A high-throughput screening method for cyclodextrin transferase catalyzing the synthesis of L-ascorbic acid-2-glucoside

Abstract

The present invention relates to a high-throughput screening method for a cyclodextrin transferase that catalyzes the synthesis of L-ascorbic acid-2-glucoside, comprising the steps of: adding β-cyclodextrin and ascorbic acid to the culture system of the strain to be screened, adjusting the pH to 5.0-6.0 for catalytic reaction; removing the strain after the catalytic reaction, adding an oxidant for oxidation reaction; adding a color developer of L-ascorbic acid-2-glucoside for color development after the oxidation reaction. The present invention removes unreacted ascorbic acid through a pre-oxidation strategy, then adds a substance that can develop color with L-ascorbic acid-2-glucoside, reacts under certain conditions, and can be easily detected in an ELISA instrument after the reaction, so as to accurately quantify the content of L-ascorbic acid-2-glucoside in the reaction solution system, bringing convenience to high-throughput screening of mutant strains.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a high-throughput screening method for a cyclodextrin transferase for catalyzing the synthesis of L-ascorbic acid-2-glucoside. Background Art

[0002] At present, the world leader in the synthesis of L-ascorbic acid-2-glucoside (AA-2G) is the Japanese company "Hayashihara Co., Ltd.", which uses CGTase, or "cyclodextrin transferase," which is used on a large scale in the industry for the synthesis of β-cyclodextrin. In order to improve the efficiency of catalytic synthesis of AA-2G, it is necessary to screen for cyclodextrin transferases with higher activity. For the screening of this enzyme, the commonly used methods are the "phenolphthalein color change circle method" and the "methyl orange method." The principle is that starch is converted into cyclodextrin by bacteria producing CGTase enzymes, and phenolphthalein or methyl orange is wrapped with cyclodextrin to fade the solution, so as to achieve the purpose of evaluating enzyme activity. Obviously, this method does not directly evaluate the synthetic activity of AA-2G, and cannot truly reflect the level of using the enzyme to synthesize AA-2G.

[0003] Therefore, how to obtain a method for directly evaluating the activity of cyclodextrin transferase in catalyzing the synthesis of AA-2G and apply it to high-throughput screening to quickly and accurately obtain beneficial mutant strains from a saturated mutation library is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0004] The purpose of the present invention is to provide a high-throughput screening method based on direct evaluation of the activity of cyclodextrin transferase catalyzing the synthesis of L-ascorbic acid-2-glucoside. In order to achieve the purpose of the present invention, the following technical scheme is proposed:

[0005] One aspect of the present invention relates to a high-throughput screening method for a cyclodextrin transferase that catalyzes the synthesis of L-ascorbic acid-2-glucoside, comprising the following steps:

[0006] Adding β-cyclodextrin and ascorbic acid to the culture system of the strain to be screened, adjusting the pH to 5.0-6.0 to carry out a catalytic reaction;

[0007] After the catalytic reaction, the strain is removed and an oxidant is added to carry out an oxidation reaction;

[0008] After the oxidation reaction, a color developing agent of L-ascorbic acid-2-glucoside was added for color development.

[0009] The present invention uses an oxidation reaction to oxidize unreacted ascorbic acid, while L-ascorbic acid-2-glucoside is not destroyed in the process. The color is developed by a color developer of L-ascorbic acid-2-glucoside, so that the content of L-ascorbic acid-2-glucoside can be quantitatively detected.

[0010] In a preferred embodiment of the present invention, the added weight ratio of β-cyclodextrin to ascorbic acid is 1:1-2.

[0011] In a preferred embodiment of the present invention, the oxidant is hydrogen peroxide, ozone, Fe 3+ A combination of one or more of salt and air or oxygen; preferably, the oxidant is hydrogen peroxide. By using hydrogen peroxide as the oxidant, its redox product has less impact on the reaction system.

[0012] In a preferred embodiment of the present invention, the color developer is phosphomolybdic acid, which is heated to 70-90° C. for color development. AA-2G is decomposed into ascorbic acid and glucose during the color development process, so that the phosphomolybdic acid appears blue-green, and since there is no free ascorbic acid in the system, it does not affect the color development process; moreover, it has been verified that glucose itself does not interfere with the reaction system.

[0013] In a preferred embodiment of the present invention, the absorbance is detected by an enzyme marker after the color development reaction, and the content of L-ascorbic acid-2-glucoside is quantitatively determined by the absorbance data.

[0014] The present invention removes unreacted ascorbic acid through a pre-oxidation strategy, then adds a substance that can develop color with AA-2G, reacts under certain conditions, and can be conveniently detected on an enzyme marker after the reaction, thereby accurately quantifying the content of AA-2G in the reaction solution system, bringing convenience to high-throughput screening of mutant strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the sample spectrum at 0 hours;

[0016] Figure 2 This is the sample spectrum after 2.0 hours of reaction;

[0017] Figure 3 This is the sample spectrum after 7.0 hours of reaction;

[0018] Figure 4 This is the sample spectrum after 24.0 hours of reaction;

[0019] Figure 5 This is a photo of the solution before heating;

[0020] Figure 6 This is a photo of the solution after heating;

[0021] Figure 7 is the relationship between AA-2G consumption and absorbance;

[0022] Figure 8This is the photo before the heating reaction;

[0023] Fig. 9 This is a photo taken after heating at 80°C. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with specific examples, but the following examples are only used to illustrate the contents of the present invention rather than to limit the present invention, and therefore any changes within the meaning and scope equivalent to the claims of the present invention should be deemed to be included in the scope of the claims.

[0025] Example 1

[0026] (1) Catalytic reaction: Escherichia coli was cultured in a 96-well plate until OD = 7, 50 μL of β-cyclodextrin (50 g / L) and 50 μL of ascorbic acid (50 g / L) were added, and the pH was adjusted to 5.0-5.5 with 0.1 M sodium hydroxide. The plate was kept at 37° C. and shaken at 200 rpm for 24 h.

[0027] (2) Pre-oxidation reaction: After removing the bacteria by centrifugation, 10 μl of 30% hydrogen peroxide was added for pre-oxidation reaction. The temperature was raised to 50°C and the reaction was continued for 24 h. The experimental results are shown in the figure. Figure 1-4 As shown, with the extension of time, L-ascorbic acid-2-glucoside in the system remained almost unchanged, while ascorbic acid gradually decreased and almost disappeared in the 24-hour sample.

[0028] (3) Color reaction: Add 80 μL of 47.62 mg / g phosphomolybdic acid and keep at 80°C for 1 hour. Transfer to a microplate reader and detect the absorbance at 660 nm. The greater the absorbance at this wavelength, the more AA-2G is generated by the system. Liquid chromatography was also performed on the samples after the color reaction. The experimental results are shown in Table 1. The experimental results show that the method of the present invention can achieve detection results similar to those of liquid chromatography, but the method of the present invention is simpler and less expensive than liquid chromatography, which facilitates high-throughput screening of mutant strains.

[0029] Detection conditions of liquid chromatography: chromatographic column: C18, 150*4.6mm, 5um; mobile phase: A: 10mMol / L dodecyltrimethylammonium chloride aqueous solution, pH3.0; B: methanol (volume ratio of A:B is 85:15); column temperature: 35°C, wavelength: 260nm; flow rate: 1ml / min.

[0030] Table 1: Comparison of ELISA results and HPLC results in the colorimetric experiment

[0031] Liquid phase test results (mg / g) Color development results using phosphomolybdic acid (mg / g) 0.924117333 0.959276 1.108176554 1.101726

[0032] Verification experiment 1: Phosphomolybdic acid heating color reaction experiment

[0033] (1) Add 5 g of AA-2G with concentrations of 0.1 mg / g, 0.25 mg / g, 0.5 mg / g, 0.75 mg / g and 1 mg / g respectively into a test tube, and add 10 g of 4.762 mg / g phosphomolybdic acid solution. The solution before heating is shown in the following figure. Figure 5 shown.

[0034] (2) Keep the reaction at 80℃ for 1 hour. The solution after the reaction is shown in the following figure. Figure 6 As shown, it can be seen that by adding phosphomolybdic acid and heating, AA-2G can react with phosphomolybdic acid to cause a color difference, which can even be recognized by the naked eye.

[0035] (3) The heated reaction samples were transferred to an ELISA instrument and the absorbance at 660 nm was detected. The experimental results are shown in Table 2 and Figure 7 As shown, the experimental results show that the phosphomolybdic acid spectrophotometer method has good linearity and accuracy and can be used for high-throughput screening of strains.

[0036] Table 2 Absorbance detection results

[0037]

[0038] Verification experiment 2: Glucose interference experiment on the reaction system:

[0039] Phosphomolybdic acid mother solution (47.62 mg / g): Dissolve 1 g of phosphomolybdic acid in 20 g of water;

[0040] Vc mother solution (2 mg / g): Take 0.1 g VC and add 50 mL water to dissolve.

[0041] 1. The mother solution of phosphomolybdic acid is diluted 10 times, and the solution is light yellow;

[0042] 2. Add ①Vc mother solution (about 10 drops), ②anhydrous glucose solid (0.03g), and ③AA-2G solid (0.014g) to the diluted phosphomolybdic acid solution in sequence; the result is as follows: Figure 8 As shown, solution ③ instantly changed from light yellow to blue-green; while the glucose and AA-2G systems remained light yellow without any change;

[0043] 3. After heating at 80℃, the results are as follows Fig. 9 As shown, No. ① changes from light yellow to blue-green, No. ② remains light yellow, and No. ③ remains blue-green. The experimental results show that AA-2G and glucose will not interfere with the reaction system.

[0044] The above describes the preferred embodiments of the present invention, but it is not intended to limit the present invention. Those skilled in the art may make improvements and changes to the embodiments disclosed herein without departing from the scope and spirit of the present invention.

Claims

1. A method for directly evaluating the activity of cyclodextrin transferase in a strain to catalyze the synthesis of L-ascorbic acid-2-glucoside, It is characterized in that The steps include: Adding β-cyclodextrin and ascorbic acid to the culture system of the strain to be screened, adjusting the pH to 5.0-6.0 to carry out a catalytic reaction; After the catalytic reaction, the strain is removed and an oxidant is added to carry out an oxidation reaction; After the oxidation reaction, phosphomolybdic acid is added and heated to 70-90° C. for color development to quantitatively detect the content of L-ascorbic acid-2-glucoside; The oxidant is hydrogen peroxide.

2. The method according to claim 1, wherein the weight ratio of β-cyclodextrin to ascorbic acid is 1:1-2.

3. The method according to claim 1, wherein the absorbance is detected by an enzyme marker after the color development reaction, and the content of L-ascorbic acid-2-glucoside is quantitatively determined by the absorbance data.

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