阅读说明：本技术 一种甾体c1,2位脱氢酶酶活检测方法 (Method for detecting enzyme activity of steroid C1, 2-dehydrogenase ) 是由 柳志强 倪叶雯 张博 陈鑫鑫 柯霞 郑裕国 于 2020-11-25 设计创作，主要内容包括：本发明涉及一种甾体C1,2位脱氢酶酶活检测方法。甾体C1,2位脱氢酶能够代谢还原WST-1,同时在PMS的联合作用下生成还原成水溶性的橙黄色甲臢产物,在440nm下测定吸光度,以WST-1还原量表示脱氢酶活性,根据标准曲线计算橙黄色甲臢的生成量,进而求出甾体C1,2位脱氢酶活性。本发明提供了一种氧化还原酶活力检测方法,特异性强、灵敏度高、操作方便、检测时间短、定量准确,适合高通量的甾体C1,2位脱氢酶活性检测。(The invention relates to a method for detecting enzyme activity of steroid C1, 2-dehydrogenase. The steroid C1, 2-dehydrogenase metabolically reduces WST-1 and produces a formazan product reduced to water-soluble orange in combination with PMS, measures the absorbance at 440nm, expresses dehydrogenase activity as the WST-1 reducing amount, calculates the formazan producing amount from the standard curve, and then determines the steroid C1, 2-dehydrogenase activity. The invention provides a method for detecting the activity of oxidoreductase, which has the advantages of strong specificity, high sensitivity, convenient operation, short detection time and accurate quantification and is suitable for high-flux activity detection of steroid C1, 2-dehydrogenase.)

1. A method for detecting activity of a steroid C1, 2-dehydrogenase enzyme, the method comprising:

(1) mixing a proper amount of steroid C1, 2-site dehydrogenase substrate, phenazine methyl sulfate and a WST-1 detection reagent in a buffer solution;

(2) weighing WST-1, adding 95% alcohol for dissolving, diluting with distilled water to constant volume to obtain 0.4% WST-1 solution, and storing in dark place; 0.4% WST-1 solution was pipetted into a volumetric flask and Na was added₂S₂O₄Shaking the powder evenly, and then fixing the volume to the scale by using ethyl acetate; respectively putting 0.25mL, 0.5mL, 1.00mL, 1.50mL and 2.00mL into a 10mL volumetric flask, and fixing the volume to a scale by using ethyl acetate to obtain standard colorimetric series of TF25 mu g, 50 mu g, 100mu g, 150 mu g and 200 mu g, measuring the absorbance of the extract at 440nm by taking a blank as a reference, and drawing a standard curve;

(3) adding a solution to be detected containing steroid C1, 2-site dehydrogenase into a buffer solution, uniformly mixing, placing the solution under the optimum reaction temperature and the optimum reaction pH value of the steroid C1, 2-site dehydrogenase for reacting for 1-4 hours, and detecting the absorbance at 440 nm;

(4) and (5) calculating to obtain the concentration of the steroid C1 and 2-site dehydrogenase in the solution to be detected by contrasting the standard curve of the steroid C1 and 2-site dehydrogenase.

2. The method of claim 1, wherein the steroid C1, dehydrogenase-2 substrate is mixed with a cosolvent and added to the buffer, the cosolvent being one of: DMSO, DMF, Tween 80 and ethanol.

3. The method according to claim 1, wherein the concentration of substrate for steroid C1, dehydrogenase at position 2 in the reaction solution is 10 to 100mM, the concentration of phenazine methosulfate is 5 to 25. mu.M, and the concentration of WST-1 is 10 to 100 mM.

4. The method of claim 1, wherein said steroid C1,2 dehydrogenase substrate is androstenedione.

5. The method according to claim 1, wherein the optimum reaction temperature of the steroid C1, 2-dehydrogenase is 30 ℃ to 40 ℃ and the optimum reaction pH is 8.0 to 10.0.

6. The method according to claim 1, wherein the test solution containing steroid C1, 2-dehydrogenase is a bacterial solution containing steroid C1, 2-dehydrogenase from the phylum actinomycetemcomitans, proteobacteria, or firmicutes.

(I) technical field

The invention relates to a method for detecting enzyme activity of steroid C1, 2-dehydrogenase.

(II) background of the invention

The steroid compound is a special polycyclic terpene compound, usually contains a cyclopentane polyhydrophenanthrene mother nucleus consisting of A, B, C and four D rings, wherein the A, B, C ring is a complete closed six-membered ring, and the D ring is a five-membered ring; steroids are widely found in animals, plant tissues and certain microorganisms. At present, the types of reactions involved mainly include hydroxylation, oxidation, hydrolysis, side chain degradation, dehydrogenation at C1, 2-position, esterification, reduction, isomerization and introduction of other groups, etc.

Sterone C1, dehydrogenase at position 2, is both an inducible enzyme, an intracellular enzyme, and a membrane protein, with its active center anchored to the intracellular membrane. Two hydrophobic amino acid segments in the protein jointly form the transmembrane region of the protein. Has a flavin adenine dinucleotide binding site. The sterone C1, 2-site dehydrogenase can catalyze the C1, 2-site dehydrogenation reaction of A ring in the transformation of steroid microorganisms. The KsdD catalytic reaction mechanism should follow the theory of two-step unimolecular conjugate base elimination (E1 cb): firstly, the ketone group at the C3 position of the 3-ketosteroid substrate interacts strongly with the electrophilic residues of the enzyme, so that the C-2 (beta) hydrogen bond is broken, and the hydrogen atom is removed in the form of proton through a generalized base to form an enolate intermediate or a carbanion intermediate; thereafter, a double bond is formed between the C1 and C2 positions, and at the same time, the hydride is transferred from the C1 position to the flavin coenzyme. After a double bond is introduced into the C1, 2-position of the ring of the steroid compound A, the anti-inflammatory activity of the steroid compound A is multiplied. For example, the anti-inflammatory activity of prednisone acetate generated by the dehydrogenation reaction of cortisone acetate through C1,2 is increased by 3-4 times; at present, the production process of a plurality of clinically important steroid compounds all involves the dehydrogenation reaction of steroid C1,2 of microorganisms, including prednisolone, dexamethasone, paramethasone, betamethasone, triamcinolone, methylprednisolone and other adrenal cortical hormones with anti-inflammatory capability.

The detection of the activity of the oxidoreductase is carried out by adding an artificial acceptor including TTC (2,3, 5-triphenyltetrandrine chloride), resazurin, methylene blue, INT (iodonitrotetraliviolet), etc. and determining the intensity of the dehydrogenation process from the rate of reductive discoloration of the artificial acceptor, among which TTC is most widely used. TTC needs to be added with an organic reagent in the operation process of the dehydrogenase activity detection method, and the solution has light color development, easy fading and long reaction time. WST-1 is a compound similar to MTT, is an upgraded substitute for MTT, and has obvious advantages compared with MTT or other MTT-like products such as XTT, MTS and the like. First, formazan (formazan) that MTT is reduced by some dehydrogenases within the mitochondria is not water-soluble and requires a specific lysis solution to solubilize; while both WST-1 and XTT, MTS produced formazan (formazan) are water soluble, the subsequent solubilization step can be omitted. Second, the formazan (formazan) produced by WST-1 is more soluble than the formazan produced by XTT and MTS. And thirdly, the WST-1 is more stable than XTT and MTS, so that the experimental result is more stable. In addition, WST-1 has a wider linear range and higher sensitivity than MTT, XTT and the like.

At present, the method for testing the activity of the sterone C1, 2-site dehydrogenase mainly comprises two reaction systems of PMS-cytochrome C and PMS-NBT. The PMS-cytochrome C system mainly comprises a PMS-cytochrome C method and a PMS-DCPIP method. The PMS-NBT system is mainly a PMS-NBT method. With the continuous upgrade of the artificial hydrogen acceptor tetrazolium salt in recent years, WST series (such as WST-1 and WST-8) are newly developed. And for some common dehydrogenases (such as succinate dehydrogenase), a related enzyme activity detection system is established by using newly developed tetrazolium salt, and the detection method for the activity of the ketosteroid C1, 2-position dehydrogenase also stays in the tetrazolium salt at the primary stage. Therefore, it is necessary to establish a detection system most suitable for the activity of dehydrogenase at position 2 of sterone C1 aiming at different tetrazolium salts.

Disclosure of the invention

The invention aims to provide a method for detecting the activity of steroid C1, 2-dehydrogenase.

The technical scheme adopted by the invention is as follows:

a method for detecting activity of a steroid C1, 2-dehydrogenase enzyme, the method comprising:

(1) mixing a proper amount of steroid C1, 2-site dehydrogenase substrate, phenazine methyl sulfate and a WST-1 detection reagent in a buffer solution;

(2) weighing WST-1, adding 95% alcohol for dissolving, diluting with distilled water to constant volume to obtain 0.4% WST-1 solution, and storing in dark place; 0.4% WST-1 solution was pipetted into a volumetric flask and Na was added₂S₂O₄Shaking the powder evenly, and then fixing the volume to the scale by using ethyl acetate; respectively putting 0.25mL, 0.5mL, 1.00mL, 1.50mL and 2.00mL into a 10mL volumetric flask,measuring volume to scale with ethyl acetate to obtain standard colorimetric series of TF25 μ g, 50 μ g, 100 μ g, 150 μ g and 200 μ g, measuring absorbance of the extractive solution at 440nm with blank as reference, and drawing standard curve;

(3) adding a solution to be detected containing steroid C1, 2-site dehydrogenase into a buffer solution, uniformly mixing, placing the solution under the optimum reaction temperature and the optimum reaction pH value of the steroid C1, 2-site dehydrogenase for reacting for 1-4 hours, and detecting the absorbance at 440 nm;

(4) and (5) calculating to obtain the concentration of the steroid C1 and 2-site dehydrogenase in the solution to be detected by contrasting the standard curve of the steroid C1 and 2-site dehydrogenase.

The invention utilizes the color change of a WST-1 detection reagent to monitor the activity of the steroid C1, 2-dehydrogenase in real time, and provides a detection method for the activity of the steroid C1, 2-dehydrogenase. A WST-1 detection reagent detection system is selected, and the enzyme activity detection of steroid C1, 2-dehydrogenase shows a good linear dose relationship; through the absorbance value of the reactant at 440nm, the activity evaluation of the steroid C1, 2-position dehydrogenase is carried out.

The reaction principle is as follows: WST-1 is a yellow dye. The steroid C1, 2-dehydrogenase metabolically reduces WST-1 and produces a formazan product reduced to water-soluble orange in combination with PMS, measures the absorbance at 440nm, expresses dehydrogenase activity as the WST-1 reducing amount, calculates the formazan producing amount from the standard curve, and then determines the steroid C1, 2-dehydrogenase activity.

The chemical formula of the WST-1 is as follows:

preferably, the steroid C1, 2-dehydrogenase substrate is mixed with a cosolvent and then added to the buffer, wherein the cosolvent is one of the following: DMSO, DMF, Tween 80 and ethanol.

The concentration of steroid C1, 2-site dehydrogenase substrate in the reaction solution is 10-100 mM, the concentration of phenazine methyl sulfate is 5-25 μ M, and the concentration of WST-1 is 10-100 mM. The detection range of the activity of the ketosteroid C1, 2-dehydrogenase in the solution to be detected is 10 mU/L-100 mU/L. Preferably, the steroid C1, 2-dehydrogenase substrate can be androstenedione or cholesterol, glycocholic acid, taurocholic acid, dioscin, and the like, and is preferably Androstenedione (AD).

The optimum reaction temperature of the steroid C1, 2-site dehydrogenase is 30-40 ℃, and the optimum reaction pH value is 8.0-10.0.

The solution to be tested containing the steroid C1, 2-site dehydrogenase can be a bacterial liquid containing the steroid C1, 2-site dehydrogenase from actinomycetemcomia, proteobacteria or firmicutes, and the like.

The invention has the following beneficial effects: aiming at the defects of cytotoxicity and false positive reaction to medicaments with oxidation resistance in the prior art, the invention provides the method for detecting the activity of the steroid C1, 2-position dehydrogenase, which has the advantages of strong specificity, high sensitivity, convenient operation, short detection time and accurate quantification and is suitable for high-flux activity detection of the steroid C1, 2-position dehydrogenase.

(IV) description of the drawings

FIG. 1 is a graph showing the detection of substrate AD (or product ADD) as a function of reaction time by the liquid phase method in example 1;

FIG. 2 is a standard curve for substrate AD (or product ADD) in the liquid phase method of example 1;

figure 3 is a graph of the absorbance change over reaction time of the MTT method detecting the water-soluble orange yellow formazan product of example 2;

figure 4 is the MTT water-soluble orange yellow formazan product standard curve of example 2;

figure 5 is a graph of the absorbance change over reaction time of the red formazan product following detection of organic solvent reconstitution by the TTC method in example 3;

figure 6 is the red formazan product standard curve following reconstitution of the TTC organic solvent in example 3;

FIG. 7 is a graph showing the change in absorbance with reaction time of Prussian blue detected by the potassium ferricyanide [ K3Fe (CN)6] method in example 4;

FIG. 8 shows potassium ferricyanide [ K3Fe (CN)6] in example 4]With Fe³⁺Acting to generate a Prussian blue standard curve;

figure 9 is a graph of the absorbance change over reaction time of the WST-1 method of example 5 in the detection of the water-soluble orange yellow formazan product;

figure 10 is the WST-1 water-soluble orange yellow formazan product standard curve of example 5.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

example 1: detection of ketosteroid C1, 2-position dehydrogenase activity by HPLC method

The specific method comprises the following steps: the bacterial cells were induced with IPTG for 16 hours (E.coli BL21(DE3) from Beijing Quanyujin Biotech Co., Ltd., the same below), centrifuged at 8000r/min at 4 ℃ for 10min to collect the cells, washed twice with 25mL of PBS buffer solution (pH7.2), and 5g of wet cells were weighed and suspended in 25mL of this buffer solution. Carrying out ultrasonic crushing with 40% power in ice bath, and working for 30min at a time interval of 10S for 5S. Centrifuging at 12,000r/min for 15min to obtain supernatant. Taking 0.2mL of supernatant, 2mmol/L of PMS, 1g/L of AD, 500mM of Tris-HCl buffer solution with pH7.5, and enabling the total reaction system to reach 10 mL. Reacting at 30 ℃, sampling at certain intervals, and adding HCl to terminate the reaction.

The liquid phase method comprises the following steps: Agilent-C18 column (particle, X), mobile phase: methanol: water ═ 6.5:3.5(v/v) and flow rate 1 mL/min. The UV absorbance was measured at 254nm and the column temperature was 35 ℃. The graph of the substrate AD as a function of the reaction time by the liquid phase method is shown in FIG. 1.

Drawing a standard curve: 1g/L of substrate AD (5% DMSO for assisting dissolution) is prepared by using a reaction system and is respectively diluted to 0.1-0.9 g/L. And (3) carrying out HPLC detection, wherein the detection method comprises the following steps: agilent C18 column, mobile phase: methanol: water 6.5:3.5(v/v), flow rate 1mL/min, wavelength 254nm, column temperature 35 ℃. The resulting standard curve is shown in FIG. 2.

The disadvantage of the HPLC method is that it has an "out-of-column effect" and is not sufficiently sensitive, and if the flow pattern of the mobile phase changes, any diffusion and retention of the separated material will significantly lead to broadening of the chromatographic peak and a decrease in column efficiency.

Example 2: MTT method for detecting activity of C1, 2-dehydrogenase of sterone

The reaction principle is as follows: MTT is a yellow dye. The dehydrogenase can metabolize and reduce MTT, blue (or blue-violet) water-insoluble formazan is generated under the action of PMS, and the formazan is dissolved and developed through the action of isopropanol. The dehydrogenase activity was determined by measuring the absorbance at 570nm, expressing the dehydrogenase activity as the MTT reducing amount, and calculating the amount of blue (or bluish-violet) precipitated formazan from the standard curve.

Preparation of MTT standard curve: 0.4g of MTT is weighed, 500 microliter of 95 percent alcohol is added for dissolution assistance, and the volume of distilled water is adjusted to 100mL to prepare 0.4 percent MTT solution. The 0.4% MTT solution was stored protected from light. Aspirate 250. mu.L of 0.4% MTT solution into a 10mL volumetric flask and add a little Na₂S₂O₄And (3) uniformly shaking the powder to generate a bluish purple crystal Triphenylmethane (TF), fixing the volume to a scale with ethyl acetate, and uniformly shaking the powder. Respectively placing 0.25mL, 0.5mL, 1.00mL, 1.50mL and 2.00mL in a 10mL volumetric flask, and metering to a scale with ethyl acetate to obtain standard colorimetric series of TF25 μ g, 50 μ g, 100 μ g, 150 μ g and 200 μ g, and measuring absorbance of the extract at 570nm with a blank as a reference to draw a standard curve, which is shown in FIG. 4.

The specific method comprises inducing bacteria solution for 16 hours with IPTG, centrifuging at 4 deg.C and 8000r/min for 10min to collect thallus, washing twice with 25mL PBS buffer solution with pH7.2, weighing 5g wet thallus, and suspending in 25mL of the buffer solution. Carrying out ultrasonic crushing with 40% power in ice bath, and working for 30min at a time interval of 10S for 5S. Centrifuging at 12,000r/min for 15min to obtain supernatant. Taking 0.1mL of supernatant, 0.1mg of PMS, 1.5mg of MTT, 0.1mL of AD with the concentration of 1g/L and 50mM of Tris-HCl buffer solution with the pH value of 7.5, wherein the total reaction system reaches 3 mL. Reacting for 60min at 30 ℃, adding HCl to terminate the reaction, detecting the change condition of the absorbance value under the wavelength of 570nm, and defining the enzyme quantity required for reducing 1 mu mol MTT in one minute as an enzyme activity unit U. The XTT method detects the absorbance change profile of the water-soluble orange-yellow formazan product over reaction time see figure 3.

And (4) conclusion: this method has the disadvantage that blue (or bluish violet) water-insoluble formazan is generated, requiring organic solvent redissolution.

Example 3: TTC method for detecting activity of sterone C1, 2-site dehydrogenase

The reaction principle is as follows: TTC (2,3, 5-triphenyltetrazolium chloride) is colorless, an artificial acceptor of hydrogen, which is reduced to Triphenylmethane (TF) as a red crystal during cellular respiration. Extracting with organic solvent (such as toluene, ethyl acetate, chloroform, acetone or ethanol). Measuring absorbance of the extract at 485nm, expressing dehydrogenase activity as TTC reducing amount, and calculating generation amount of triphenylmethane meal according to the standard curve to obtain dehydrogenase activity.

Preparing a TTC standard curve: 0.4g of TTC is weighed, 500 microliter of 95 percent alcohol is added for dissolution assistance, and distilled water is added to the volume of 100mL to prepare 0.4 percent TTC solution. 0.4% TTC solution is protected from light and cannot be used after being reddened. Aspirate 250. mu.L of 0.4% TTC solution into a 10mL volumetric flask and add a little Na₂S₂O₄And (3) powder is shaken up to generate red crystal Triphenylmethane (TF), and then ethyl acetate is used for fixing the volume to the scale and shaking up. Respectively placing 0.25mL, 0.5mL, 1.00mL, 1.50mL and 2.00mL in a 10mL volumetric flask, and metering to a scale with ethyl acetate to obtain standard colorimetric series of TF25 μ g, 50 μ g, 100 μ g, 150 μ g and 200 μ g, and taking blank as reference, measuring absorbance of the extract at 485nm to draw a standard curve, see FIG. 6.

The specific method comprises inducing bacteria solution for 16 hours with IPTG, centrifuging at 4 deg.C and 8000r/min for 10min to collect thallus, washing twice with 25mL PBS buffer solution with pH7.2, weighing 5g wet thallus, and suspending in 25mL of the buffer solution. Carrying out ultrasonic crushing with 40% power in ice bath, and working for 30min at a time interval of 10S for 5S. Centrifuging at 12,000r/min for 15min to obtain supernatant. Taking 0.1mL of supernatant, adding 0.002mL of PMS with the concentration of 1M/L, 0.5mL of TTC solution with the concentration of 1mM/L, 0.1mL of AD with the concentration of 1g/L and 50mM Tris-HCl buffer solution with the pH value of 7.5, and leading the total reaction system to reach 1 mL. Reacting for 60min at 30 ℃, adding HCl to terminate the reaction, detecting the change condition of an absorbance value under the wavelength of 485nm, and defining the enzyme quantity required for reducing 1 mu mol of TTC in one minute as an enzyme activity unit U. The TTC reduction method detects the red formazan product absorbance change profile with catalytic reaction time see figure 5.

And (4) conclusion: the method has the defects that the red crystal Triphenylmethane (TF) is generated, and the redissolution of an organic solvent is needed, so that the operation is complicated.

Example 4: potassium ferricyanide [ K ]₃Fe(CN)₆]Detection of sterone C1, 2-site dehydrogenase activity by reduction method

The reaction principle is as follows: potassium ferricyanide [ K ]₃Fe(CN)₆]Is red fixedThe body salt is soluble in water, and the aqueous solution has yellow green fluorescence. Dehydrogenases catalyze the reaction of reducing potassium ferricyanide to potassium ferrocyanide, K₄Fe(CN)₆Then with Fe³⁺The Prussian blue is generated by the action, the light absorption value is measured at the wavelength of 700nm to detect the generation amount of the Prussian blue, the generation amount is used as the reducing force of dehydrogenase, and the higher the light absorption value is, the stronger the activity of the dehydrogenase is.

K₃Fe(CN)₆And (3) standard curve preparation: weighing K₃Fe(CN)₆0.4g, adding distilled water to constant volume of 100mL to prepare 0.4% K₃Fe(CN)₆And (3) solution. Aspirate 250. mu.L of 0.4% K₃Fe(CN)₆The solution was placed in a 10mL volumetric flask and a little Na was added₂S₂O₄Powdering, shaking to obtain K₄Fe(CN)₆Then distilled water is added to the constant volume to the scale mark, and the mixture is shaken up. Respectively putting 0.25mL, 0.5mL, 1.00mL, 1.50mL and 2.00mL into a 10mL volumetric flask, and fixing the volume to the scale with distilled water to obtain K₄Fe(CN)₆25 μ g, 50 μ g, 100 μ g, 150 μ g, 200 μ g of standard colorimetric series with addition of Fe³⁺The Prussian blue is generated by the action, the blank is used as a reference, the absorbance of the extraction liquid is measured at 700nm, and a standard curve is drawn, which is shown in figure 8.

The specific method comprises inducing bacteria solution for 16 hours with IPTG, centrifuging at 4 deg.C and 8000r/min for 10min to collect thallus, washing twice with 25mL PBS buffer solution with pH7.2, weighing 5g wet thallus, and suspending in 25mL of the buffer solution. Carrying out ultrasonic crushing with 40% power in ice bath, and working for 30min at a time interval of 10S for 5S. Centrifuging at 12,000r/min for 15min to obtain supernatant. Collecting 0.1mL of supernatant, 0.1mg of PMS, and 0.21mL of 61Mm/L potassium ferricyanide [ K ]₃Fe(CN)₆]0.1mL of 1g/L AD, 500mM Tris-HCl buffer pH7.5, and 3mL of total reaction system. Reacting at 30 deg.C for 60min, adding HCl to terminate the reaction, detecting the change of absorbance at 700nm, and reducing 1 μmol potassium ferricyanide [ K3Fe (CN)6] in one minute]The amount of enzyme required is defined as one unit of enzyme activity U. Potassium ferricyanide [ K3Fe (CN)6]The diagram of the absorbance change of the prussian blue along with the reaction time is shown in figure 7.

In conclusion, this method has the disadvantage of having a toxic effect on the enzyme. Under the same system, the low-concentration potassium ferricyanide reduces the enzyme activity by at least half; high concentrations of potassium ferricyanide inactivate the enzyme, making the detection results inaccurate.

Example 5: method for detecting activity of C1, 2-dehydrogenase of sterone by using WST-1 method

The reaction principle is as follows: when two hydrogen atoms on a substrate (AD) are removed by ketosteroid C1 and 2-site dehydrogenase to generate ADD, FAD + receives the hydrogen atoms and is converted into FADH2, the latter reduces a chromogenic reagent through a hydrogen-transferring body PMS, and the chromogenic reagent has a characteristic absorption peak under ultraviolet wavelength, so that the enzyme activity of the ketosteroid C1 and 2-site dehydrogenase can be detected by detecting the change of an absorbance value under the ultraviolet wavelength. Therefore, the ketosteroid C1, 2-dehydrogenase can be detected by adding an artificial acceptor. The detection reagent adopts a WST-1 detection reagent system, and has an absorption value substance at 440nm generated by an electron acceptor PMS.

And (3) preparing a WST-1 standard curve: weighing WST-10.4 g, adding 500 μ L95% (v/v) alcohol for dissolution, and adding distilled water to 100mL to obtain 0.4% (w/w) WST-1 solution. 0.4% WST-1 solution was stored protected from light. Aspirate 250. mu.L of 0.4% WST-1 solution into a 10mL volumetric flask and add a little Na₂S₂O₄And (3) powder is shaken up to generate soluble Triphenylmethane (TF), and then ethyl acetate is used for fixing the volume to the scale and shaking up. Respectively placing 0.25mL, 0.5mL, 1.00mL, 1.50mL and 2.00mL in a 10mL volumetric flask, and metering to a scale with ethyl acetate to obtain standard colorimetric series of TF25 μ g, 50 μ g, 100 μ g, 150 μ g and 200 μ g, and measuring absorbance of the extract at 440nm with blank as reference to draw a standard curve, which is shown in FIG. 10.

The specific method comprises inducing bacteria solution for 16 hours with IPTG, centrifuging at 4 deg.C and 8000r/min for 10min to collect thallus, washing twice with 25mL PBS buffer solution with pH7.2, weighing 5g wet thallus, and suspending in 25mL of the buffer solution. Carrying out ultrasonic crushing with 40% power in ice bath, and working for 30min at a time interval of 10S for 5S. Centrifuging at 12,000r/min for 15min to obtain supernatant. Taking 0.1mL of supernatant, 25 μ M/L PMS, 40 μ M/L WST-1, 50 μ M/L AD, 50mM Tris-HCl buffer solution with pH7.5, and making the total reaction system reach 1 mL. Reacting for 60min at 30 ℃, adding HCl to terminate the reaction, detecting the change condition of the absorbance value under the wavelength of 440nm, and defining the enzyme quantity required for reducing 1 mu mol of WST-1 in one minute as an enzyme activity unit U.

The WST-1 method detects the absorbance change profile of the water-soluble orange yellow formazan product with reaction time see figure 9.

The advantages and disadvantages of the various processes are seen in table 1 below:

and (4) conclusion: in the presence of an electronic coupling reagent phenazine methosulfate, a WST-1 detection reagent can be reduced by ketosteroid C1, 2-site dehydrogenase to generate a highly water-soluble orange-yellow formazan product (formazan), ultraviolet absorption is realized at 440nm, the specificity is strong (the result for steroid 1, 2-site dehydrogenase and substrates thereof is more stable), the sensitivity is high (OD600 can be detected under 0.2, other methods have inaccurate detection results), the operation is convenient, the detection time is short, the quantification is accurate, and the method is suitable for high-flux detection of the activity of the ketosteroid C1, 2-site dehydrogenase.