16-20 h to induce the expression of the target protein. Then the bacterial cells were collected by centrifugation and the bacterial cells were suspended with 10 mM imidazole aqueous solution, the bacterial cells were lysed by ultrasonic crushing, the supernatant containing protein was loaded onto a Ni-NTA purification column and purified, and the purified protein was eluted using a 40-400 mM imidazole gradient, and the expression and purity of the target Bs-YjiC were analyzed by SDS-PAGE protein electrophoresis. As shown in the SDS-PAGE protein gel ( Figure 4 ), the recombinant glycosyltransferase Bs-YjiC was expressed in a soluble form, and the target protein was mainly located in the supernatant of the cell lysate, and the size of the glycosyltransferase Bs-YjiC was about 44.8 kD. In addition, the results of SDS-PAGE protein gel showed that the glycosyltransferase Bs-YjiC with a purity of more than 95% could be purified by nickel column.

[0031] Example 2

[0032] Glycosyltransferase Bs-YjiC catalyzes the synthesis of ginsenoside Rg220 hydroxylglucosylation

[0033] The reaction system catalyzed by the glycosyltransferase Bs-YjiC obtained in Example 1 includes:

[0034] The glycosylation includes 0.5 mM ginsenoside Rg2, 5 mM uridine diphosphate glucose, 100 mM phosphate buffer (pH 7.5), 1 (v / v) % Tween-20, 10 ug glycosyltransferase Bs-YjiC, 37 °C reaction for 12 h in 100 uL reaction system.

[0035] After the reaction is completed, an equal volume of methanol is added to terminate the reaction, and then centrifuged at 12000 rpm for 10 min. The supernatant is taken and added to a liquid phase bottle, and the glycosylation product is analyzed and identified by high performance liquid chromatography and high performance liquid chromatography-mass spectrometry. The analysis conditions of high performance liquid chromatography are as follows: mobile phase A is distilled water, mobile phase B is acetonitrile, the chromatographic column is a C18 chromatographic column (4.6 mm x 250 mm, 5 um particle) purchased from Kromasil, the injection amount is 20 uL, the gradient elution conditions are 0-35 min, 20 % B, 35 min-40 min, 20 % B-30 % B, 40 min-50 min, 30 % B-31 % B, 50 min-60 min, 31 % B-32 % B, 60 min-70 min, 32 % B-45 % B, 70 min-100 min, 45 % B-60 % B, 100 min-110 min, 60 % B, 110 min-111 min, 60 % B-20 % B, 111 min-120 min, 20 % B. The ultraviolet detector has a detection wavelength of 203 nm, the flow rate of the mobile phase is 1 mL / min, and the column oven is 35 °C. The mass spectrometer is an Agilent mass spectrometer equipped with an ESI ion source in positive ion mode.

[0036] As shown in Figure 2 A, Figure 2 B, high performance liquid chromatography analysis shows that the glycosyltransferase Bs-YjiC can catalyze ginsenoside Rg2 to generate product (Product) 1, which has the same peak position as ginsenoside Re; further analysis of the product by high performance liquid chromatography-mass spectrometry shows that the molecular weight of the product is: ([M+Na-H2O] + m / z + ~ 969.5000), and the molecular formula of ginsenoside Re is C 48 H 82 O 18 , and the theoretical molecular weight is: ([M+Na-H2O] + m / z +~ 969.5393), indicating that product 1 is ginsenoside Re.

[0037] Example 3

[0038] Glycosyltransferase Bs-YjiC catalyzes gypenoside R2C 20 Hydroxyl glucosylation

[0039] The reaction system in which the glycosyltransferase Bs-YjiC obtained in Example 1 catalyzes gypenoside R2 glucosylation comprises:

[0040] Glycosylation in 100 μL of the reaction system comprises 0.5 mM gypenoside R2, 5 mM uridine diphosphate glucose, 100 mM phosphate buffer (pH 7.5), 1 (v / v) % Tween-20, 10 ug glycosyltransferase Bs-YjiC, and reaction at 37°C for 12 h.

[0041] After the reaction is completed, an equal volume of methanol is added to terminate the reaction, and then centrifugation is performed at 12000 rpm for 10 min, the supernatant is taken, and then added to a liquid phase bottle. The glycosylation product is analyzed and identified by high performance liquid chromatography and high performance liquid chromatography-mass spectrometry. The analysis conditions of high performance liquid chromatography are as follows: mobile phase A is distilled water, mobile phase B is acetonitrile, the chromatographic column is a C18 chromatographic column (4.6 mm x 250 mm, 5 μm particle) purchased from Kromasil, the injection amount is 20 μL, the gradient elution conditions are 0-35 min, 20% B, 35 min-40 min, 20% B-30% B, 40 min-50 min, 30% B-31% B, 50 min-60 min, 31% B-32% B, 60 min-70 min, 32% B-45% B, 70 min-100 min, 45% B-60% B, 100 min-110 min, 60% B, 110 min-111 min, 60% B-20% B, 111 min-120 min, 20% B. The ultraviolet detector is used, the detection wavelength is 203 nm, the flow rate of the mobile phase is 1 mL / min, and the column oven is 35°C. The mass spectrometer is an Agilent mass spectrometer equipped with an ESI ion source, and the positive ion mode is used.

[0042] As shown in Figure 3 A, Figure 3 B, high performance liquid chromatography analysis shows that the glycosyltransferase Bs-YjiC can catalyze gypenoside R2 to generate product (Product) 2, and the peak position is consistent with that of gypenoside R1. Further analysis of the product by high performance liquid chromatography-mass spectrometry shows that the molecular weight of the product is: ([M+Na-H2O] + m / z + ~ 955.52140), and the molecular formula of gypenoside R1 is C47 H 80 O 18 , theoretical molecular weight of ([M+Na-H2O] + m / z + ~ 955.5237), indicating that the product 2 is R1.

Claims

1. Use of the glycosyltransferase Bs-YjiC from Bacillus subtilis 168, characterized in that comprising the steps of: Catalyzing reaction of ginsenoside Rg2 with glycosyltransferase Bs-YjiC and uridine diphosphate glucose, C 20 hydroxyl group of ginsenoside Rg2 is glucosylated to generate ginsenoside Re; or catalyzing the reaction of panaxsaponin R2 with glycosyltransferase Bs-YjiC and uridine diphosphate glucose, so that the C 20 of panaxsaponin R2 is hydroxylglucosylated to generate panaxsaponin R1; The amino acid sequence of the glycosyltransferase Bs-YjiC is shown in SEQ ID NO.

1. The amino acid sequence of the glycosyltransferase Bs-YjiC is shown in SEQ ID NO. 1.

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