CN107254506A - The assay method of the enzyme activity of cysteine dioxygenase in a kind of marine organisms body - Google Patents

Abstract

The invention belongs to the technical field of biological detection, and in particular relates to a method for measuring the enzyme activity of cysteine dioxygenase in marine organisms. solution, under appropriate reaction conditions, the added cysteine is converted into cysteine sulfinic acid, and a suitable derivatization reagent is added to the reaction solution to measure the amount of cysteine sulfinic acid generated, and then the cysteine is calculated The enzyme activity of dioxygenase is obtained by dissolving o-phthalaldehyde in methanol, adding ethanethiol to the derivatization reagent, and then constant volume with sodium borate buffer, which can accurately, concisely and safely determine the half The enzyme activity of the cysteine dioxygenase has low detection cost, and can also be used for the determination of the enzyme activity of the cysteine dioxygenase in other animals, and has universal applicability.

Description

A method for measuring the enzyme activity of cysteine dioxygenase in marine organisms

technical field

The invention belongs to the technical field of biological detection, in particular to a method for measuring the enzyme activity of cysteine dioxygenase in marine organisms.

Background technique

Cysteine dioxygenase COD is a mononuclear non-heme metalloprotease found in animal cells, especially abundant in liver cells, and plays a key role in metabolism and biotransformation. Cysteine dioxygenase catalyzes the oxidation reaction of cysteine Cys to generate cysteine sulfinic acid, which is the first step in the catabolic process of cysteine, and hypotaurine is generated in the next oxidation process, And further converted into taurine. Taurine is a sulfur-containing non-protein amino acid that exists in a free state in animals and is an active substance that regulates the normal physiological activities of the body. It has a wide range of physiological functions and can enhance the antioxidant capacity of cells, protect cardiomyocytes, improve Nerve conduction and visual function, prevention of cardiovascular disease, and improvement of endocrine status are widely used clinically in the treatment of a series of diseases such as cardiovascular disease, diabetes, digestive tract disease, nervous system disease, and eye disease. Main organs, and marine organisms, especially marine fish and shellfish are widely distributed. However, the biosynthetic pathway of taurine in animals is relatively complicated, especially compared with mammals, although the content of taurine in marine organisms is very rich, the mechanism of the biosynthetic pathway is still unclear. Therefore, taking the cysteine dioxygenase in the main pathway of taurine as the object, the activity of cysteine dioxygenase in different types of marine organisms is evaluated by establishing a rapid and sensitive analysis method, which is useful for further research in the future. The synthetic pathway of taurine in marine organisms plays an important role, but there is no research on the enzymatic activity of cysteine dioxygenase.

Chinese patent CN201410369496X, the patent name is a method for measuring the activities of three deiodinases in fish tissue, and the application date is July 31, 2014. After a period of incubation, measure the radioactive intensity of the iodide ions removed by the deiodinase, and calculate the enzymatic activity of the deiodinase through the formula to determine three types of deiodinase ID1, ID2, and ID3. However, this method has specific applicability and is suitable for the determination of the activity of deiodinase, and the determination of the radioactive intensity of iodide ions has high requirements on the instrument, so it is not suitable for the determination of the enzyme activity of cysteine dioxygenase.

Contents of the invention

For cysteine dioxygenase, it is of great significance to clarify the synthetic mechanism of taurine, but there is no research on the enzyme activity of cysteine dioxygenase at present. The purpose of the present invention is to provide a The method for measuring the enzyme activity of cysteine dioxygenase in marine organisms can accurately, concisely and safely measure the enzyme activity of cysteine dioxygenase in marine organisms, and the detection cost is low, and it can also be used The determination of the enzyme activity of cysteine dioxygenase in other animals has universal applicability.

The present invention provides following technical scheme:

A method for measuring the enzyme activity of cysteine dioxygenase in marine organisms, isolating a solution containing cysteine dioxygenase from tissue samples of marine organisms, adding The cysteine is converted into cysteine sulfinic acid, the amount of cysteine sulfinic acid generated is measured, and then the enzyme activity of cysteine dioxygenase is calculated.

In the method for measuring the enzyme activity of cysteine dioxygenase in marine organisms of the present invention, first the tissue samples of marine organisms are processed, and the solution containing cysteine dioxygenase is separated, and then the cysteine dioxygenase is used to Cystine is used as a substrate, cysteine is quantitatively added to the solution containing cysteine dioxygenase, and converted into cysteine sulfinic acid by cysteine dioxygenase, and cysteine The amount of sulfonic acid is used to calculate the enzyme activity of cysteine dioxygenase, where the enzyme activity unit U is defined as the amount of enzyme required to catalyze the generation of 1 μg of cysteine sulfinic acid per minute, so the formula for calculating the enzyme activity is: EA=M/T, where EA refers to the enzyme activity U, M refers to the mass μg of cysteine sulfinic acid, and T refers to the reaction time min. In this way, by converting cysteine that is compatible with cysteine dioxygenase, the enzyme activity of cysteine dioxygenase can be calculated accurately and quantitatively, and it is not only applicable to marine organisms, but also to general animal body.

As an improvement of the method of the present invention, the determination of the amount of cysteine sulfinic acid generated comprises the following steps, first adding a suitable derivatization reagent to the solution containing cysteine sulfinic acid, and then determining the amount of derivatization by high performance liquid chromatography. concentration of the substance. Cysteine sulfinic acid is an intermediate in the oxidation of cysteine in vivo, which has poor stability and short existence time. The stability of cysteine sulfinic acid can be improved by derivatization reagents to improve the accuracy of detection.

As an improvement of the method of the present invention, the derivatization reagent is prepared through the following process: dissolve 0.01g of o-phthalaldehyde in 1mL of methanol, then add 0.01mL of ethanethiol to mix, and then use 0.4mol /L of sodium borate buffer to 10mL to obtain the derivatization reagent. Amino acid derivative reagents include phenyl isothiocyanate and monosulfonyl chloride reagents, but phenyl isothiocyanate is highly toxic and harmful to the chromatographic column, while monosulfonyl chloride reagents are expensive, while o-phthalaldehyde is safe and Convenient and low cost.

As an improvement of the method of the present invention, the assay method of the enzyme activity of cysteine dioxygenase in the above-mentioned marine organism comprises the following steps:

(1) Washing, homogenizing, centrifuging and retaining the supernatant of the tissue sample of the marine organism;

(2) Add 1 to 1.2 times the volume of cysteine solution and 0.2 to 0.24 times the volume of sodium sulfide solution to the supernatant of a quantitative volume, then shake and react at 35 to 39°C for 1 to 3 hours to obtain a reaction solution, and The reaction solution was boiled to inactivate the enzyme activity for 5-10 minutes;

(3) Add an equal volume of derivatization reagent to the reaction solution of quantitative volume, heat and shake to react, and filter to obtain the test solution;

(4) establish the standard curve of the concentration and peak area of cysteine sulfinic acid through high performance liquid chromatography;

(5) the test solution is analyzed by high performance liquid chromatography and obtains the concentration of cysteine sulfinic acid by the standard curve in the step (4);

(6) Calculate the enzyme activity of cysteine dioxygenase from the concentration of cysteine sulfinic acid in step (5).

The cysteine dioxygenase in the supernatant fully converts cysteine into cysteine sulfinic acid in the presence of sodium sulfide, and establishes the relationship between the concentration and absorbance of cysteine sulfinic acid by high performance liquid chromatography. standard curve, and then calculate the amount of cysteine sulfinic acid converted in the reaction solution. Among them, marine organisms include fish, shellfish, snails, shrimps and crabs, and cephalopods, and their tissue samples include meat, liver, intestines, gills and other tissues.

As an improvement of the method of the present invention, the preparation process of the supernatant in step (1) is as follows: the tissue samples of marine organisms are washed and soaked in 2 to 3 times the volume of phosphate buffered saline, and the phosphate buffered saline The temperature is 0-8°C, and then it is homogenized by a homogenizer for 25-35 minutes and centrifuged at 2-6°C, and the speed of the centrifuge is 8000-10000rpm/min. Fully isolate and maintain cysteine dioxygenase activity in tissue samples of marine organisms.

As an improvement of the method of the present invention, the oscillation frequency in step (3) is 100-140 rpm/min, the reaction temperature is 35-39° C., the reaction time is 1-5 min, and the pore size of the filter membrane is 0.22 μm. If the reaction time is short, the reaction between cysteine sulfinic acid and the derivatization reagent will be incomplete, and if the reaction time is long, the derivative product of cysteine sulfinic acid will be decomposed, resulting in test deviation.

As an improvement of the method of the present invention, the standard curve of setting up cysteine sulfinic acid concentration and peak area in step (4) comprises the following steps:

Step 1: prepare the standard solution of cysteine sulfinic acid;

Step 2: adding quantitative derivatization reagents to the standard solution of cysteine sulfinic acid and shaking to obtain a mixed solution;

Step 3: After the mixed solution is filtered through a filter membrane, it is sent to high-performance liquid chromatography for analysis to obtain a standard curve with the concentration of cysteine sulfinic acid as the abscissa and the peak area as the ordinate.

Select the appropriate concentration of the standard solution of cysteine sulfinic acid so that the concentration of cysteine sulfinic acid has a good linear relationship with the peak area, and the coefficient of fit of the obtained linear equation is not less than 0.995.

As an improvement of the method of the present invention, the standard solution of cysteine sulfinic acid is prepared by dissolving quantitative cysteine sulfinic acid in phosphate buffer solution to a constant volume. Same as the preparation of the supernatant.

As an improvement of the method of the present invention, the molar ratio of cysteine sulfinic acid to o-phthalaldehyde in step 2 is 0.5-2:1. The derivatization reaction of cysteine sulfinic acid and o-phthalaldehyde within this ratio range is complete, and the peak area is good.

As an improvement of the method of the present invention, the parameters detected by high performance liquid chromatography are as follows: the chromatographic column is AgilentEclipse XDB-C18, and the mobile phase is methanol and 0.05mol/L sodium acetate aqueous solution, wherein the volume of methanol is 30% to 80%. , the flow rate of the mobile phase is 0.8-2.0 mL/min, and the wavelength is 315 nm. Cysteine sulfinic acid has a good separation effect, the peak eluting time is short, 1.3-1.5 minutes, and the peak shape of cysteine sulfinic acid can be completely separated from adjacent peaks.

The beneficial effects of the present invention are as follows:

The method for measuring the enzyme activity of cysteine dioxygenase in marine organisms of the present invention can accurately, concisely and safely measure the enzyme activity of cysteine dioxygenase in marine organisms, and the detection cost is low, and at the same time It can also be used for the determination of cysteine dioxygenase enzyme activity in other animals, and has universal applicability.

detailed description

The specific embodiments of the present invention will be further described below.

Unless otherwise specified, the raw materials used in the present invention can be purchased from the market or commonly used in the field. If not specified, the methods in the following examples are all conventional methods in the field.

Example 1

A method for measuring the enzyme activity of cysteine dioxygenase in marine organisms, isolating a solution containing cysteine dioxygenase from tissue samples of marine organisms, adding The cysteine is converted into cysteine sulfinic acid, the amount of cysteine sulfinic acid generated is measured, and then the enzyme activity of cysteine dioxygenase is calculated.

Determining the amount of cysteine sulfinic acid produced preferably comprises the steps of first adding a suitable derivatization reagent to the solution containing cysteine sulfinic acid, and then determining the concentration of the derivative by high performance liquid chromatography, wherein the derivatization reagent It is preferably prepared by the following process: dissolve 0.01g of o-phthalaldehyde in 1mL of methanol, then add 0.01mL of ethanethiol to mix, and then dilute to 10mL with 0.4mol/L sodium borate buffer to obtain derivatization reagent.

The assay method of the enzyme activity of cysteine dioxygenase in the above-mentioned marine organism preferably comprises the following steps:

(1) The tissue samples of marine organisms are washed, homogenized, centrifuged and the supernatant is retained. The preferred supernatant preparation process is as follows: the tissue samples of marine organisms are washed and placed in 2 times the volume of phosphate buffer The pH value of the phosphate buffer solution is 7.6, the temperature of the phosphate buffer solution is 0°C, and then it is homogenized by a homogenizer for 30 minutes and centrifuged at 4°C, and the speed of the centrifuge is 9000rpm/min;

(2) Add 1 volume of cysteine solution and 0.2 volume of sodium sulfide solution to a quantitative volume of supernatant, then shake and react at 37°C for 2 hours to obtain a reaction solution, and boil the reaction solution for 5 minutes to inactivate the enzyme. Wherein the concentration of cysteine solution is preferably 50mmol/L, and the concentration of sodium sulfide solution is preferably 50mmol/L;

(3) Add an equal volume of derivatization reagent to a quantitative volume of the reaction solution to mix, then heat and shake at 100 rpm/min for 3 minutes, the reaction temperature is 37°C, and filter through a 0.22 μm filter membrane to obtain the test solution;

(4) establish the standard curve of the concentration and peak area of cysteine sulfinic acid through high performance liquid chromatography, preferably comprise the following steps:

Step 1: Prepare the standard solution of cysteine sulfinic acid, preferably the standard solution of cysteine sulfinic acid is made by dissolving quantitative cysteine sulfinic acid in phosphate buffer solution to constant volume, and the prepared cysteine sulfinic acid The concentration of the standard solution is: 0.005, 0.01, 0.015, 0.025, 0.035, 0.05, 0.1, 0.25, 0.5, unit μg/mL;

Step 2: Add quantitative derivatization reagents to the standard solution of cysteine sulfinic acid and shake to react to obtain a mixed solution. The preferred molar ratio of cysteine sulfinic acid to o-phthalaldehyde is 0.5:1;

Step 3: After the mixed solution is filtered through a filter membrane, it is sent to high-performance liquid chromatography for analysis, and a standard curve with the concentration of cysteine sulfinic acid as the abscissa and the peak area as the ordinate is obtained;

(5) the test solution is analyzed by high performance liquid chromatography and the concentration of cysteine sulfinic acid in the reaction solution is obtained from the standard curve in step (4), and the concentration of cysteine sulfinic acid in the test solution is deducted from the concentration of the blank control group. The result is the concentration of cysteine sulfinic acid in the reaction solution;

(6) Calculate the enzyme activity of cysteine dioxygenase from the concentration of cysteine sulfinic acid in step (5).

The parameters of the preferred HPLC detection of the enzyme activity of cysteine sulfinate decarboxylase in the above-mentioned marine organisms are as follows: the chromatographic column is Agilent Eclipse XDB-C18, and the mobile phase is methanol and 0.05mol/L sodium acetate aqueous solution , wherein methanol accounts for 80% of the volume, and the rest is sodium acetate aqueous solution, the flow rate of the mobile phase is 1.0 mL/min, and the wavelength is 315 nm.

The blank control group was obtained by taking the same volume of supernatant and treating it according to the process of step (2) and step (3), but no cysteine solution was added in step (2).

Example 2

A method for measuring the enzyme activity of cysteine dioxygenase in marine organisms, isolating a solution containing cysteine dioxygenase from tissue samples of marine organisms, adding The cysteine is converted into cysteine sulfinic acid, the amount of cysteine sulfinic acid generated is measured, and then the enzyme activity of cysteine dioxygenase is calculated.

Determining the amount of cysteine sulfinic acid produced preferably comprises the steps of first adding a suitable derivatization reagent to the solution containing cysteine sulfinic acid, and then determining the concentration of the derivative by high performance liquid chromatography, wherein the derivatization reagent It is preferably prepared by the following process: dissolve 0.01g of o-phthalaldehyde in 1mL of methanol, then add 0.01mL of ethanethiol to mix, and then dilute to 10mL with 0.4mol/L sodium borate buffer to obtain derivatization reagent.

The assay method of the enzyme activity of cysteine dioxygenase in the above-mentioned marine organism preferably comprises the following steps:

(1) The tissue samples of marine organisms are washed, homogenized, centrifuged and the supernatant is retained. The preferred supernatant preparation process is as follows: the tissue samples of marine organisms are washed and placed in 2.5 times the volume of phosphate buffer The pH value of the phosphate buffer is 7.6, the temperature of the phosphate buffer is 4°C, and then homogenized by a homogenizer for 25 minutes and centrifuged at 2°C, the centrifuge speed is 8000rpm/min;

(2) Add 1.1 times volume of cysteine solution and 0.22 times volume of sodium sulfide solution to a quantitative volume of supernatant, then shake and react at 35°C for 1 hour to obtain a reaction solution, and boil the reaction solution to inactivate the enzyme activity for 7.5 minutes , wherein the concentration of cysteine solution is preferably 50mmol/L, and the concentration of sodium sulfide solution is preferably 50mmol/L;

(3) Add an equal volume of derivatization reagent to a quantitative volume of the reaction solution and mix, then heat and shake at 120rpm/min for 1min, the reaction temperature is 35°C, and filter through a 0.22μm filter membrane to obtain the test solution;

(4) establish the standard curve of the concentration and peak area of cysteine sulfinic acid through high performance liquid chromatography, preferably comprise the following steps:

Step 1: Prepare the standard solution of cysteine sulfinic acid, preferably the standard solution of cysteine sulfinic acid is prepared by dissolving quantitative cysteine sulfinic acid in phosphate buffer solution to constant volume, and the prepared cysteine sulfinic acid The concentration of the standard solution is: 0.005, 0.01, 0.015, 0.025, 0.035, 0.05, 0.1, 0.25, 0.5, unit μg/mL;

Step 2: Add quantitative derivatization reagents to the standard solution of cysteine sulfinic acid and shake the reaction to obtain a mixed solution. The preferred molar ratio of cysteine sulfinic acid to o-phthalaldehyde is 1:1;

Step 3: After the mixed solution is filtered through a filter membrane, it is sent to high-performance liquid chromatography for analysis, and a standard curve with the concentration of cysteine sulfinic acid as the abscissa and the peak area as the ordinate is obtained;

(5) the test solution is analyzed by high performance liquid chromatography and the concentration of cysteine sulfinic acid in the reaction solution is obtained from the standard curve in step (4), and the concentration of cysteine sulfinic acid in the test solution is deducted from the concentration of the blank control group. The result is the concentration of cysteine sulfinic acid in the reaction solution;

(6) Calculate the enzyme activity of cysteine dioxygenase from the concentration of cysteine sulfinic acid in step (5).

The parameters of the preferred HPLC detection of the enzyme activity of cysteine sulfinate decarboxylase in the above-mentioned marine organisms are as follows: the chromatographic column is Agilent Eclipse XDB-C18, and the mobile phase is methanol and 0.05mol/L sodium acetate aqueous solution , where methanol accounts for 80% of the volume, the rest is sodium acetate aqueous solution, the flow rate of the mobile phase is 2.0mL/min, and the wavelength is 315nm.

The blank control group was obtained by taking the same volume of supernatant and treating it according to the process of step (2) and step (3), but no cysteine solution was added in step (2).

Example 3

A method for measuring the enzyme activity of cysteine dioxygenase in marine organisms, isolating a solution containing cysteine dioxygenase from tissue samples of marine organisms, adding The cysteine is converted into cysteine sulfinic acid, the amount of cysteine sulfinic acid generated is measured, and then the enzyme activity of cysteine dioxygenase is calculated.

Determining the amount of cysteine sulfinic acid produced preferably comprises the steps of first adding a suitable derivatization reagent to the solution containing cysteine sulfinic acid, and then determining the concentration of the derivative by high performance liquid chromatography, wherein the derivatization reagent It is preferably prepared by the following process: dissolve 0.01g of o-phthalaldehyde in 1mL of methanol, then add 0.01mL of ethanethiol to mix, and then dilute to 10mL with 0.4mol/L sodium borate buffer to obtain derivatization reagent.

The assay method of the enzyme activity of cysteine dioxygenase in the above-mentioned marine organism preferably comprises the following steps:

(1) The tissue samples of marine organisms are washed, homogenized, centrifuged and the supernatant is retained. The preferred supernatant preparation process is as follows: the tissue samples of marine organisms are washed and placed in 3 times the volume of phosphate buffer The pH value of the phosphate buffer solution is 7.6, the temperature of the phosphate buffer solution is 8°C, and then it is homogenized by a homogenizer for 35 minutes and centrifuged at 6°C, and the speed of the centrifuge is 10,000rpm/min;

(2) Add 1.2 times volume of cysteine solution and 0.24 times volume of sodium sulfide solution to a quantitative volume of supernatant, then shake and react at 39°C for 3 hours to obtain a reaction solution, boil the reaction solution to inactivate the enzyme activity for 10 minutes, Wherein the concentration of cysteine solution is preferably 50mmol/L, and the concentration of sodium sulfide solution is preferably 50mmol/L;

(3) Add an equal volume of derivatization reagent to a quantitative volume of the reaction solution and mix, then heat and shake at 140rpm/min for 5min, the reaction temperature is 39°C, and filter through a 0.22μm filter membrane to obtain the test solution;

(4) establish the standard curve of the concentration and peak area of cysteine sulfinic acid through high performance liquid chromatography, preferably comprise the following steps:

Step 1: Prepare the standard solution of cysteine sulfinic acid, preferably the standard solution of cysteine sulfinic acid is prepared by dissolving quantitative cysteine sulfinic acid in phosphate buffer solution to constant volume, and the prepared cysteine sulfinic acid The concentration of the standard solution is: 0.005, 0.01, 0.015, 0.025, 0.035, 0.05, 0.1, 0.25, 0.5, unit μg/mL;

Step 2: Add quantitative derivatization reagents to the standard solution of cysteine sulfinic acid and shake to react to obtain a mixed solution. The preferred molar ratio of cysteine sulfinic acid to o-phthalaldehyde is 2:1;

Step 3: After the mixed solution is filtered through a filter membrane, it is sent to high-performance liquid chromatography for analysis, and a standard curve with the concentration of cysteine sulfinic acid as the abscissa and the peak area as the ordinate is obtained;

(5) the test solution is analyzed by high performance liquid chromatography and the concentration of cysteine sulfinic acid in the reaction solution is obtained from the standard curve in step (4), and the concentration of cysteine sulfinic acid in the test solution is deducted from the concentration of the blank control group. The result is the concentration of cysteine sulfinic acid in the reaction solution;

(6) Calculate the enzyme activity of cysteine dioxygenase from the concentration of cysteine sulfinic acid in step (5).

The parameters of the preferred HPLC detection of the enzyme activity of cysteine sulfinate decarboxylase in the above-mentioned marine organisms are as follows: the chromatographic column is Agilent Eclipse XDB-C18, and the mobile phase is methanol and 0.05mol/L sodium acetate aqueous solution , where methanol accounts for 80% of the volume, the rest is sodium acetate aqueous solution, the flow rate of the mobile phase is 0.8mL/min, and the wavelength is 315nm.

The blank control group was obtained by taking the same volume of supernatant and treating it according to the process of step (2) and step (3), but no cysteine solution was added in step (2).

The results of this assay method

1. Deviation

Prepare 3 different concentrations of cysteine sulfinic acid standard solutions, respectively 10 μg/mL, 100 μg/mL, and 500 μg/mL, and then repeat the test 3 times after processing according to step (3). The test results are shown in Table 1.

Table 1

The RSDs of the three concentrations of cysteine sulfinic acid standard samples were 3.06%, 2.94% and 0.84%, respectively, indicating that the method has high precision and good reproducibility.

2. Testing of marine organisms

Select four representative marine organisms such as Penaeus vannamei, swimming crab, large yellow croaker, clam, etc., and detect the enzyme activity of cysteine dioxygenase in its different tissues according to the assay method of the present invention, and the results are shown in the table 2. Not detected means that this part of the tissue cannot be clearly distinguished and separated and cannot be detected.

It can be seen from the table that there is a large difference in the enzyme activity of cysteine dioxygenase in different marine organisms, and the enzyme activity of cysteine dioxygenase in Penaeus vannamei is as high as 32U. In addition, there are differences in the enzyme activity of cysteine dioxygenase in different tissues. The activity of cysteine dioxygenase in the liver of large yellow croaker was significantly higher than that in other tissues, while the activity of cysteine dioxygenase in the gills was the lowest.

Table 2

Claims (10)

1. a method for measuring the enzyme activity of cysteine dioxygenase in marine organisms, is characterized in that, from the tissue sample of marine organisms, isolate the solution containing cysteine dioxygenase, in suitable Under certain reaction conditions, the added cysteine is converted into cysteine sulfinic acid, the amount of cysteine sulfinic acid generated is measured, and then the enzymatic activity of cysteine dioxygenase is calculated. 2. the assay method of the enzymatic activity of cysteine dioxygenase in marine organisms according to claim 1, is characterized in that, the amount of the cysteine sulfinic acid that measures generation comprises the following steps, at first to containing cysteine sulfinic acid A suitable derivatization reagent is added to the reaction solution of sulfinic acid, and then the concentration of the derivative is determined by high performance liquid chromatography. 3. the assay method of the enzymatic activity of cysteine dioxygenase in marine organisms according to claim 2, is characterized in that, described derivatization reagent is prepared through the following process: dissolve in the methanol of 1mL Add 0.01 g of o-phthalaldehyde and 0.01 mL of ethanethiol to mix, and then use 0.4 mol/L sodium borate buffer to make up to 10 mL to obtain a derivatization reagent. 4. the assay method of the enzyme activity of cysteine dioxygenase in marine organisms according to claim 1 or 2 or 3, comprises the following steps: (1) Wash, homogenize and centrifuge the tissue samples of marine organisms and retain the supernatant; (2) Add 1 to 1.2 times the volume of cysteine solution and 0.2 to 0.24 times the volume of sodium sulfide solution to a quantitative volume of supernatant, then shake and react at 35 to 39 °C for 1 to 3 hours to obtain a reaction solution, Boil the reaction solution to inactivate the enzyme activity for 5-10 minutes; (3) Add an equal volume of derivatization reagent to a quantitative volume of the reaction solution, heat and shake to react, and filter to obtain the test solution; (4) Establish a standard curve of cysteine sulfinic acid concentration and peak area by high performance liquid chromatography; (5) Analyze the test solution by high performance liquid chromatography and obtain the concentration of cysteine sulfinic acid from the standard curve in step (4); (6) Calculate the enzyme activity of cysteine dioxygenase from the concentration of cysteine sulfinic acid in step (5). 5. the assay method of the enzymatic activity of cysteine dioxygenase in marine organisms according to claim 4, is characterized in that, The preparation process of the supernatant in step (1) is as follows: the tissue samples of marine organisms are washed and infiltrated in 2 to 3 times the volume of phosphate buffer, the temperature of the phosphate buffer is 0 to 8 ° C, and then Homogenize with a homogenizer for 25 to 35 minutes and centrifuge at 2 to 6 °C, and the speed of the centrifuge is 8000 to 10000 rpm/min. 6. The method for measuring the enzyme activity of cysteine dioxygenase in marine organisms according to claim 4, characterized in that the oscillation frequency in step (3) is 100-140 rpm/min, and the reaction temperature is 35 ～39℃, the reaction time is 1～5min, and the pore size of the filter membrane is 0.22 μm. 7. The method for measuring the enzyme activity of cysteine dioxygenase in marine organisms according to claim 4, wherein the standard curve of cysteine sulfinic acid concentration and peak area established in step (4) comprises The following steps: Step 1: prepare the standard solution of cysteine sulfinic acid; Step 2: adding quantitative derivatization reagents to the standard solution of cysteine sulfinic acid and shaking to obtain a mixed solution; Step 3: After the mixed solution is filtered through a filter membrane, it is sent to high-performance liquid chromatography for analysis to obtain a standard curve with the concentration of cysteine sulfinic acid as the abscissa and the peak area as the ordinate. 8. the assay method of the enzymatic activity of cysteine dioxygenase in marine organisms according to claim 7, is characterized in that, the cysteine sulfinic acid standard solution in the step 1 is through quantitative cysteine sulfinic acid The acid is dissolved in phosphate buffered saline at constant volume. 9. the assay method of the enzymatic activity of cysteine dioxygenase in marine organisms according to claim 7, is characterized in that, in step 2, the mol ratio of cysteine sulfinic acid and o-phthalaldehyde is 0.5～ 2:1. 10. the assay method of the enzymatic activity of cysteine dioxygenase in marine organisms according to claim 4, is characterized in that, the parameter that high performance liquid chromatography detects is as follows: chromatographic column is Agilent Eclipse XDB-C18, flow The phase is methanol and 0.05mol/L sodium acetate aqueous solution, wherein the volume of methanol is 30%-80%, the flow rate of the mobile phase is 0.8-2.0mL/min, and the wavelength is 315nm.