High-flux glycosidase activity detection system based on micro reaction system

Abstract

A high-flux glycosidase activity detection system based on a miniature reaction system is characterized in that a 4-hydroxybenzoyl hydrazine (pHBH) color development method is adopted, a 200 microliters miniature reaction liquid system is combined, an enzyme activity reaction is completed through a PCR instrument, absorbance is measured through a microplate reader, and rapid and accurate calculation of glycosidase activity is achieved. The method has the characteristics of simplicity and convenience in operation, miniaturization of a reaction system, high detection sensitivity and the like, and is particularly suitable for activity screening and detection of large-scale glycosidase samples.

Description

Technical Field

[0001] The invention relates to the field of enzyme activity determination, and in particular to a method for realizing efficient and accurate determination of glycosidase activity by utilizing a PCR instrument and a micro-reaction system. Background Art

[0002] Glycosidase is an important enzyme that can hydrolyze glycosidic bonds and is widely used in the food industry, bioenergy, medicine and biotechnology. Accurate determination of glycosidase activity is crucial for the production, application and research of glycosidase. With the rapid development of biotechnology, especially in the field of enzyme engineering and metabolic engineering, the demand for high-throughput screening and activity determination methods is becoming increasingly urgent.

[0003] At present, the commonly used methods for determining glycosidase activity mainly include: 1. Spectrophotometry: such as the 3,5-dinitrosalicylic acid (DNS) method, which reflects enzyme activity by detecting reducing sugars in enzymatic hydrolysis products. This method is simple and rapid, but traditional operations often require a large amount of reagents, and the temperature control using a water bath is not accurate enough, and it is difficult to achieve high-throughput detection.

[0004] 2. High performance liquid chromatography (HPLC): It has high resolution and accuracy, but the operation is complicated, the equipment is expensive, and the sample processing is time-consuming, so it is not suitable for large-scale screening.

[0005] 3. Fluorescence and chemiluminescence methods: Using fluorescent or chemiluminescent substrates, they have high sensitivity, but the reagents are expensive and the analysis conditions are harsh, and it is also difficult to achieve high-throughput detection.

[0006] These traditional methods have the following shortcomings when conducting large-scale glycosidase activity screening: 1. Complicated operation: Most methods require complex sample pretreatment and multi-step operations, which are time-consuming and prone to errors.

[0007] 2. High reagent consumption: Traditional methods usually require milliliter-level reaction systems, resulting in high reagent costs.

[0008] 3. Difficulty in high throughput: Most methods have difficulty processing a large number of samples simultaneously, which limits their application in areas such as enzyme engineering.

[0009] 4. High equipment requirements: Some methods such as HPLC require expensive specialized equipment, which increases the cost of testing.

[0010] 5. Different sensitivity and accuracy: The sensitivity and accuracy of different methods vary greatly, making it difficult to meet diverse testing needs.

[0011] In view of the above problems, it is of great practical significance to develop a simple, rapid, accurate, efficient and high-throughput detection method for glycosidase activity determination. This can not only improve the efficiency of glycosidase activity determination, but also provide strong support for research in the fields of enzyme engineering, metabolic engineering, etc., and promote the development of related industries. Summary of the invention

[0012] The present invention aims to provide a high-throughput glycosidase activity assay method. The method aims at the demand for rapid and efficient detection of glycosidase activity, adopts a 200-microliter micro-reaction system, combines the pHBAH colorimetric method, accurately controls the enzymatic reaction and colorimetric reaction conditions in a PCR instrument, and uses an enzyme marker to measure the absorbance, thereby ensuring the detection accuracy while realizing the automated detection of large quantities of glycosidase samples.

[0013] Compared with the traditional milliliter system, the 200 microliter micro-reaction system used in the present invention has significant advantages: miniaturization can shorten the diffusion path of enzyme molecules and substrate molecules, effectively accelerate the rate of enzymatic reaction, and reduce the difference caused by the temperature gradient in the reaction system. With the help of the PCR instrument, the reaction temperature and duration of each stage can be flexibly set, thereby optimizing the enzymatic reaction and color reaction process, and ensuring uniform reaction conditions between each microwell. At the same time, the micro-system greatly reduces the amount of reagents used, thereby reducing the experimental cost and being suitable for high-throughput applications. The principle of the present invention is based on the redox reaction between pHBAH and glycoside products: when glycosidase catalyzes glycoside substrates to generate reducing glycoside products, it can be colored with pHBAH under alkaline conditions, and the color intensity (absorbance at 410 nm) is positively correlated with the amount of reducing sugar generated. By matching this absorbance value with the glycoside product standard curve, the product concentration of the enzymatic reaction can be accurately calculated, and then the glycosidase activity value can be calculated.

[0014] The present invention is achieved through the following technical solutions: A high-throughput glycosidase activity detection method based on a micro-reaction system adopts the pHBAH colorimetric method, combined with a 200-microliter micro-reaction liquid system, using a PCR instrument to perform enzymatic reaction and colorimetric reaction, and measuring the absorbance with an enzyme marker to quickly and accurately calculate glycosidase activity.

[0015] The 200 microliter micro-reaction liquid system is: 150 μL pHBAH solution, 40 μL glycoside substrate solution, and 10 μL glycosidase solution. Miniaturization allows the enzyme and substrate to be fully mixed in a small space, thereby rapidly catalyzing the reaction under uniform temperature control conditions and performing high-sensitivity colorimetric detection with pHBAH.

[0016] The specific steps include: (1) Reaction system preparation •Use 200 μL micro-reaction liquid system, including pHBAH solution, glycoside substrate solution and glycosidase solution. The small volume system can significantly reduce reagent consumption, and the protein and substrate are mixed more fully in the reaction.

[0017] (2) Temperature and time control •Use the PCR instrument to control the enzymatic reaction and color development reaction, heat the temperature to the target range accurately and timely (for example, 50°C for enzymatic reaction and 100°C for color development), and strictly control the reaction time. The PCR instrument can provide a consistent temperature control environment for each well in the microplate, making high-throughput detection highly reproducible.

[0018] (3) Enzyme-catalyzed reaction • pHBAH is specifically designed for the detection of reducing sugars. In the enzymatic reaction stage, glycosidase catalyzes the production of reducing sugars from glycoside substrates and reacts at 50°C for 30 minutes in a mixture of 40 μL substrate and 10 μL enzyme solution. Rapid cooling to 4°C after the reaction helps terminate the reaction and stabilize the generated product.

[0019] (4) Color development and determination • Add 150 μL of pHBAH solution to each well and react for 10 minutes in a 100°C water bath or PCR instrument. Reducing sugars undergo redox reactions with pHBAH in an alkaline environment to form a brown-red product with a characteristic absorption peak at 410 nm.

[0020] •Use a microplate reader to measure the absorbance at 410 nm, and use the standard curve regression equation prepared with the glycoside product standard to calculate the reducing sugar content of each sample and then estimate the enzyme activity.

[0021] Based on the above-mentioned micro-reaction system and its mechanism, the present invention can quickly realize closed-loop control from enzymatic reaction to color development reaction on the same platform, and can automatically obtain glycosidase activity mechanics data, which is suitable for large-scale enzyme activity determination needs in scientific research and production fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a schematic diagram of the method flow of the detection process of the present invention; Figure 2 Comparison of relative enzyme activity between microliter system and milliliter system; Figure 3 The standard curve equation of Example 1; Figure 4 This is a graphical representation of the glycosidase (pectinase) activity of Example 1. DETAILED DESCRIPTION

[0023] The reagents required for use in the present invention include: galacturonic acid (glycoside product) standard solution, phosphate buffer solution (PBS), pHBAH solution, and corresponding glycoside substrate solution.

[0024] The present invention requires the use of instruments: a multi-channel pipette, a PCR instrument, and an enzyme-labeling instrument.

[0025] The micro-reaction system used: 150 μL pHBAH solution, 40 μL substrate, 10 μL enzyme reaction solution.

[0026] The present invention is further described below in conjunction with the accompanying drawings (embodiments): The specific steps of the high-throughput glycosidase activity detection method based on the micro-reaction system of the present invention are as follows (see Appendix Figure 1 ): a. Sample preparation: Dissolve glycosidase and galacturonic acid (glycoside product) in PBS buffer, and pHBAH in NaOH-sodium citrate buffer (0.4 M NaOH, 0.1 M sodium citrate). The concentration of the glycosidase dissolved in PBS buffer is 2 mg / mL.

[0027] The concentration of the galacturonic acid (glycoside product) dissolved in PBS buffer is 10 mg / mL.

[0028] The pHBAH is dissolved in deionized water at a concentration of 1 mg / mL 。

[0029] b. Preparation of standard curve: A known galacturonic acid (glycoside product) standard solution is reacted with pHBAH, the absorbance at 410 nm is measured with an enzyme reader, and a standard curve equation is drawn; the amount of the galacturonic acid (glycoside product) standard solution used is: 0, 0.1, 0.2, 0.3, 0.4, 0.5 mg.

[0030] c. Enzymatic reaction: Mix the glycosidase solution and glycoside substrate solution in a 96-well PCR plate, react at 50°C for 30 minutes, and quickly cool to 4°C after the reaction. The mixing ratio of the glycosidase solution and the glycoside substrate solution is 10 μL:40 μL.

[0031] d. Color reaction: Add 150 μL of pHBAH solution to each well of the PCR plate, mix well and react at 100°C for 10 minutes; e. Absorbance determination: Use an ELISA reader to measure the absorbance at 410 nm, calculate the reducing sugar content in each sample based on the standard curve, and then calculate the glycosidase activity based on the definition of unit enzyme activity U. After testing the micro system, there is no difference in activity between the micro system and the milliliter system (see Appendix Figure 2 ).

[0032] Example 1 1. Preparation of standard curve: react the prepared known concentration of galacturonic acid (glycoside product) standard solution with pHBAH. After the reaction, use an enzyme marker to measure the absorbance at 410 nm. Draw the standard curve equation according to the concentration of galacturonic acid (glycoside product) and the corresponding absorbance value; the linear regression equation is: y=1.0907x+0.0325, R 2 = 0.9869, x is the detection value at OD 410 nm, y is the concentration of galacturonic acid (glycoside product) (see Appendix Figure 3 ).

[0033] 2. Glycosidase E104 enzyme activity detection: Add 10 μL of prepared glycosidase solution and 40 μL of glycoside substrate solution to a 96-well PCR plate, mix well, react at 50 °C for 30 minutes, and quickly take out the plate from the PCR instrument after the reaction is completed, and cool it on ice; add 150 μL of pHBAH solution to each well of the PCR plate, mix well, place the PCR plate in the PCR instrument, and react at 100 °C for 10 minutes; then use a microplate reader to measure the absorbance at 410 nm, and the three repeated values ​​are 0.1316, 0.1343, and 0.1328, respectively.

[0034] 3. According to the standard curve, calculate the content of galacturonic acid (glycoside product) in each sample. Then, under specific conditions, one enzyme activity unit (U) is defined as the amount of enzyme required to generate 1 μmoL galacturonic acid in 1 min. The corresponding glycosidase (pectinase) activities are calculated to be 6666.66 U / g, 7230.658 U / g, and 7317.9656 U / g (see Appendix Figure 4 ).

Claims

1. A high-throughput glycosidase activity detection system based on a micro-reaction system, characterized in that: The 4-hydroxybenzoyl hydrazide (pHBAH) colorimetric method was used in combination with a 200-μl micro-reaction liquid system. The enzyme activity reaction was completed using a PCR instrument, and the absorbance was measured using a microplate reader to achieve rapid and accurate calculation of glycosidase activity.

2. The high-throughput glycosidase activity detection system based on a micro-reaction system according to claim 1, characterized in that: The total reaction system is a 200 microliter micro-reaction liquid system, and a high-throughput device is used to complete heating, mixing and color development to complete the detection reaction.

3. The high-throughput glycosidase activity detection system based on a micro-reaction system according to claim 1, characterized in that: The method includes: 1) Using a 200 μL micro-reaction liquid system, including pHBAH solution, glycoside substrate solution, and glycosidase solution; 2) Use a PCR instrument to control the temperature and time of the enzymatic reaction and the color development reaction; 3) Use 4-hydroxybenzoyl hydrazide (pHBAH) to react with the glycoside product, and measure the absorbance at 410 nm using an enzyme reader; 4) Calculate the corresponding enzyme activity of glycosidase based on the linear relationship regression equation of the glycoside product standard solution.

4. The high-throughput glycosidase activity detection system based on a micro-reaction system according to claim 3, characterized in that: The specific steps are as follows: a. Sample preparation: Dissolve glycosidase and glycoside product in PBS buffer, and pHBAH in NaOH-sodium citrate buffer (0.4 M NaOH, 0.1 M sodium citrate). ； b. Preparation of standard curve: react the known concentration of glycoside product standard solution with pHBAH, measure the absorbance at 410 nm with an enzyme reader, and draw the standard curve equation; c. Enzymatic reaction: Add glycosidase solution and glycoside substrate solution to a 96-well PCR plate, react at 50°C for 30 minutes, and cool rapidly after the reaction. d. Color reaction: Add 150 μL of pHBAH solution to each well of the PCR plate, mix well and react at 100°C for 10 minutes; e. Absorbance determination: Use a microplate reader to measure the absorbance value at 410 nm, calculate the reducing sugar content in each sample according to the standard curve, and then calculate the glycosidase activity according to the definition of unit enzyme activity U.

5. The high-throughput glycosidase activity detection system based on a micro-reactor system according to claim 4, characterized in that: In step a, the concentration of glycosidase dissolved in PBS buffer is 1-10 mg / mL.

6. The high-throughput glycosidase activity detection system based on a micro-reactor system according to claim 4, characterized in that: In step a, the concentration of the glycoside product dissolved in PBS buffer is 5-15 mg / mL to prepare a glycoside product standard solution.

7. The high-throughput glycosidase activity detection system based on a micro-reaction system according to claim 4, characterized in that: In step a, pHBAH is dissolved in NaOH-sodium citrate buffer (0.4 M NaOH, 0.1 M sodium citrate) at a concentration of 1-2 mg / mL 。 8. The high-throughput glycosidase activity detection system based on a micro-reactor system according to claim 4, characterized in that: In step c, the mixing ratio of the glycosidase solution and the glycoside substrate solution is 10 μL:40 μL.

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