Preparation method of QS21 intermediate

Abstract

The invention provides a preparation method of a QS21 intermediate, which comprises the following steps: S1, mixing QA soapberry acid, UDP-D-GlcA and E1-CSLM2 enzyme, reacting, and purifying to obtain a QA-GlcA intermediate; s2, the QA-GlcA intermediate, UDP-D-Gal and E2-GmSGT2 enzyme are subjected to a mixed reaction and then purified, and the QS21 intermediate QA-GlcA-Gal is obtained. According to the method, an in-vitro enzyme method is adopted, the QA-GlcA intermediate is synthesized through catalysis of the E1-CSLM2 enzyme, the QA-GlcA intermediate is catalyzed through the E2-GmSGT2 enzyme to obtain the QS21 intermediate QA-GlcA-Gal, the steps are simple and convenient, and the yield is high.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a preparation method of QS21 intermediate. BACKGROUND

[0002] QS21 intermediate is a derivative of quillic acid, and these compounds are the core components of QS21 structure. QS21 belongs to triterpene saponins, and its molecular structure comprises four key regions: a lipophilic triterpene core as a main skeleton, a branched trisaccharide at the C3 position, a linear tetrasaccharide chain at the C28 position, and an acyl side chain connected by an ester bond. Currently, the methods for synthesizing QS21 include chemical synthesis, plant cell culture and extraction, but these synthesis methods are complex and have low extraction rate. SUMMARY

[0003] Therefore, the present application provides a preparation method of QS21 intermediate, which adopts in-vitro enzyme method, and synthesizes QA-GlcA intermediate through E1-CSLM2 enzyme catalysis, and obtains QS21 intermediate (QA-GlcA-Gal) through E2-GmSGT2 enzyme catalysis, so that the steps are simple and the yield is high.

[0004] To achieve the above-mentioned purpose, the present application adopts the following technical solutions: The present application provides a preparation method of QS21 intermediate, comprising the following steps: S1, mixing QA quillaja saponin, UDP-D-GlcA and E1-CSLM2 enzyme for reaction and then purifying to obtain QA-GlcA intermediate; S2, mixing QA-GlcA intermediate, UDP-D-Gal and E2-GmSGT2 enzyme for reaction and then purifying to obtain QS21 intermediate QA-GlcA-Gal.

[0005] In some embodiments, the enzyme catalysis reaction of step S1 is as follows:

[0006] In some embodiments, the enzyme catalysis reaction of step S2 is as follows:

[0007] Preferably, in step S1, the E1-CSLM2 enzyme is obtained by insect sf9 expression.

[0008] Preferably, in step S1, the mixed reaction substances further comprise pH 7.5 Tris-HCl, MgCl2 and DMSO.

[0009] Preferably, in step S1, after mixing, the final concentration of QA quillaic acid is 1 mM, the final concentration of UDP-D-GlcA is 2 mM, the final concentration of Tris-HCl is 50 mM, and the final concentration of MgCl2 is 10 mM, and the volume ratio of DMSO is 5%.

[0010] Preferably, in step S1, the temperature of the mixed reaction is 30℃.

[0011] Preferably, in step S2, the E2-GmSGT2 enzyme is obtained by expression of E. coli.

[0012] Preferably, in step S2, the mixed reaction further includes pH 7.5 Tris-HCl and MgCl2.

[0013] Preferably, in step S2, after mixing, the final concentration of QA-GlcA intermediate is 1 mM, the final concentration of UDP-D-Gal is 2 mM, the final concentration of Tris-HCl is 50 mM, and the final concentration of MgCl2 is 10 mM.

[0014] Preferably, the temperature of the mixed reaction is 30℃.

[0015] Compared with the prior art, the present application has the following beneficial effects: (1) The present application catalyzes the reaction of QA quillaic acid by E1-CSLM2 enzyme to obtain QA-GlcA intermediate, and then catalyzes the reaction of the QA-GlcA intermediate by E2-GmSGT2 enzyme to obtain intermediate QS21-GlcA-Gal. The steps are simple and the yield is high.

[0016] (2) In order to improve the catalytic efficiency of E1-CSLM2 enzyme and E2-GmSGT2 enzyme, the present application uses insect sf9 to express E1-CSLM2 enzyme and uses E. coli to express E2-GmSGT2. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The activity detection mass spectrum of the enzyme E1-CSLM2 provided in Example 3 of the present application (sf9 expression); E. coli The activity detection mass spectrum of the enzyme E1-CSLM2 provided in Example 3 of the present application (sf9 expression); Figure 2 The activity detection mass spectrum of the enzyme E1-CSLM2 provided in Example 3 of the present application (sf9 expression); Figure 3 The HPLC detection chart of the intermediate product QA-GlcA provided in Example 3 of the present application; Figure 4 The activity detection mass spectrum of the enzyme E2-GmSGT2 provided in Example 3 of the present application (sf9 expression); E. coli The activity detection mass spectrum of the enzyme E2-GmSGT2 provided in Example 3 of the present application (sf9 expression); Figure 5The mass spectrum of activity detection of the enzyme E2-UGT73CU3 (sf9 expression) provided in Embodiment 3 of the present application; Figure 6 The mass spectrum of activity detection of the enzyme E2-UGT73CU3 (sf9 expression) provided in Embodiment 3 of the present application; E. coli The mass spectrum of activity detection of the enzyme E2-UGT73CU3 (sf9 expression) provided in Embodiment 3 of the present application; Figure 7 The HPLC detection chart of the intermediate product QA-GlcA-Gal provided in Embodiment 3 of the present application; Figure 8 The NMR detection chart of the intermediate product QA-GlcA-Gal provided in Embodiment 3 of the present application. DETAILED DESCRIPTION

[0018] The present application will be further described in detail below with specific embodiments, so that those skilled in the art can more clearly understand the present application.

[0019] QS21 intermediates are derivatives of quillic acid, which are the core components of QS21 structure. QS21 belongs to triterpene saponins, and its molecular structure contains four key regions: a lipophilic triterpene core as the main skeleton, a branched trisaccharide at the C3 position, a linear tetrasaccharide chain at the C28 position, and an acyl side chain connected by an ester bond. Currently, the methods for synthesizing QS21 include chemical synthesis, plant cell culture and extraction, but these synthesis methods are complex and have low extraction rates.

[0020] To solve the above technical problems, the present application provides a preparation method of QS21 intermediates, comprising the following steps: S1, purifying the mixed reaction of QA quillic acid, UDP-D-GlcA and E1-CSLM2 enzyme to obtain a QA-GlcA intermediate; S2, purifying the mixed reaction of the QA-GlcA intermediate, UDP-D-Gal and E2-GmSGT2 enzyme to obtain a QS21 intermediate QA-GlcA-Gal.

[0021] Further, in step S1, the E1-CSLM2 enzyme is obtained by sf9 expression of insects.

[0022] Further, in step S1, the mixed reaction also includes: pH 7.5 Tris-HCl, MgCl2 and DMSO.

[0023] Further, in step S1, after mixing, the final concentration of QA quillaic acid is 1 mM, the final concentration of UDP-D-GlcA is 2 mM, the final concentration of Tris-HCl is 50 mM, the final concentration of MgCl2 is 10 mM, and the volume ratio of DMSO is 5%.

[0024] Further, in step S1, the temperature of the mixed reaction is 30°C.

[0025] Further, in step S2, the E2-GmSGT2 enzyme is obtained by expression in E. coli.

[0026] Further, in step S2, the mixed reaction also includes: pH 7.5 Tris-HCl and MgCl2.

[0027] Further, in step S2, after mixing, the final concentration of QA-GlcA intermediate is 1 mM, the final concentration of UDP-D-Gal is 2 mM, the final concentration of Tris-HCl is 50 mM, and the final concentration of MgCl2 is 10 mM.

[0028] Further, the temperature of the mixed reaction is 30°C.

[0029] Example 1 Insect cell expression of E1-CSLM2, E2-UGT73CU3 After the target gene (CSLM2, UGT73CU3) is synthesized, it is cloned into a baculovirus transfer vector (such as pFastBac1) and transformed into DH10Bac competent cells. A white single colony is inoculated in 5 mL of LB medium containing 50 μg / mL of kanamycin and incubated at 37°C for 16 hours. Recombinant Bacmid DNA is extracted using an alkaline lysis method. 5 μg of recombinant Bacmid and 10 μL of Cellfectin II reagent are each diluted in 100 μL of antibiotic-free SFM, mixed, and incubated at room temperature for 20 minutes. Discard the cell culture medium, add the DNA-liposome complex, and incubate at 27°C for 5 hours. Replace the fresh medium and continue to culture for 96 hours to harvest the P1 virus supernatant. Inoculate the P1 virus into sf9 cells (density 2 x 10 6 cells / mL) at a ratio of 1 / 200, and incubate at 27°C for 96 hours. Collect the cell culture medium at 4°C at 7000 rpm for 10 min, collect the precipitate, resuspend the precipitate with PBS, and ultrasonicate (15% 3s / 3s 10 min) to collect the crude enzyme broken solution for use.

[0030] Example 2 E. coli expression of E1-CSLM2, E2-UGT73CU3, E2-GmSGT2 The E1-CSLM2, E2-UGT73CU3, and E2-GmSGT2 genes are synthesized and cloned into the pET28a(+) vector. The recombinant plasmid is transformed into E. coli BL21 (DE3) to obtain the recombinant E. coli BL21 (DE3) containing the E1-CSLM2, E2-UGT73CU3, and E2-GmSGT2 genes. Quillaja saponariaThe CSLM2 gene, UGT73CU3 gene, and GmSGT2 gene derived from soybean were cloned into pMAL-c2X, transformed into 100 μL of BL21(DE3) competent cells, and plated on ampicillin plates for overnight culture. Single colonies were picked and transferred to 10 mL of medium containing 100 μg / mL ampicillin, cultured at 37°C for 6 hours, and then transferred at a 1% (v / v) ratio to 400 mL of LB medium containing 100 μg / mL ampicillin, and cultured at 37°C until OD500. 600 =0.7, add 0.2mM IPTG to a final concentration, incubate overnight at 20℃, and collect bacterial cells by centrifugation at 5000g. Resuspend the bacterial cells in 10 volumes of 20mM Tris-HCl (pH 7.5), 500mM NaCl, and 0.1% Triton X-100 solution, and disrupt the cells using an ultrasonic cell disruptor. After centrifugation at 12000g, aspirate the supernatant, filter through a 0.45μm filter membrane, and purify the fusion protein in one step using an Amylose resin column.

[0031] Example 3 Synthesis of QS21 intermediate QA-GlcA-Gal 1. Catalytic synthesis of QA-GlcA intermediate The catalytic reaction system is shown in Table 1.

[0032] Table 1

[0033] The enzyme activity was detected using the reaction system shown in Table 1, and the purity of the catalytically obtained product was also tested. The results are shown in the table below. Figures 1-3 .

[0034] from Figure 1 It can be seen that, E. coli The E1-CSLM2 protein expressed in *E. coli* showed no activity (substrate molecular weight 661 was not detected) as the reaction did not occur according to mass spectrometry. Figure 2 As can be seen, the E1-CSLM2 protein expressed by SF9 showed that the reaction proceeded normally (the substrate molecular weight was detected as 661).

[0035] Therefore, the E1-CSLM2 protein expressed by SF9 was subjected to a scale-up reaction using the system shown in Table 1. The obtained product was purified by liquid chromatography, lyophilized, and then its purity was determined by HPLC. Figure 3 As can be seen, the purity of the product detected by HPLC is 99% (excluding solvent peaks).

[0036] 2. Catalytic synthesis of QA-GlcA-Gal intermediate The catalytic reaction system is shown in Table 2.

[0037] Table 2

[0038] The reaction was carried out according to the reaction system in Table 2, and the enzyme activity was detected. The results are shown in the table below. Figures 4-6 .

[0039] from Figures 4-6 It can be seen that, E. coli For the expressed E2-GmSGT2 protein, mass spectrometry showed that the reaction proceeded normally (product molecular weight 823 was detected, substrate molecular weight 661 was not detected); for the SF9 expressed E2-UGT73CU3 protein, mass spectrometry showed that the reaction proceeded normally, but the reaction efficiency was low (both product and substrate molecular weights were detected). E. coli If the expressed E2-UGT73CU3 protein does not undergo the reaction (no product molecular weight is detected) according to mass spectrometry, then the protein is inactive.

[0040] Will E. coli The expressed E2-GmSGT2 protein was subjected to a scale-up reaction using the system shown in Table 2. The obtained product was purified by liquid chromatography, lyophilized, and then analyzed to determine the purity and yield of the product. Figure 7 As can be seen, the purity determined by HPLC was 97%, and the product yield of the scaled-up reaction was 81%. We also performed NMR analysis on the product structure, and the results are as follows... Figure 8 As shown.

[0041] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0042] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A process for the preparation of a QS21 intermediate, characterised in that, The method comprises the following steps: S1, mixing QA-betulinic acid, UDP-D-GlcA and E1-CSLM2 enzyme to react and purifying to obtain QA-GlcA intermediate; S2, mixing QA-GlcA intermediate, UDP-D-Gal and E2-GmSGT2 enzyme to react and purifying to obtain QS21 intermediate QA-GlcA-Gal.

2. A process for the preparation of a QS21 intermediate according to claim 1 characterised in that, In step S1, the E1-CSLM2 enzyme is obtained by insect sf9 expression.

3. A process for the preparation of a QS21 intermediate according to claim 1 characterised in that, In step S1, the mixed reaction further comprises pH 7.5 Tris-HCl, MgCl2 and DMSO.

4. A process for the preparation of a QS21 intermediate according to claim 3, characterised in that, In step S1, after mixing, the final concentration of QA-betulinic acid is 1 mM, the final concentration of UDP-D-GlcA is 2 mM, the final concentration of Tris-HCl is 50 mM, the final concentration of MgCl2 is 10 mM, and the volume ratio of DMSO is 5%.

5. A process for the preparation of a QS21 intermediate according to claim 1 characterised in that, In step S1, the temperature of the mixed reaction is 30℃.

6. A process for the preparation of a QS21 intermediate according to claim 1 characterised in that, In step S2, the E2-GmSGT2 enzyme is obtained by E. coli expression.

7. A process for the preparation of a QS21 intermediate according to claim 1 characterised in that, In step S2, the mixed reaction further comprises pH 7.5 Tris-HCl and MgCl2.

8. A process for the preparation of a QS21 intermediate according to claim 7, characterised in that, In step S2, after mixing, the final concentration of QA-GlcA intermediate is 1 mM, the final concentration of UDP-D-Gal is 2 mM, the final concentration of Tris-HCl is 50 mM, and the final concentration of MgCl2 is 10 mM.

9. A process for the preparation of a QS21 intermediate according to claim 1 characterised in that, The temperature of the mixed reaction is 30℃.

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