阅读说明：本技术 经改善的用于检测磷脂酶c的酶活力的方法及产品 (Improved method and product for detecting enzymatic activity of phospholipase C ) 是由 潘思惠 顾思天 牛其文 于 2019-09-18 设计创作，主要内容包括：本申请提供了经改善的用于检测磷脂酶C的酶活力的方法以及用于检测磷脂酶C的酶活力的产品。使用本申请的方法或产品可以更精确地定量酶活力,检测成本低,便于高通量地进行筛选或检测,能够满足规模化工业生产的检测需求。(The present application provides improved methods for detecting enzymatic activity of phospholipase C and products for detecting enzymatic activity of phospholipase C. The method or the product can be used for more accurately quantifying the enzyme activity, has low detection cost, is convenient for high-throughput screening or detection, and can meet the detection requirement of large-scale industrial production.)

1. A method for detecting enzymatic activity of phospholipase C, comprising the steps of:

1) enzymatically hydrolyzing a phospholipid, preferably Phosphatidylcholine (PC) or Phosphatidylethanolamine (PE), with phospholipase C in the reaction system;

2) adding a Tris-HCl buffer and then a water-immiscible organic solvent, preferably chloroform, to the reaction system;

3) after phase separation, contacting the aqueous solution with a phosphatase, then with molybdic acid or molybdate, preferably ammonium molybdate, under acidic conditions, preferably pH 4.8, and further reacted with a reducing agent, preferably ascorbic acid;

4) and measuring an absorbance value at 600-810nm, and calculating the enzyme activity of the phospholipase C by using a standard curve of phosphomolybdic blue detection.

2. A method for screening a strain expressing phospholipase C comprising the steps of:

1) enzymatically hydrolyzing a phospholipid with the phospholipase C in a reaction system;

2) adding a Tris-HCl buffer solution to the reaction system and then adding a water-immiscible organic solvent;

3) after phase separation, contacting the aqueous solution with phosphatase, then with molybdic acid or molybdate under acidic conditions, and further reacting with a reducing agent;

4) the absorbance was measured at 600 and 810 nm.

3. A method for detecting the enzymatic activity of phospholipase C in a fermentation broth comprising the steps of:

1) contacting the fermentation broth with a phospholipid in a reaction system and allowing the phospholipase C to enzymatically hydrolyze the phospholipid;

2) adding a Tris-HCl buffer solution to the reaction system and then adding a water-immiscible organic solvent;

3) after phase separation, contacting the aqueous solution with phosphatase, then with molybdic acid or molybdate under acidic conditions, and further reacting with a reducing agent;

4) and measuring an absorbance value at 600-810nm, and calculating the enzyme activity of the phospholipase C by using a standard curve of phosphomolybdic blue detection.

4. The method of any one of claims 1-3, wherein the Tris-HCl buffer is 0.15-1.0M at pH8.0-9.0, or 0.25M at pH 9.0.

5. The method according to any one of claims 1 to 4, wherein the reaction system in step 1) comprises an active metal ion required for the phospholipase C reaction, such as Ca²⁺And/or Zn²⁺And Triton x-100, and the pH of the reaction system is 4.0-6.0, 4.4-5.8, 4.4-4.8, or 4.8.

6. The method according to any one of claims 1 to 5, wherein the concentration of the phospholipid in the reaction system of step 1) is 0.3% to 6% (w/v), 0.5% to 3%, 0.8% to 3%, or 1%.

7. The method according to claim 5 or 6, wherein the Triton x-100 concentration in the reaction system of step 1) is 10 mM-100 mM, 10 mM-80 mM, 20 mM-80 mM, 40 mM-80 mM, or 40 mM.

8. The method of any one of claims 1-7, wherein the reaction temperature for enzymatic hydrolysis of phospholipids by phospholipase C is 25-55 ℃, 30-50 ℃, 35-45 ℃ or 40 ℃.

9. A product for detecting enzymatic activity of phospholipase C, comprising: a first vessel comprising a phospholipid, a second vessel comprising a Tris-HCl buffer, a third vessel comprising a water-immiscible organic solvent, a fourth vessel comprising a phosphatase reaction system solution, a fifth vessel comprising a molybdenum blue chromogenic reactant solution, and optionally a sixth vessel comprising a phospholipase C reaction system solution, preferably the Tris-HCl buffer is 0.15-1.0M Tris-HCl buffer at pH8.0-9.0, or 0.25M Tris-HCl buffer at pH 9.0.

10. A method of providing a phospholipase C product, comprising detecting the enzymatic activity of the phospholipase C using the method of any of claims 1 to 8 or the product of claim 9, preferably the method of providing a phospholipase C product further comprises one or more steps selected from dilution, shaking, standing, centrifugation, split charging, and colorimetry.

Technical Field

The present application relates to the field of phospholipase C preparations, and more particularly, to improved methods for detecting enzymatic activity of phospholipase C and related products and methods for providing phospholipase C products.

Background

Crude oils obtained from oil stocks usually contain a certain amount of gums, mainly phospholipids, the presence of which can seriously affect the quality of the finished oil. Therefore, degumming in the vegetable oil refining process is very important, and degumming is a difficult problem in the refining process. Compared with the traditional chemical degumming method, the continuously improved enzymatic degumming has the advantages of mild reaction conditions, wide applicability, acid and alkali chemicals and energy consumption saving in production, less pollutant discharge, almost no wastewater generation and the like, can effectively improve the refining rate, and has very wide application prospect.

Enzyme bag for enzymatic degummingPhospholipase A₁(PLA₁) Phospholipase A₂(PLA₂) And phospholipase c (plc), the sites of action of the three phospholipases are shown below.

Wherein X represents choline, ethanolamine, inositol, serine, hydrogen, etc.

PLA can specifically hydrolyze ester bonds on Sn-1 site or Sn-2 site of glycerophospholipid to generate lysophospholipid and free fatty acid with better hydrophilicity. The PLC is capable of specifically hydrolyzing the glycerophosphoester bond at position C3 of glycerophospholipids to produce diglycerides and organophosphates (e.g., phosphocholine, phosphoinositide, phosphoethanolamine, phosphoserine, etc.). The PLC can not hydrolyze neutral oil such as Triglyceride (TAG), Diglyceride (DAG) and Monoglyceride (MAG), so that other byproducts can not be generated when the PLC is used for degumming, the diglyceride obtained by hydrolyzing phospholipid can be dissolved in the oil and can be used as a component of oil together with the TAG without being removed, the adverse effect on detection indexes of products is avoided, the oil yield is obviously improved, and the economic benefit is increased.

Phospholipase C can be classified by substrate type into nonspecific phospholipase C, phosphatidylcholine-specific phospholipase C, phosphatidylinositol-specific phospholipase C, phosphatidylethanolamine-specific phospholipase C, phosphatidic acid-specific phospholipase C, and the like. Phospholipase C is widely existed in animals, plants and microorganisms, and PLC derived from animals and plants is generally located on cell membranes, has a complex structure, belongs to endogenous phospholipase C, and is difficult to separate. The phospholipase C derived from microorganisms is generally simple in structure and can be isolated from bacteria or fungi, including but not limited to Clostridium perfringens, Clostridium bifidum, Bacillus cereus, Bacillus mycoides, Bacillus thuringiensis, Listeria monocytogenes, Pseudomonas aeruginosa, Pseudomonas fluorescens, Staphylococcus, Acinetobacter baumannii, Streptomyces, Burkholderia, Streptomyces shijokutaensis, Candida albicans, Saccharomyces cerevisiae, etc.

The reported methods for detecting the activity of PLC enzyme include a yolk plate method, an acid-base titration method, a turbidity analysis method, an NPPC method, an HPLC method, a radioisotope labeling method, a fluorescent labeling method, a chemiluminescence detection method, a spectrophotometry method, an inorganic phosphorus determination method and the like.

(1) The yolk plate method is to judge the enzyme activity by the diameter of milky halo formed around the oxford cup after hydrolyzing lecithin in the yolk agar plate by PLC, and can only be used for qualitative detection of PLC activity, and has more factors influencing the size of the milky halo.

(2) The acid-base titration method utilizes the characteristic that the PLC hydrolyzes lecithin to release acidic phosphorylcholine to react with NaOH, and calculates the enzyme activity by measuring the amount of sodium hydroxide consumed by enzyme reaction under a certain condition and in a certain time.

(3) The turbidity analysis method reflects the enzyme activity by measuring the turbidity change of the lecithin emulsion before and after PLC hydrolysis, and the method has convenient operation, relatively long reaction time and lack of specificity.

(4) The NPPC method uses a structural analogue of phosphatidylcholine, namely p-Nitrophosphocholine (NPPC), as a substrate for PLC reaction, p-nitrophenol generated by PLC hydrolysis has a maximum absorption value at 410nm, and the enzyme activity can be calculated by a colorimetric method.

(5) The HPLC method is used for separating and measuring the product of PLC hydrolysis of hydrophilic phospholipid, and has high accuracy, high requirements on technical level of equipment and experimenters and slow detection speed, so that the application is limited.

(6) The radioactive isotope labeling method is to label various phospholipid substances by using different radioactive isotopes, and measure the quantity of isotope-labeled PLC reaction hydrolysate by using a scintillation counter, so as to calculate the enzyme activity of a sample. The method has the advantages of high sensitivity, accurate and reliable measurement result, strong specificity, higher requirements on instruments and equipment and higher cost.

(7) The strategies adopted by a fluorescence labeling method and a chemiluminescence detection method are similar, a special phospholipid substrate or an analogue is labeled by utilizing a fluorescent group, a luminescent group and the like respectively, and a product is detected by utilizing fluorescence detection or negative light sensitization after PLC hydrolysis.

(8) The strategy of the spectrophotometry is similar to that of the NPPC method, the PLC specifically hydrolyzes the phosphorothioate structural analogue of the dioctanoyl lecithin to generate the thioglycerol diester, the phosphorothioate structural analogue and the thioglycerol diester can generate color forming reaction, and the enzyme activity can be defined by using the change of absorbance.

(9) The molybdenum blue phosphorus determination method is characterized in that a phosphoric acid compound generated by hydrolysis of a phospholipid substrate by PLC is hydrolyzed by alkaline phosphatase to release inorganic phosphate ions, a complex formed by the inorganic phosphate ions and molybdate is reduced by ascorbic acid under an acidic condition to be blue, and the maximum absorbance value is obtained at the position of 680-800nm, so that the enzyme activity of the PLC is calculated.

The molybdenum blue phosphorus determination method has high sensitivity, but involves two enzyme reactions and a color reaction, so the requirements on reaction conditions and a reaction system are high. Although different documents report methods for measuring the activity of the PLC enzyme by using phosphomolybdic blue, the methods have certain defects. For example, in the phospholipase C activity measurement methods reported by Markus and Uwe (A. Durban, Markus & Bornschele, Uwe. an improved assay for the determination of phosphoipase C activity [ J ]. European Journal of Lipid Science and Technology,2007,109: 469-. Since the optimum pH range of alkaline phosphatase is usually 8-10, the method can significantly inhibit the activity of acid phospholipase C. Furthermore, Krug et al (Edward L.Krug, Nancy J.Truesdale, Claudia Kent.A Simplified assay for phospholipases C [ J ]. Analytical Biochemistry,1979,97(1):43-47) reported that EDTA was selected to terminate the phospholipase C reaction, but alkaline phosphatase usually requires activation by zinc and divalent magnesium or calcium ions, and thus EDTA as a metal chelator also inhibits alkaline phosphatase activity. The hydrochloric acid solution employed in the process of Markus and Uwe (A. Durban, Markus & Bornscheler, Uwe. an improved assay for the determination of phosphoipase C activity [ J ]. European Journal of Lipid Science and Technology,2007,109: 469-.

In addition, for phosphomolybdic blue color development, although the introduction of blank samples can counteract the influence of additives such as ions on color development, the introduction of blank samples cannot counteract the influence of residual phospholipid substrates (the substrate content of the blank samples is different from the residual substrate content of the experimental samples); in Krug et al (Edward L.Krug, Nancy J.Truesdale, Claudia Kent.A amplified assisted for phospholipases C [ J ]. Analytical Biochemistry,1979,97(1):43-47) and Hergenerotherand Martin (Paul J.Hergenerotherand F.Martin, Determination of the kinetic parameters for phospholipases C (Bacillus cereus) on differential phospholipases use a chromatographic analysis on the quantification of organic phospholipases [ J ]. Analytical Biochemistry,1997,251 (1)) reported that SDS coloration methods could not be used to prevent precipitation of the proteins, and that precipitation of the phospholipids could not be prevented.

Therefore, there is still a need for a detection method that can accurately quantify the enzymatic activity of phospholipase C.

Disclosure of Invention

In one aspect, the present application provides a method for detecting enzymatic activity of phospholipase C, comprising the steps of:

1) enzymatically hydrolyzing a phospholipid with phospholipase C in a reaction system;

2) adding a Tris-HCl buffer solution to the reaction system and then adding a water-immiscible organic solvent;

3) after phase separation, contacting the aqueous solution with phosphatase, then with molybdic acid or molybdate under acidic conditions, and further reacting with a reducing agent;

4) and measuring an absorbance value at 600-810nm, and calculating the enzyme activity of the phospholipase C by using a standard curve of phosphomolybdic blue detection.

In one aspect, the present application provides a method for screening a strain expressing phospholipase C, comprising the steps of:

1) enzymatically hydrolyzing a phospholipid with the phospholipase C in a reaction system;

2) adding a Tris-HCl buffer solution to the reaction system and then adding a water-immiscible organic solvent;

3) after phase separation, contacting the aqueous solution with phosphatase, then with molybdic acid or molybdate under acidic conditions, and further reacting with a reducing agent;

4) the absorbance was measured at 600 and 810 nm.

In another aspect, the present application provides a method for detecting the enzymatic activity of phospholipase C in a fermentation broth, comprising the steps of:

1) contacting the fermentation broth with a phospholipid in a reaction system to cause the phospholipase C to enzymatically hydrolyze the phospholipid;

2) adding a Tris-HCl buffer solution to the reaction system and then adding a water-immiscible organic solvent;

3) after phase separation, contacting the aqueous solution with phosphatase, then with molybdic acid or molybdate under acidic conditions, and further reacting with a reducing agent;

4) and measuring an absorbance value at 600-810nm, and calculating the enzyme activity of the phospholipase C by using a standard curve of phosphomolybdic blue detection.

In yet another aspect, the present application provides a product for detecting enzymatic activity of phospholipase C, comprising: a first vessel containing a phospholipid, a second vessel containing a Tris-HCl buffer, a third vessel containing a water-immiscible organic solvent, a fourth vessel containing a phosphatase reaction system solution, and a fifth vessel containing a molybdenum blue chromogenic reactant solution.

In a further aspect, the present application provides a method of providing a phospholipase C product, comprising detecting the enzyme activity of phospholipase C using the method of the above aspect, or a product for detecting the enzyme activity of phospholipase C.

The method for detecting the enzyme activity of phospholipase C of the present application can be applied to various phospholipids as substrates of phospholipase C, and can allow various types of phospholipase C to measure the enzyme activity thereof at its optimum temperature and pH. Without being bound by any theory, the detection method of the present application uses a Tris-HCl buffer as a termination reagent, which can effectively inhibit the reaction of phospholipase C without affecting the subsequent enzymatic hydrolysis reaction of phosphatase. The detection method of the present application also uses a water-immiscible organic solvent for extraction, thereby eliminating interference of residual phospholipid substrate with color development. The detection method can more accurately and stably detect the enzyme activity of the phospholipase C, is simple and convenient to operate, has low detection cost, is convenient for screening and detecting at high flux, can meet the detection requirement of large-scale industrial production, and has very important production practice significance.

Drawings

FIG. 1 shows the enzyme activities of phospholipase C enzymes at different reaction temperatures.

FIG. 2 shows the enzyme activities of phospholipase C enzymes at different reaction pH.

FIG. 3 shows the effect of phospholipid substrate concentration on the enzymatic activity of phospholipase C.

FIG. 4 shows the effect of Triton x-100 concentration on the enzymatic activity of phospholipase C.

FIG. 5 shows the relationship between phospholipase C protein concentration and absorbance.

FIG. 6 shows the effect of phospholipid substrate on phosphomolybdenum blue coloration.

Detailed Description

The inventors of the present application found that enzymatic hydrolysis of a single phospholipid or a complex phospholipid using a phospholipase C (e.g., a solution containing a phosphatidylcholine-specific phospholipase C, a phosphatidylinositol-specific phospholipase C, a phosphatidylethanolamine-specific phospholipase C, a phosphatidylserine-specific phospholipase C, and/or a phosphatidic acid-specific phospholipase C, including, but not limited to, a phosphatidylcholine-specific phospholipase C, a phosphatidylinositol-specific phospholipase C, a phosphatidylethanolamine-specific phospholipase C, a fermentation broth of a phosphatidylserine-specific phospholipase C, and/or a phosphatidic acid-specific phospholipase C, a fermentation supernatant, an enzyme broth, etc.), then adding Tris-HCl buffer solution as a termination reagent to effectively inhibit the reaction of the phospholipase C, then adding an organic solvent which is not miscible with water to extract, adding the phase-separated aqueous phase solution into a phosphatase reaction system to react, then contacting the reaction solution with molybdic acid or molybdate (including but not limited to ammonium molybdate, sodium molybdate and potassium molybdate) solution under an acidic condition, further reducing the reaction solution by a reducing agent, and detecting the absorbance at 600-810nm, thus being capable of more accurately and stably detecting the enzyme activity of the phospholipase C. The detection method can be directly used for detecting the enzyme activity of the phospholipase C in the fermentation liquor, and can be used for quickly screening the bacterial strain expressing the phospholipase C or quickly selecting the fermentation conditions.

Thus, in one aspect, the present application provides a method for detecting enzymatic activity of phospholipase C, comprising the steps of:

1) enzymatically hydrolyzing a phospholipid with phospholipase C in a reaction system;

2) adding a Tris-HCl buffer solution to the reaction system and then adding a water-immiscible organic solvent;

3) after phase separation, contacting the aqueous solution with phosphatase, then with molybdic acid or molybdate under acidic conditions, and further reacting with a reducing agent;

4) and measuring an absorbance value at 600-810nm, and calculating the enzyme activity of the phospholipase C by using a standard curve of phosphomolybdic blue detection.

In another aspect, the present application provides a method for screening a strain expressing phospholipase C, comprising the steps of:

1) enzymatically hydrolyzing a phospholipid with the phospholipase C in a reaction system;

2) adding a Tris-HCl buffer solution to the reaction system and then adding a water-immiscible organic solvent;

3) after phase separation, contacting the aqueous solution with phosphatase, then with molybdic acid or molybdate under acidic conditions, and further reacting with a reducing agent;

4) the absorbance was measured at 600 and 810 nm.

In yet another aspect, the present application provides a method for detecting the enzymatic activity of phospholipase C in a fermentation broth, comprising the steps of:

1) contacting the fermentation broth with a phospholipid in a reaction system and allowing the phospholipase C to enzymatically hydrolyze the phospholipid;

2) adding a Tris-HCl buffer solution to the reaction system and then adding a water-immiscible organic solvent;

3) after phase separation, contacting the aqueous solution with phosphatase, then with molybdic acid or molybdate under acidic conditions, and further reacting with a reducing agent;

4) and measuring an absorbance value at 600-810nm, and calculating the enzyme activity of the phospholipase C by using a standard curve of phosphomolybdic blue detection.

In one embodiment of the present application, the phospholipase C includes, but is not limited to, phosphatidylcholine-specific phospholipase C, phosphatidylinositol-specific phospholipase C, phosphatidylethanolamine-specific phospholipase C, phosphatidylserine-specific phospholipase C, and/or phosphatidic acid-specific phospholipase C. In one embodiment of the present application, the phospholipase C may be used as such or in the form of a dilution thereof or a solution thereof.

In one embodiment of the present application, the fermentation broth may be contacted with the phospholipid as such or as a dilution thereof.

In one embodiment of the present application, the phospholipid may be the phospholipid itself or a liquid containing the phospholipid, such as a dilution of the phospholipid. In one embodiment of the present application, the phospholipid may be a single phospholipid or a complex phospholipid commonly used in the art. In one embodiment of the present application, the phospholipid may be a phosphoglyceride, such as, but not limited to, phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, and the like. In one embodiment of the present application, the phospholipid may be a phospholipid derived from oilseeds and cereals, such as a complex phospholipid including, but not limited to, phospholipids derived from one or more of soy, peanut, sesame, castor, corn, cottonseed, rapeseed, sunflower.

In one embodiment of the present application, the reaction system for enzymatic hydrolysis of phospholipid by phospholipase C (hereinafter also referred to as "phospholipase C reaction system") contains, in addition to phospholipase C and a phospholipid substrate, an active metal ion, such as Ca, required for the phospholipase C reaction²⁺And/or Zn²⁺And Triton x-100.

In one embodiment of the present application, the phospholipase C reaction system has a pH of 4.0 to 6.0. In one embodiment of the present application, the phospholipase C reaction system has a pH of 4.4 to 5.8. In one embodiment of the present application, the phospholipase C reaction system has a pH of 4.4 to 4.8. In one embodiment of the present application, the phospholipase C reaction system has a pH of 4.8.

In one embodiment of the present application, the pH of the phospholipase C reaction system can be adjusted to 4.0-6.0, 4.4-5.8, 4.4-4.8, or 4.8 using buffers or pH adjusting agents well known to those skilled in the art, as long as the buffers and pH adjusting agents do not affect the reaction of phospholipase C and the subsequent phosphatase and do not interfere with color development, according to the disclosure or actual needs of the present application. For example, the pH of the phospholipase C reaction system can be adjusted to 4.0-6.0, 4.4-5.8, 4.4-4.8, or 4.8 using NaAc-HAc buffer at a final concentration of 25 mM.

In one embodiment of the present application, Ca in the phospholipase C reaction system²⁺The concentration is 0.2-15 mM, 2-5mM or 5 mM.

In one embodiment of the present application, Zn in the phospholipase C reaction system²⁺The concentration is 20-160 μ M or 80 μ M.

In one embodiment of the present application, the concentration of the phospholipid substrate in the phospholipase C reaction system is between 0.3% and 6% (w/v). In one embodiment of the present application, the concentration of the phospholipid substrate in the phospholipase C reaction system is between 0.5% and 3%. In one embodiment of the present application, the concentration of the phospholipid substrate in the phospholipase C reaction system is between 0.8% and 3%. In one embodiment of the present application, the concentration of the phospholipid substrate in the phospholipase C reaction system is 1%.

In one embodiment of the present application, the concentration of Triton x-100 in the phospholipase C reaction system is in the range of 10mM to 100 mM. In one embodiment of the present application, the concentration of Triton x-100 in the phospholipase C reaction system is in the range of 10mM to 80 mM. In one embodiment of the application, the concentration of Triton x-100 in the phospholipase C reaction system is between 20mM and 80mM or between 40mM and 80 mM. In one embodiment of the present application, the concentration of Triton x-100 in the phospholipase C reaction system is 40 mM.

In one embodiment of the application, the protein concentration of the phospholipase C in the phospholipase C reaction system before being added into the reaction system is 4.0-46.7 mug/mL. In one embodiment of the application, the protein concentration of the phospholipase C in the phospholipase C reaction system before being added into the reaction system is 4.8-46.7 mug/mL or 17.5-46.7 mug/mL. In one embodiment of the application, the protein concentration of the phospholipase C in the phospholipase C reaction system before being added into the reaction system is 4.8-30.0 [ mu ] g/mL. In one embodiment of the present application, the protein concentration of phospholipase C in the phospholipase C reaction system prior to addition to the reaction system is 4.8. mu.g/mL or 30.0. mu.g/mL.

In one embodiment of the present application, the reaction temperature for enzymatic hydrolysis of phospholipids by phospholipase C is from 25 ℃ to 55 ℃. In one embodiment of the present application, the reaction temperature for enzymatic hydrolysis of phospholipids by phospholipase C is in the range of 30 ℃ to 50 ℃. In one embodiment of the present application, the reaction temperature for enzymatic hydrolysis of phospholipids by phospholipase C is 35 ℃ to 50 ℃. In one embodiment of the present application, the reaction temperature for enzymatic hydrolysis of phospholipids by phospholipase C is 35 ℃ to 45 ℃. In one embodiment of the present application, the reaction temperature for enzymatic hydrolysis of phospholipids by phospholipase C is 40 ℃.

In one embodiment of the present application, Tris-HCl buffer solution of 0.15-1.0M and pH8.0-9.0 is added to the phospholipase C reaction system as a stopping reagent. In one embodiment of the present application, Tris-HCl buffer solution of 0.25-1.0M and pH8.0-9.0 is added to the phospholipase C reaction system as a stopping reagent. In one embodiment of the present application, Tris-HCl buffer at pH 9.0 at 0.25M is added to the phospholipase C reaction system as a stop reagent.

In one embodiment of the present application, after adding Tris-HCl buffer as a stop reagent, a water-immiscible organic solvent is added for extraction. In one embodiment of the present application, the water immiscible organic solvent includes, but is not limited to, chloroform.

In one embodiment of the present application, after adding a water-immiscible organic solvent such as chloroform to the phospholipase C reaction system, isoamyl alcohol may optionally be added to the system.

In one embodiment of the present application, the reaction system in which the aqueous solution is contacted with phosphatase (hereinafter referred to as phosphatase reaction system) comprises phosphatase, Mg²⁺And a buffer. In one embodiment of the present application, the phosphatase reaction system comprises phosphatase, Ca²⁺And a buffer.

In one embodiment of the present application, the concentration of phosphatase in the phosphatase reaction system is 0.01U/. mu.L to 0.06U/. mu.L. In one embodiment of the present application, the concentration of phosphatase in the phosphatase reaction system is 0.01U/. mu.L to 0.015U/. mu.L. In one embodiment of the present application, the concentration of phosphatase in the phosphatase reaction system is 0.01U/. mu.L or 0.06U/. mu.L.

In one embodiment of the present application, Mg in the phosphatase reaction system²⁺The concentration is 2-20 mM. In one embodiment of the present application, Mg in the phosphatase reaction system²⁺The concentration was 10 mM.

In one embodiment of the present application, Ca in the phosphatase reaction system²⁺The concentration is 2-20 mM. In one embodiment of the present application, Ca in the phosphatase reaction system²⁺The concentration was 10 mM.

In one embodiment of the present application, the phosphatase reaction system is buffered with 40-100mM Tris-HCl (pH 8.0-9.0). In one embodiment of the present application, the phosphatase reaction system is buffered with 40-50mM Tris-HCl (pH 8.0-9.0). In one embodiment of the present application, the buffer of the phosphatase reaction system is 50mM Tris-HCl (pH 9.0).

In one embodiment of the present application, the phosphatase reaction system comprises 0.01-0.015U/. mu.L phosphatase, 2-20mM Mg²⁺And 40-100mM Tris-HCl (pH 8.0-9.0). In one embodiment of the present application, the phosphatase reaction system comprises 0.01-0.015U/. mu.L phosphatase, 2-20mM Ca²⁺And 40-100mM Tris-HCl (pH 8.0-9.0). In one embodiment of the present application, the phosphatase reaction system comprises 50mM Tris-HCl (pH 9.0), 10mM MgCl₂And 0.01U/. mu.L alkaline phosphatase. In one embodiment of the present application, the phosphatase reaction system comprises 50mM Tris-HCl (pH 9.0), 10mM MgCl₂And 0.015U/. mu.L alkaline phosphatase. In one embodiment of the present application, the phosphatase reaction system comprises 50mM Tris-HCl (pH 9.0), 10mM Mg²⁺And 0.06U/. mu.L alkaline phosphatase.

In one embodiment of the present application, the aqueous phase solution after phase separation is contacted with phosphatase at 30 ℃ to 45 ℃ for at least 30 min. In one embodiment of the present application, the aqueous phase solution is contacted with phosphatase at 35 ℃ to 42 ℃. In one embodiment of the present application, the aqueous phase solution is contacted with phosphatase at 37 ℃ to 40 ℃. In one embodiment of the present application, the aqueous solution is contacted with phosphatase at a temperature of: 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃ or 45 ℃. In one embodiment of the present application, the aqueous solution is contacted with phosphatase at 37 ℃.

In one embodiment of the present application, the aqueous solution after phase separation is contacted with phosphatase for 0.5h to 2 h. In one embodiment of the present application, the aqueous solution is contacted with phosphatase for 0.5h or 1 h.

In one embodiment of the present application, the molybdate used includes, but is not limited to, one or more of ammonium molybdate, sodium molybdate, potassium molybdate. In one embodiment of the present application, the molybdate used is ammonium molybdate.

In one embodiment of the present application, molybdate is used at a concentration of 2-3% (w/v) or 2.4-2.6% (w/v). In one embodiment of the present application, the molybdate is used at a concentration of 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9% or 3.0% (w/v). In one embodiment of the present application, molybdate is used at a concentration of 2.5% (w/v). In one embodiment of the present application, ammonium molybdate is used at a concentration of 2.5% (w/v).

In one embodiment of the present application, the reducing agent used is, for example, ascorbic acid, stannous chloride or phenol. One skilled in the art can use a suitable concentration of a reducing agent, such as ascorbic acid, stannous chloride or phenol, in accordance with the disclosure or actual needs of the present application. In one embodiment herein, the reducing agent is 8% to 10% (w/v) ascorbic acid, 1% to 2% (w/v) stannous chloride or 0.5% to 1% (w/v) phenol. In one embodiment of the present application, the reducing agent is 10% (w/v) ascorbic acid.

In one embodiment of the present application, the absorbance is measured at 600-810 nm. In one embodiment of the present application, the absorbance is measured at 650-750 nm. In one embodiment of the application, the absorbance is detected at 600nm, 610nm, 620nm, 630nm, 640nm, 650nm, 660nm, 670nm, 680nm, 690nm, 700nm, 710nm, 720nm, 730nm, 740nm, 750nm, 760nm, 770nm, 780nm, 790nm, 800nm or 810 nm. In one embodiment of the present application, the absorbance is measured at 700 nm.

In yet another aspect, the present application provides a product for detecting enzymatic activity of phospholipase C, comprising: a first vessel containing a phospholipid, a second vessel containing a Tris-HCl buffer, a third vessel containing a water-immiscible organic solvent, a fourth vessel containing a phosphatase reaction system solution, and a fifth vessel containing a molybdenum blue chromogenic reactant solution.

In one embodiment of the present application, the product for detecting enzymatic activity of phospholipase C further comprises a sixth container comprising a phospholipase C reaction system solution.

In one embodiment of the present application, the phospholipase C reaction system solution contains an active metal ion required for the phospholipase C reaction, such as Ca²⁺And/or Zn²⁺And Triton x-100. In one embodiment of the present application, the phospholipase C reaction system solution has a pH of 4.0 to 6.0, 4.4 to 5.8, 4.4 to 4.8, or 4.8. In one embodiment of the present application, the pH of the phospholipase C reaction system solution is adjusted to 4.0-6.0, 4.4-5.8, 4.4-4.8 or 4.8 using NaAc-HAc buffer at a final concentration of 25 mM. In one embodiment of the present application, Ca in the phospholipase C reaction system solution²⁺The concentration is 0.2-15 mM, 2-5mM or 5 mM. In one embodiment of the present application, the phospholipase C reaction system comprises Zn in solution²⁺The concentration is 20-160 μ M or 80 μ M. In one embodiment of the present application, the concentration of Triton x-100 in the phospholipase C reaction system solution is 10mM to 100mM, 10mM to 80mM, 20mM to 80mM, or 40 mM. In one embodiment of the present application, the phospholipase C reaction system solution comprises 5mM Ca²⁺80 μ M Zn²⁺And 40mM Triton x-100 and pH 4.8.

In one embodiment of the present application, the phospholipid in the first container may be a phospholipid itself or a liquid containing a phospholipid. In one embodiment of the present application, the phospholipid in the first container may be a single phospholipid or a complex phospholipid as commonly used in the art. In one embodiment of the present application, the phospholipid may be a phosphoglyceride, such as, but not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, and the like. In one embodiment of the present application, the phospholipid may be a phospholipid derived from oilseeds and cereals, such as a complex phospholipid including, but not limited to, phospholipids derived from one or more of soy, peanut, sesame, castor, corn, cottonseed, rapeseed, sunflower.

In one embodiment of the application, the phospholipid concentration in the first container is 0.3% to 6% (w/v), 0.5% to 3%, 0.8% to 3%, or 1%.

In one embodiment of the present application, the Tris-HCl buffer in the second container is 0.15-1.0M and pH8.0-9.0 Tris-HCl buffer, or 0.25M and pH 9.0 Tris-HCl buffer.

In one embodiment of the present application, the water immiscible organic solvent in the third vessel includes, but is not limited to, chloroform.

In one embodiment of the present application, the phosphatase reaction system solution in the fourth container comprises phosphatase, Tris-HCl buffer and Mg²⁺Or Ca²⁺. In one embodiment of the present application, the concentration of phosphatase in the phosphatase reaction system solution in the fourth container is 0.01U/. mu.L to 0.06U/. mu.L, 0.01U/. mu.L to 0.015U/. mu.L, 0.01U/. mu.L, or 0.06U/. mu.L. In one embodiment of the present application, Mg in the phosphatase reaction system solution in the fourth container²⁺The concentration is 2-20mM or 10 mM. In one embodiment of the present application, Ca in the phosphatase reaction system solution in the fourth container²⁺The concentration is 2-20mM or 10 mM. In one embodiment of the present application, the Tris-HCl buffer in the phosphatase reaction system solution in the fourth container is 40-100mM Tris-HCl buffer (pH8.0-9.0), or 40-50mM Tris-HCl buffer (pH8.0-9.0), or 50mM Tris-HCl buffer (pH 9.0).

In one embodiment of the present application, the phosphatase reaction system solution in the fourth container comprises 0.01-0.015U/. mu.L phosphatase, 2-20mM Mg²⁺And 40-100mM Tris-HCl (pH8.0-9.0), or 0.01-0.015U/. mu.L phosphatase, 2-20mM Ca²⁺And 40-100mM Tris-HCl (pH8.0-9.0), or 50mM Tris-HCl (pH 9.0), 10mM MgCl₂And 0.01U/. mu.L alkaline phosphatase, or 50mM Tris-HCl (pH 9.0), 10mM Mg²⁺And 0.06U/. mu.L alkaline phosphatase, or 50mM Tris-HCl (pH 9.0), 10mM Mg²⁺And 0.015U/. mu.L alkaline phosphatase.

In one embodiment of the present application, the molybdenum blue chromogenic reactant solution in the fifth vessel comprises molybdic acid or molybdate. In one embodiment of the present application, the molybdate is one or more of ammonium molybdate, sodium molybdate and potassium molybdate. In one embodiment of the present application, the molybdenum blue chromogenic reactant solution comprises 2-3% (w/v), 2.4-2.6% (w/v), or 2.5% (w/v) molybdate. In one embodiment of the present application, the molybdenum blue chromogenic reactant solution comprises 2.5% (w/v) ammonium molybdate.

In one embodiment of the present application, the product for detecting the enzymatic activity of phospholipase C is in the form of a kit.

In one embodiment of the present application, the product for detecting enzyme activity of phospholipase C comprises:

a first container comprising a phospholipid, wherein the phospholipid is, for example, a single phospholipid or a complex phospholipid and is present at a concentration of, for example, 1% (w/v).

A second container comprising a Tris-HCl buffer, such as 0.25M Tris-HCl buffer at pH 9.0;

a third vessel containing a water-immiscible organic solvent, such as chloroform;

a fourth container containing a phosphatase reaction system solution, for example, containing 50mM Tris-HCl (pH 9.0), 10mM Mg²⁺And 0.06U/. mu.L alkaline phosphatase;

a fifth vessel containing a molybdenum blue chromogenic reactant solution, for example, containing 2.5% (w/v) ammonium molybdate; and

a sixth vessel containing a phospholipase C reaction system solution, wherein the phospholipase C reaction system solution, for example, contains 5mM Ca²⁺80 μ M Zn²⁺And 40mM Triton x-100 and pH 4.8.

In one embodiment of the present application, the phosphatase contained in the phosphatase reaction system solution in the fourth container may be separately stored as a phosphatase storage system solution in the seventh container together with a solution stabilizing the phosphatase. In one embodiment of the present application, the solution stabilizing the phosphatase comprises 200-500mM Tris-HCl buffer (pH8.0-9.0) and 10-50mM MgCl₂. In one embodiment of the present application, the phosphatase isThe storage system solution contains 50-75U/mL or 50-300U/mL alkaline phosphatase. In one embodiment of the present application, the phosphatase storage system solution comprises 200-500mM Tris-HCl buffer (pH8.0-9.0), 10-50mM MgCl₂And 50-75U/mL or 50-300U/mL alkaline phosphatase.

In a further aspect, the present application provides a method of providing a phospholipase C product, comprising detecting the enzyme activity of phospholipase C using the method of the above aspect, or a product for detecting the enzyme activity of phospholipase C. In one embodiment of the application, the method of providing a phospholipase C product further comprises one or more steps selected from the group consisting of dilution, shaking, standing, centrifugation, split charging, and colorimetry.

When the product for detecting the enzyme activity of the phospholipase C is used, a reducing agent required by molybdenum blue color development reaction is required to be added. For better use of the reducing agent, fresh formulation is often required. Thus, in most cases, it is self-made by the user, and it is also possible to include a reducing agent in the product. In one embodiment of the use of the product of the present application, the reducing agent is 8% to 10% (w/v) ascorbic acid, 1% to 2% stannous chloride or 0.5% to 1% phenol. In one embodiment of the use of the product of the present application, the reducing agent is 10% (w/v) ascorbic acid.

The following examples are provided by way of illustration and not by way of limitation.

Examples

In the following examples of the present application, the phospholipase is phosphatidylcholine-specific phospholipase C from bacillus cereus, expressed by fermentation of laboratory-constructed recombinant pichia pastoris and purchased from impermans; alkaline phosphatase was purchased from Takara or NEB.

Preparation example preparation of Standard Curve for phosphomolybdic blue assay and measurement of enzymatic Activity of phospholipase C

1.1 formulation of Standard Curve for phosphomolybdic blue detection

From 1mg/mL PO₄ ^3-KH of₂PO₄Taking out 0, 1, 2, 4, 5, 6, 7, 8 μ L of the solution, adding deionized waterAdding the son water to 940 mu L, adding 20 mu L of 10% (w/v) ascorbic acid solution, mixing uniformly, adding 40 mu L of 2.5% (w/v) ammonium molybdate solution, and finally obtaining PO₄ ^3-The concentrations were 0, 1, 2, 4, 5, 6, 7, 8. mu.g/mL. Each concentration point was sampled in parallel in 3 replicates. Measuring light absorption value A at 700nm after water bath at 50 deg.C for 20min₇₀₀And performing linear fitting on the obtained data to obtain a standard curve for detecting the phosphomolybdic blue.

1.2 measurement of enzyme Activity of phospholipase C

In a 400 mu L phospholipase C reaction system, the concentration of phospholipid substrate is 0.5-3% (w/v), the concentration of Triton x-100 is 40-80 mM, and Ca is added²⁺A concentration of 0.2 to 15mM, and Zn²⁺Concentration of 20-160. mu.M, and adjusting pH to the optimum reaction pH of phospholipase C, such as pH 4.8, with NaAc-HAc at a final concentration of 25mM, wherein the addition volume of phospholipase C is 20. mu.L (the protein concentration of phospholipase C is 17.5-46.7. mu.g/mL before addition to the reaction system), and the temperature of the water bath is adjusted to a suitable temperature, such as 40 ℃. After the reaction is carried out for 5min, 100 mu L of 0.25-1.0M Tris-HCl buffer solution with the pH value of 8.0-9.0 is added, 500 mu L chloroform is added and evenly shaken for 30s, 10 mu L isoamyl alcohol is added, the mixture is kept stand for a few minutes and then centrifuged, and the supernatant is taken for the next reaction. Blank control water was used instead of inactivated enzyme.

Collecting 80 μ L supernatant, adding 20 μ L Mg²⁺Reacting with an excessive amount of Tris-HCl buffer solution (50mM, pH8.0-9.0) of alkaline phosphatase at 37 ℃ for more than 1h, adding 840 mu L of deionized water, adding 20 mu L of 10% (w/v) ascorbic acid and 40 mu L of 2.5% (w/v) ammonium molybdate solution, developing the solution in a water bath at 50 ℃ for 20min, and measuring the absorbance A at 700nm₇₀₀. And (4) calculating the enzyme activity value of the phospholipase C by combining the prepared standard curve for detecting the phosphomolybdic blue.

Calculation of enzyme Activity of phospholipase C

The enzyme activity calculation formula is as follows:

(1) standard curve: y ═ a × x (y: absorbance A of 700 nm)₇₀₀；x：PO₄ ^3-(ii) concentration of (g/. mu.l); a: regression coefficient)

(2) Obtaining a light absorption value A after each sample is developed₇₀₀：

Phospholipase C enzyme activity (A/a) X1/95/0.08X 0.5/5/0.02X dilution of phospholipase C (. mu.mol/min/mL)

Remarking:

(A/a): PO measured for each sample₄ ^3-(ii) concentration of (g/. mu.l);

1: the total volume is 1mL during color development;

95：PO₄ ^3-the molar mass of (a) is 95. mu.g/. mu.mol;

0.08: 0.08mL of the solution taken from the phospholipase C reaction was subjected to the next reaction, after which all PO was measured₄ ^3-From this volume;

0.5: the total volume of the water phase after the reaction of the phospholipase C in the first step is 0.5 mL;

5: time of phospholipase C reaction, min;

0.02: the volume of the enzyme solution added to the first reaction was 0.02 mL.

Example 1

In a 400. mu.L phospholipase C reaction system, the phospholipid substrate concentration was 1%, the Triton x-100 concentration was 40mM, and Ca was added²⁺Concentration 5mM, Zn²⁺The concentration was 80. mu.M, the pH was adjusted to 4.8 with NaAc-HAc of 25mM final concentration, the volume of phospholipase C added was 20. mu.L (the enzyme solution was diluted with NaAc-HAc buffer of 10mM pH 5.6 to a protein concentration of 30. mu.g/mL before addition to the reaction system), 100. mu.L of a terminator (Tris-HCl buffer of 0.25M, pH 9.0.0) was added after 5min of reaction in a 35-50 ℃ water bath, 500. mu.L of chloroform was added and shaken for 30s, 10. mu.L of isoamyl alcohol was added and left to stand for several minutes, and the supernatant was centrifuged to conduct the next reaction. The blank uses water instead of inactivated enzyme.

80 μ L of the supernatant was added to 20 μ L of 50mM Mg²⁺And 0.3U/. mu.L of alkaline phosphatase Tris-HCl buffer (pH 9.0) at 37 ℃ for 1 hour, adding 840. mu.L of deionized water, adding 20. mu.L of 10% (w/v) ascorbic acid and 40. mu.L of 2.5% (w/v) ammonium molybdate solution, developing in a water bath at 50 ℃ for 20min, and determining A₇₀₀. And (4) calculating an enzyme activity value by combining a standard curve of phosphomolybdic blue detection. The enzyme activity of phospholipase C at different reaction temperatures is shown in figure 1. Can be seen inThe phospholipase C has higher enzyme activity at 35-45 ℃.

Example 2

In a 400. mu.L phospholipase C reaction system, the phospholipid substrate concentration was 1%, the Triton x-100 concentration was 40mM, and Ca was added²⁺Concentration 5mM, Zn²⁺The concentration is 80 mu M, the pH is adjusted to 4.4-5.8 by NaAc-HAc with the final concentration of 25mM, the volume of the added phospholipase C is 20 mu L (the protein concentration is 30 mu g/mL before being added into the reaction system), 100 mu L of termination reagent (Tris-HCl buffer solution of 0.25M, pH 9.0.0) is added after the reaction is carried out for 5min in a water bath kettle at 40 ℃, 500 mu L of chloroform is added for shaking for 30s, 10 mu L of isoamyl alcohol is added for standing for a few minutes, and the supernatant is centrifuged to carry out the next reaction. The blank uses water instead of inactivated enzyme.

The subsequent treatment was the same as that described in example 1. The enzyme activity of phospholipase C under different reaction pH is shown in FIG. 2. Therefore, the phospholipase C has higher enzyme activity under the condition of pH 4.4-4.8.

Example 3

In a 400 mu L phospholipase C reaction system, the concentration of phospholipid substrate is 0.5-3%, the concentration of Triton x-100 is 40mM, and Ca is²⁺Concentration 5mM, Zn²⁺The concentration was 80. mu.M, the pH was adjusted to 4.8 with NaAc-HAc of 25mM final concentration, the volume of phospholipase C added was 20. mu.L (protein concentration was 30. mu.g/mL before addition to the reaction system), 100. mu.L of a terminator (Tris-HCl buffer solution of 0.25M, pH 9.0.0) was added after 5min of reaction in a water bath at 40 ℃, 500. mu.L of chloroform was added and shaken for 30s, 10. mu.L of isoamyl alcohol was added and left to stand for several minutes, and the supernatant was centrifuged to conduct the next reaction. The blank uses water instead of inactivated enzyme.

The subsequent treatment was the same as that described in example 1. The relative enzyme activity of phospholipase C at different substrate concentrations is shown in FIG. 3. Therefore, the enzyme activity of the phospholipase C is higher when the concentration of the substrate is 0.8-3%.

Example 4

In a 400-mu-L phospholipase C reaction system, the concentration of phospholipid substrate is 1%, the concentration of Triton x-100 is 10-80 mM, and Ca is²⁺Concentration 5mM, Zn²⁺The concentration was 80. mu.M, the pH was adjusted to 4.8 with NaAc-HAc at a final concentration of 25mM, and the phospholipase C was added in a volume of 20. mu.ML (protein concentration 30. mu.g/mL before addition to the reaction system), after 5min in a 40 ℃ water bath, 100. mu.L of a terminator (0.25M, pH 9.0.0 in Tris-HCl buffer), 500. mu.L of chloroform was added and shaken for 30s, 10. mu.L of isoamyl alcohol was added and left to stand for several minutes, and the supernatant was centrifuged to conduct the next reaction. The blank uses water instead of inactivated enzyme.

The subsequent treatment was the same as that described in example 1. The relative enzyme activity of phospholipase C at different Triton x-100 concentrations is shown in FIG. 4. It can be seen that the enzyme activity of the phospholipase C is higher when the Triton x-100 concentration is 20-80 mM.

Example 5

In a 400. mu.L phospholipase C reaction system, the phospholipid substrate concentration was 1%, the Triton x-100 concentration was 40mM, and Ca was added²⁺Concentration 5mM, Zn²⁺The concentration is 80 mu M, the pH is adjusted to 4.8 by NaAc-HAc with the final concentration of 25mM, the volume of the added phospholipase C is 20 mu L (the protein concentration is 17.5-46.7 mu g/mL before being added into the reaction system), 100 mu L of termination reagent (Tris-HCl buffer solution of 0.25M, pH 9.0.0) is added after the reaction is carried out for 5min in a water bath kettle at the temperature of 40 ℃, 500 mu L of chloroform is added for shaking for 30s, 10 mu L of isoamyl alcohol is added for standing for a few minutes, and the supernatant is centrifuged to carry out the next reaction. The blank uses water instead of inactivated enzyme.

The subsequent treatment was the same as that described in example 1. The absorbance at 700nm of the samples measured at different phospholipase C protein concentrations is shown in FIG. 5. Therefore, the protein concentration of the phospholipase C and the light absorption value of the sample have a good linear relation under the method.

Example 6

In a 400. mu.L phospholipase C reaction system, the phospholipid substrate concentration was 1%, the Triton x-100 concentration was 40mM, and Ca was added²⁺Concentration 5mM, Zn²⁺Adjusting the pH to 4.8 by NaAc-HAc with the final concentration of 25mM, adding 20 mu L of phospholipase C (the protein concentration of an enzyme solution is 30 mu g/mL before the phospholipase C is added into a reaction system), reacting for 5min in a water bath at 40 ℃, adding 100 mu L of termination reagent (0.05-5M, pH 8.0.0-9.0 Tris-HCl buffer), standing for 0 or 15min, adding 500 mu L of chloroform, shaking for 30s, adding 10 mu L of isoamyl alcohol, standing for a few minutes, centrifuging, taking the supernatant, and carrying out the next reaction. Substituting water for blankAnd (4) replacing the inactivated enzyme.

The subsequent treatment was the same as that described in example 1. The results of enzyme activity measurements of samples corresponding to different concentrations and pH of the stop reagent are shown in Table 1. Wherein the enzyme activity of the sample with the standing time of 0 corresponding to each termination reagent is 100%, and the increase of the relative enzyme activity of the sample after standing for 15min is less than 5%, so that the phospholipase C reaction is effectively terminated.

TABLE 1 Effect of different concentrations and pH of Tris-HCl solutions on the termination of phospholipase C reactions

As can be seen from Table 1, Tris-HCl buffer at a concentration and pH range was effective in terminating the phospholipase C reaction.

Example 7

In a 400. mu.L system, the phospholipid substrate concentration was 1%, the Triton x-100 concentration was 40mM, and Ca was²⁺Concentration 5mM, Zn²⁺The concentration was 80. mu.M, the pH was adjusted to 4.8 with NaAc-HAc of 25mM final concentration, 20. mu.L of deionized water was added, the mixture was allowed to stand in a water bath at 40 ℃ for 5 minutes, 100. mu.L of a terminator (Tris-HCl buffer solution of 0.25, pH 9.0) was added thereto and shaken well, and 80. mu.L of the mixture was reacted with alkaline phosphatase and developed (the same procedure as described in example 1). The results showed that the sample had an absorbance at 700nm of 0.4143 and was blue-green, while the control group, which did not contain the phospholipid substrate, exhibited substantially no color, as shown in FIG. 6. It can be seen that interference with the removal of residual phospholipid substrate prior to development has a significant effect.

Example 8

In a 400. mu.L phospholipase C reaction system, the phospholipid substrate concentration was 1%, the Triton x-100 concentration was 40mM, and Ca was added²⁺Concentration 5mM, Zn²⁺Adjusting pH to 4.8 with NaAc-HAc with final concentration of 25mM and phospholipase C with volume of 20 μ L (diluting enzyme solution 5000 times to concentration of 4.8 μ g/mL before adding into reaction system), reacting in 40 deg.C water bath for 5min, adding 400 μ L chloroform, shaking for 30s, adding 10 μ L isoamyl alcohol, standing for 0, 15, 60min, centrifuging, collecting supernatant, and performing next stepAnd (5) carrying out reaction. The blank uses water instead of inactivated enzyme. The subsequent treatment was the same as that described in example 1. The enzyme activity measurement results of different samples are shown in Table 2, wherein the enzyme activity of the sample with the standing time of 0 is 100%, and the increase of the relative enzyme activity of the sample with the standing time of 15min is less than 5%, and the phospholipase C reaction is considered to be effectively stopped.

TABLE 2 Effect of terminating phospholipase C reaction by chloroform extraction

As can be seen from Table 2, the phospholipase C reaction was not efficiently terminated by only adding chloroform to extract the residual phospholipid substrate.

Example 9

In a 400. mu.L phospholipase C reaction system, the phospholipid substrate concentration was 1%, the Triton x-100 concentration was 40mM, and Ca was added²⁺Concentration 5mM, Zn²⁺The concentration is 80 mu M, the pH is adjusted to 4.8 by NaAc-HAc with the final concentration of 25mM, the volume of the added phospholipase C is 20 mu L (the enzyme solution is diluted by 5000 times to the concentration of 4.8 mu g/mL before being added into the reaction system), 400 mu L of absolute ethyl alcohol is added after 5min of reaction in a water bath kettle at 40 ℃, 800 mu L of chloroform is added after 0, 15 and 60min of standing for shaking for 30s, 10 mu L of isoamyl alcohol is added, the mixture is centrifuged after slight standing, and the supernatant is taken for next reaction. The blank uses water instead of inactivated enzyme. The subsequent treatment was the same as that described in example 1. The results of the enzyme activity measurements of the different samples are shown in Table 3. Wherein the enzyme activity of the sample with the standing time of 0 is 100 percent, and the increase of the relative enzyme activity of the sample with the standing time of 15min is less than 5 percent, so that the phospholipase C reaction is effectively stopped.

TABLE 3 Effect of ethanol termination of phospholipase C reaction

As can be seen from Table 3, the phospholipase C reaction was not effectively terminated by the addition of ethanol, and if the final concentration of ethanol was increased, the subsequent alkaline phosphatase reaction could be affected.

Example 10

In a 400. mu.L phospholipase C reaction system, the phospholipid substrate concentration was 1%, the Triton x-100 concentration was 40mM, and Ca was added²⁺Concentration 5mM, Zn²⁺The concentration is 80 mu M, the pH is adjusted to 4.8 by NaAc-HAc with the final concentration of 25mM, the volume of the phospholipase C is 20 mu L (the enzyme solution is diluted by 800 times to the concentration of 30 mu g/mL before being added into the reaction system), 100 mu L of termination reagent (Gly-NaOH buffer solution with different concentrations and pH ranges shown in the table 4) is added after the reaction is carried out for 5min in a water bath kettle at 40 ℃, the mixture is kept still for 0 or 15min, 500 mu L of chloroform is added for shaking for 30s, 10 mu L of isoamyl alcohol is added for keeping still for a few minutes, and the supernatant is centrifuged to carry out the next reaction. The blank uses water instead of inactivated enzyme.

The subsequent treatment was the same as that described in example 1. The results of enzyme activity measurements for samples corresponding to different concentrations and pH of the stop reagent are shown in Table 4. Wherein the enzyme activity of the sample with the standing time of 0 corresponding to each termination reagent is 100%, and the increase of the relative enzyme activity of the sample after standing for 15min is less than 5%, so that the phospholipase C reaction is effectively terminated.

TABLE 4 Effect of Gly-NaOH solutions of varying concentrations and pH on terminating the phospholipase C reaction

As can be seen from Table 4, Gly-NaOH was not effective in terminating the reaction of phospholipase C even at the same pH and concentration (pH 9, 0.25M) as described in example 6. If the concentration and pH of Gly-NaOH are increased, the subsequent enzyme activity of alkaline phosphatase may be affected.

Example 11

Experimental groups: referring to the two-phase reaction system of the reference document CN201310430394.X, 200. mu.L of phospholipase C was added to the two-phase reaction system containing phospholipid at a final concentration of 0.5% (w/v), 25mM Tris-HCl (pH 7.5), 5mM CaCl₂. Phospholipase C was diluted to 43.6. mu.g/mL and 21.8. mu.g/mL, and 20. mu.L of each solution was added to the reaction system, and the mixture was reacted at 37 ℃ and 150rpm for 15min, followed by centrifugation at 12000rpmAnd 2 min. The blank uses water instead of inactivated enzyme. After centrifugation, 25. mu.L of Tris-HCl (250mM, pH 9.0) was added to the supernatant.

Control group: in a 200. mu.L phospholipase C biphasic reaction system, phospholipid was present at a final concentration of 0.5% (w/v), 25mM Tris-HCl (pH 7.5), 5mM CaCl₂. Phospholipase C was diluted to 43.6. mu.g/mL and 21.8. mu.g/mL, respectively, and then 20. mu.L of each was added to the reaction system, and after reaction at 37 ℃ and 150rpm for 15min, 25. mu.L of Tris-HCl (250mM, pH 9.0) was added to the supernatant, followed by centrifugation at 12000rpm for 2 min. The blank uses water instead of inactivated enzyme.

mu.L of the centrifuged supernatant was aspirated into 100. mu.L of an alkaline phosphatase reaction system containing 50mM Tris-HCl (pH 9.0), 10mM MgCl₂20U/mL alkaline phosphatase. The reaction was carried out in a water bath at 37 ℃ for 30 min. The subsequent color development treatment was the same as that described in example 1. The results of enzyme activity measurement of the samples of the control group and the experimental group are shown in Table 5. Wherein the relative enzyme activity of the sample of the control group under each phospholipase C protein concentration is 100%, and the enzyme activity of the sample corresponding to the experimental group is more than 10% of that of the control group, so that the phospholipase C still reacts in the centrifugal separation process in the experimental group, and the enzyme activity cannot be more accurately measured.

TABLE 5 verification experiment of two-phase reaction system during phase separation without reaction termination

The foregoing is merely a preferred embodiment of the present application and is not intended to limit the scope of the present application, which is defined broadly in the claims appended hereto, and any entity or method that is calculated by another person and is equivalent to or even equivalent to that defined by the claims appended hereto.

All documents mentioned in this application are incorporated by reference into this application as if each were individually incorporated by reference. Further, it should be understood that various changes or modifications can be made to the present application by those skilled in the art after reading the above-mentioned contents of the present application, and these equivalents also fall within the scope defined by the claims appended to the present application.