CN110618203A - Method for accurately, qualitatively and quantitatively determining activity of glucose dehydrogenase - Google Patents
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Abstract
The invention relates to the technical field of enzyme activity detection, in particular to a method for accurately, qualitatively and quantitatively detecting the activity of glucose dehydrogenase. The invention firstly provides an HPLC detection method of a product NADPH of glucose dehydrogenase enzymatic reaction, which adopts a reversed phase chromatographic column taking a silane bonding phase as a stationary phase, a mobile phase consists of buffer salt and an organic solvent, the ultraviolet detection wavelength is 330-350 nm, and the flow rate is 0.8-1.2 mL/min; further provides a method for accurately, qualitatively and quantitatively determining the activity of the glucose dehydrogenase. The detection method provided by the invention is simple to operate, high in sensitivity and strong in accuracy, is not easily influenced by insoluble particles and some pigments in a complex sample, and is easy for standardized operation.
Description
Technical Field
The invention relates to the technical field of enzyme activity detection, in particular to a method for accurately, qualitatively and quantitatively detecting the activity of glucose dehydrogenase.
Background
Glucose Dehydrogenase (GDH) is mainly present in animal liver and weak acetobacter oxydans, and can catalyze beta-D-Glucose to generate gluconic acid (D-gluconic acid-delta-lactone) accompanied by the generation of NADPH (reduced coenzyme). As early as 1975, Banauch et al used glucose dehydrogenase as a diagnostic enzyme for clinical blood glucose determination to determine the concentration of glucose in human body fluids. Currently, glucose dehydrogenase is mainly used in clinical blood glucose monitoring sensors and NADPH regeneration mediated by NADP-dependent glucose dehydrogenase.
At present, the main detection methods of the activity of the glucose dehydrogenase include a spectrophotometry method, an ion chromatography method, a continuous monitoring method and the like. The principle of the spectrophotometry is that when NADP + is a hydrogen acceptor, glucose dehydrogenase can catalyze beta-D-glucose to generate gluconic acid (D-gluconic acid-delta-lactone), and simultaneously along with the generation of reductive coenzyme NADPH, the generated NADPH can be quantified by a spectrophotometer, so that the activity of the glucose dehydrogenase can be calculated. Although the method is simple and easy to implement and low in cost, the method is easily affected by insoluble particles and related impurities in a sample, so that a result has larger error. The principle of the ion chromatography is that glucose dehydrogenase can catalyze beta-D-glucose to generate gluconic acid, and the enzyme activity of the glucose dehydrogenase can be measured by detecting the amount of potassium gluconate. The method needs ion chromatography, and the instrument is poor in universality and cannot meet the requirement of wide use. The continuous monitoring method mainly adopts an automatic biochemical analyzer to monitor the change process of the reaction product NADPH along with time, thereby measuring the activity of the glucose dehydrogenase. The method needs to use an automatic biochemical analyzer, has poor popularity, needs to investigate various parameters, reaction conditions and the like of the analyzer, and cannot meet the requirements of conventional detection.
The method for measuring the activity of the glucose dehydrogenase enzyme, which is mentioned in the Chinese patent CN 104388373A, adopts a spectrophotometry to detect the absorbance value of NADPH (nicotinamide adenine dinucleotide) which is a product of glucose dehydrogenase enzymatic reaction in a sample to measure the activity of the glucose dehydrogenase, and the method is easily influenced by insoluble particles and related impurities in the sample, so that the result error is large. The HPLC detection method of NADPH mentioned in Chinese patent CN108132318A adopts C18 column and gradient elution mode, which has long time and complex procedure. Therefore, the establishment of a rapid, simple, intuitive and accurate GDH enzyme activity detection method is a fundamental problem to be solved urgently. The invention is based on the purpose of researching and providing a method for quickly and accurately measuring the activity of the glucose dehydrogenase.
Disclosure of Invention
The invention aims to solve the problem of accurate qualitative and quantitative determination of the activity of the glucose dehydrogenase, and firstly solves the problem of detection of NADPH (nicotinamide adenine dinucleotide) which is a product of enzymatic reaction of the glucose dehydrogenase. In order to overcome the defects and shortcomings in the prior art, the invention firstly provides an HPLC detection method of NADPH, and further provides a method for accurately, qualitatively and quantitatively determining the enzyme activity of glucose dehydrogenase. The method can quickly, simply, visually and accurately detect the amount of the NADPH and calculate the activity of the glucose dehydrogenase, and is not easily influenced by insoluble particles and some pigments in a complex sample.
The purpose of the invention is realized by the following technical scheme: the invention provides an HPLC method for measuring NADPH, which adopts a reversed phase chromatographic column with a silane bonding phase as a stationary phase, a mobile phase consists of buffer salt and an organic solvent, the ultraviolet detection wavelength is 330-350 nm, and the flow rate is 0.8-1.2 mL/min.
In some embodiments of the invention, the chromatographic column used in the HPLC method is a C30 column.
In some embodiments of the invention, the buffer salt used in the HPLC method is selected from one or more of sodium phosphate dibasic, sodium acetate, and sodium phosphate; the organic solvent is selected from one or more of methanol, ethanol, isopropanol and acetonitrile; in some embodiments, the buffer salt used in the HPLC method is sodium acetate and the organic solvent is methanol.
In some embodiments of the invention, the HPLC method uses a buffered sodium acetate solution having a pH of 5.0 to 6.0; in some embodiments, the HPLC method uses a buffered sodium acetate solution having a pH of 5.0; in some embodiments, the HPLC method uses a buffered sodium acetate solution having a pH of 5.5; in some embodiments, the HPLC method uses a buffered sodium acetate solution having a pH of 6.0.
In some embodiments of the invention, the HPLC method uses a buffered sodium acetate solution at a concentration of 43 to 129 mM; in some embodiments, the HPLC method uses a buffered sodium acetate solution at a concentration of 43 mM; in some embodiments, the HPLC method uses a buffered sodium acetate solution at a concentration of 86 mM; in some embodiments, the HPLC method uses a concentration of the buffered sodium acetate solution of 129 mM.
In some embodiments of the invention, the percentage of the organic solvent used in the HPLC method to the total volume of the mobile phase is 8-12%; in some embodiments, the HPLC method uses 8% organic solvent based on the total volume of the mobile phase; in some embodiments, the organic solvent comprises 10% of the total volume of the mobile phase; in some embodiments, the organic solvent comprises 12% of the total volume of the mobile phase.
In some embodiments of the invention, the HPLC method uses an ultraviolet detection wavelength of 330-350 nm; in some embodiments, the HPLC method uses an ultraviolet detection wavelength of 340 nm; in some embodiments, the HPLC method uses an ultraviolet detection wavelength of 340 nm; in some embodiments, the HPLC method uses an ultraviolet detection wavelength of 350 nm.
In some embodiments of the invention, the HPLC method uses a flow rate of 0.8 to 1.2 mL/min; in some embodiments, the HPLC method uses a flow rate of 0.8 mL/min; in some embodiments, the HPLC method uses a flow rate of 1.0 mL/min; in some embodiments, the HPLC method uses a flow rate of 1.2 mL/min.
In some embodiments of the invention, the HPLC method uses a column temperature of 23-33 ℃; in some embodiments, the HPLC method uses a column temperature of 23 ℃; in some embodiments, the HPLC method uses a column temperature of 28 ℃; in some embodiments, the HPLC method uses a column temperature of 33 ℃.
The invention also provides a method for accurately, qualitatively and quantitatively determining the enzyme activity of the glucose dehydrogenase, which adopts an HPLC method to detect the NADPH product of the enzymatic reaction of the glucose dehydrogenase so as to calculate the enzyme activity of the glucose dehydrogenase in a sample, and specifically comprises the following steps:
(1) drawing a standard curve of NADPH peak area-NADPH concentration;
(2) the occurrence of a glucose dehydrogenase-mediated enzymatic reaction;
mixing Tris-HCl buffer solution with pH of 8.0, glucose solution and NADP+After the solutions are uniformly mixed, adding a glucose dehydrogenase sample diluted by a Tris-HCl buffer solution, reacting at room temperature, heating to boil and cooling to room temperature;
(3) detecting the generation amount of NADPH which is a product of an enzymatic reaction by an HPLC method;
(4) it is known that the enzyme activity of glucose dehydrogenase is calculated from the amount of NADPH produced.
In some embodiments of the present invention, the sample is diluted with Tris-HCl buffer solution until the enzyme activity of glucose dehydrogenase is in the range of 0.01-1.25U/mL before the enzymatic reaction in step (2) is performed. The reaction time at room temperature in step (2) was 4 min.
In some embodiments of the invention, the HPLC chromatographic conditions described in step (3) are:
a chromatographic column: c30 column (4.6 × 250 mm);
detection wavelength: 340 nm;
column temperature: 28 ℃;
flow rate: 1.0 ml/min;
operating time: 15 min;
mobile phase: sodium acetate solution (pH 5.5) and methanol, the percentage of methanol to the total volume of the mobile phase being 10%.
In some embodiments of the present invention, the enzyme activity in step (4) is defined as an amount of an enzyme catalyzing the production of 1. mu. mol of NADPH per minute of glucose at a temperature of 25 ℃ and a pH of 8.0 as defined as one International Unit (IU). The calculation formula is as follows:
in the formula: a: peak area of the liquid to be detected; k: the slope of the standard curve;
c: intercept of the standard curve; n: dilution factor of the enzyme;
m: weighing the weight of the enzyme powder to be detected; t: reaction time at room temperature;
Usolid body: enzyme activity of the solid sample; u shapeLiquid, method for producing the same and use thereof: enzymatic activity of the liquid sample;
Vtotal volume: total volume of reaction; vSample volume: sample reaction volume.
In some embodiments of the invention, the room temperature reaction time T is 4 min.
The "glucose dehydrogenase activity" according to the present invention means that the amount of an enzyme catalyzing the production of 1. mu. mol of NADPH per minute of glucose at a temperature of 25 ℃ and a pH of 8.0 is defined as one International Unit (IU).
The NADPH reduced coenzyme II is named as reduced nicotinamide adenine dinucleotide phosphate, participates in various anabolic reactions, such as synthesis of lipids, fatty acids and nucleotides, and can also supply energy for fixing carbon dioxide in dark reaction. These reactions require NADPH as a reducing agent, a donor of hydride, NADPH being NADP+In reduced form.
The "NADP" of the invention+"is nicotinamide adenine dinucleotide phosphate, the oxidized form of reduced coenzyme II (NADPH), meaning that one electron is lost and a positive charge is taken. Mainly used as a coenzyme of dehydrogenase, plays a role of a hydrogen donor in an enzymatic reaction and is a single hydrogen donor.
The Tris-HCl is hydrochloric Tris (hydroxymethyl) aminomethane, which is widely used as a solvent for nucleic acid and protein and can be used as a buffer system for stabilizing pH value.
The "stationary phase" of the present invention is a phase that remains in the sample and remains in the chromatographic separation.
The "mobile phase" of the present invention refers to a substance which carries the component to be measured to move forward during the chromatography process and is called as the mobile phase. And the other phase which is in an equilibrium state with the stationary phase and drives the sample to move forwards.
Compared with the prior art, the invention has the following beneficial effects:
1. the HPLC determination method established by the invention has the characteristics of intuition and accuracy, can intuitively reflect the peak area of the NADPH of the reaction product so as to realize accurate, qualitative and quantitative determination of the NADPH, has short reaction time and good product stability, and can simultaneously detect and analyze a large number of samples.
2. In the invention, the reaction condition is mild, the enzymatic reaction product NADPH is measured by HPLC without being influenced by insoluble particles and related pigments, the accuracy is strong, the method has good stability, the standardized operation is easy, and the application range is wide. Meanwhile, the amount of reagents and standard products used in the detection is small, and the cost is low.
3. The C30 column used in the HPLC method of the invention has better retention and separation for analysis of product peaks than other columns.
Drawings
FIG. 1 is the NADPH concentration-NADPH peak area standard curve of example 1;
FIG. 2 is a chromatogram for measuring NADPH by the HPLC method of example 1;
FIG. 3 is a comparative example 1 absorbance-NADPH standard curve;
FIG. 4 is a chromatogram for measuring NADPH by the HPLC method of comparative example 2.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
Example 1 glucose dehydrogenase Activity measurement procedure
(1) Drawing of NADPH concentration-NADPH Peak area Standard Curve
Weighing about 25mg of NADPH standard (buntah biological) into a 25mL volumetric flask, dissolving with Tris-HCl buffer solution (100mmol/L, pH 8.0), fixing the volume to scale, and sequentially diluting to 0.0068. mu. mol/mL, 0.03402. mu. mol/mL, 0.06804. mu. mol/mL, 0.3402. mu. mol/mL, 0.6804. mu. mol/mLMu. mol/mL of the standard strain train solution, 320. mu.L of Tris-HCl buffer (100mmol/L, pH 8.0), 20. mu.L of glucose solution (0.754mmol/mL), and 20. mu.L of NADP were added to a 500. mu.L centrifuge tube in this order+(10mg/mL) of the solution was mixed uniformly, 5. mu.L of each of the above NADPH standard solutions having different concentrations was added, and after mixing, the mixture was immediately poured into boiling water and boiled for 30 seconds, immediately cooled, equilibrated to room temperature, passed through a 0.45 μm filter, and the NADPH peak area was measured at 340 nm. A standard curve was plotted with the peak area of NADPH as the abscissa and the concentration of NADPH as the ordinate, as shown in FIG. 1.
(2) Sample pretreatment
Precisely weighing 100mg of a solid sample in a 25mL volumetric flask, dissolving the solid sample by adopting Tris-HCl buffer solution (100mmol/L, pH 8.0) and fixing the volume to scale, and diluting the solid sample to a proper time by adopting Tris-HCl buffer solution (100mmol/L, pH 8.0) when the solid sample is used temporarily; the liquid sample was directly diluted to an appropriate fold with Tris-HCl buffer (100mmol/L, pH 8.0). The activity of the glucose dehydrogenase in the diluted sample is controlled to be 0.01-1.25U/mL.
(3) Glucose dehydrogenase enzymatic reaction
To a 500. mu.L centrifuge tube were added 320. mu.L of LTris-HCl (100mmol/L, pH 8.0) buffer, 20. mu.L of glucose solution (0.754mmol/mL), and 20. mu.L of NADP in this order+(10mg/mL) solution, after mixing uniformly, adding 5 mu L of solution to be tested, reacting at room temperature for 4min, immediately putting into boiling water, heating and boiling for 30s, immediately cooling, and balancing to room temperature.
(4) HPLC detection of enzymatic reaction product NADPH
After the sample after the enzymatic reaction was filtered through a 0.45 μm filter, the NADPH content was measured by HPLC, and the chromatogram was shown in FIG. 2.
HPLC detection conditions:
a chromatographic column: Welch-C30(4.6 x 250 mm);
wavelength: 340 nm;
column temperature: 28 ℃;
flow rate: 1.0 ml/min;
operating time: 15 min;
elution conditions: sodium acetate solution (pH 5.5): pure methanol 90: 10
(5) Calculation of glucose dehydrogenase Activity
And calculating the enzyme activity of the corresponding solution to be detected according to the content of the NADPH generated in the reaction process of the sample. The enzyme activity calculation formula is as follows:
in the formula: a: peak area of the liquid to be detected; k: the slope of the standard curve; c: intercept of the standard curve; n: dilution factor of the enzyme; m: weighing the weight of the enzyme powder to be detected; u shapeSolid body: enzyme activity of the solid sample; u shapeLiquid, method for producing the same and use thereof: enzymatic activity of the liquid sample; t: reaction time at room temperature, 4 min; vTotal volume: total volume of reaction; vSample volume: sample reaction volume. The results of the enzyme activity measurements are shown in Table 1.
TABLE 1 measurement results of enzyme activity by HPLC method
Example 2 durability examination of HPLC detection method
Normal conditions for HPLC:
a chromatographic column: Welch-C30(4.6 x 250 mm); wavelength: 340 nm;
column temperature: 28 ℃; flow rate: 1.0 ml/min; operating time: 15 min;
elution conditions: sodium acetate solution (pH 5.5): pure methanol 90: 10.
sample information: GDH-BO 009.
Under the condition of properly changing the column temperature, flow rate, detection wavelength, sodium acetate solution concentration, mobile phase proportion and pH of the sodium acetate solution, evaluating the influence of the change of the single-factor chromatographic condition on the detection result, respectively entering 1 needle of NADPH standard solution, reaction liquid of enzyme and substrate and blank solution under each condition, recording a chromatogram, reporting the main peak area, and calculating the enzyme activity.
The results of the enzyme activity measurements are shown in Table 2.
TABLE 2 results of durability examination of HPLC method
The results show that under the condition of ensuring that other chromatographic conditions are the same, the deviation of the measured value of the enzyme activity and the normal condition is less than 5 percent after the above one chromatographic condition is changed. Namely, the HPLC method is durable in the range of 28. + -. 5 ℃ of column temperature, 1.0. + -. 0.2mL/min of flow rate of mobile phase, 86. + -. 43mM of concentration of sodium acetate solution, 340. + -. 10nm of detection wavelength, and 5.5. + -. 0.5 pH.
In the following comparative experiments, the inventors briefly described part of comparative experimental data during the development of a method for determining the activity of glucose dehydrogenase.
Comparative example 1 detection of NADPH by UV method
The inventors used the same sample pretreatment and glucose dehydrogenase enzymatic reaction procedure as in example 1, except that the content of NADPH in the sample after the enzymatic reaction was measured by the UV method:
and (3) taking a 1cm cuvette at the wavelength of 340nm, taking a reaction solution without enzyme solution as a reference, after blank zero adjustment, adding 0.05mL of sample solution to be detected, rapidly timing, reacting for 4min, and recording the final absorbance value A.
A calibration curve was prepared with the absorbance of NADPH (Abs) as the abscissa and the concentration of NADPH (. mu. mol/mL) as the ordinate, and this is shown in FIG. 3. Substituting the peak area of the liquid to be detected after reaction into the standard curve to obtain the content of NADPH generated in the reaction process, thereby calculating the enzyme activity of the corresponding liquid to be detected. The enzyme activity determination formula is as follows:
in the formula: a: absorbance of the solution to be detected; k: the slope of the standard curve; c: intercept of the standard curve; n: dilution factor of the enzyme; m: weighing the weight of the enzyme powder to be detected; u shapeSolid body: enzyme activity of the solid sample; u shapeLiquid, method for producing the same and use thereof: enzymatic activity of the liquid sample; t: reaction time at room temperature, 4 min; vTotal volume: total volume of reaction; vSample volume: sample reaction volume. The results of the enzyme activity measurements are shown in Table 3.
TABLE 3 results of enzyme activity measurement by UV method
Comparative example 2 detection of NADPH by HPLC (C18 column)
The inventors used the same sample pretreatment and enzymatic reaction procedures as in example 1, and then determined the NADPH content of the sample by HPLC, except that two C18 chromatographic columns (Agilent-SBC18, Waters-XB C18) were used. A comparison chromatogram of NADPH detected using a C30 column and two C18 columns is shown in FIG. 4. The retention time of the product NADPH in different columns is shown in Table 4 together with the degree of separation of the blank solvent.
TABLE 4 retention time of NADPH in different columns from the degree of separation of the blank solvent
Under the same conditions of the rest of the chromatograms, the separation effect of Welch-XB C30 was significantly better than that of the rest of the two columns in terms of degree of separation and retention time. Under the separation of Welch-XB C30, the product peak NADPH not only has better separation degree with the solvent peak, but also has stronger retention.
And (4) conclusion: compared with a UV method, the HPLC method is adopted to determine the product without being influenced by insoluble particles and related pigments, and the application range is wide; HPLC (C30 column) has better retention and resolution for analysis of the product NADPH peak than HPLC (C18 column) for determination of NADPH content.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (9)
1. An HPLC detection method of NADPH is characterized in that a reversed phase chromatographic column with a silane bonding phase as a stationary phase is adopted, a mobile phase consists of buffer salt and an organic solvent, the ultraviolet detection wavelength is 330-350 nm, and the flow rate is 0.8-1.2 mL/min.
2. The method of claim 1, wherein the reverse phase chromatography column is a C30 column.
3. The method according to claim 1, wherein the buffer salt is selected from one or more of disodium hydrogen phosphate, sodium acetate, and sodium phosphate; the organic solvent is selected from one or more of methanol, ethanol, isopropanol and acetonitrile.
4. The method of claim 1, wherein the buffer salt has a pH of 5.0 to 6.0.
5. The method according to claim 1, wherein the organic solvent accounts for 8-12% of the total volume of the mobile phase.
6. A method for accurately, qualitatively and quantitatively determining the enzyme activity of glucose dehydrogenase is characterized in that a product NADPH of enzymatic reaction of the glucose dehydrogenase is detected by an HPLC method, and then the enzyme activity of the glucose dehydrogenase in a sample is calculated, and the method specifically comprises the following steps:
(1) drawing a standard curve of NADPH peak area-NADPH concentration;
(2) the occurrence of a glucose dehydrogenase-mediated enzymatic reaction;
mixing Tris-HCl buffer solution with pH of 8.0, glucose solution and NADP+After the solutions are uniformly mixed, adding a glucose dehydrogenase sample diluted by a Tris-HCl buffer solution, reacting at room temperature, heating to boil and cooling to room temperature;
(3) detecting the generation amount of NADPH which is a product of an enzymatic reaction by an HPLC method;
(4) the enzymatic activity of glucose dehydrogenase was calculated from the amount of NADPH produced.
7. The method of claim 6, wherein the HPLC chromatographic conditions in step (3) are as follows:
a chromatographic column: c30 column (4.6 × 250 mm);
detection wavelength: 340 nm; column temperature: 28 ℃;
flow rate: 1.0 ml/min; operating time: 15 min;
mobile phase: sodium acetate solution (pH 5.5) and methanol, the percentage of methanol to the total volume of the mobile phase being 10%.
8. The method according to claim 6, wherein the enzyme activity in step (4) is calculated by the following formula:
in the formula: a: peak area of the liquid to be detected; k: the slope of the standard curve;
c: intercept of the standard curve; n: dilution factor of the enzyme;
m: weighing the weight of the enzyme powder to be detected; t: reaction time;
Usolid body: enzyme activity of the solid sample; u shapeLiquid, method for producing the same and use thereof: enzymatic activity of the liquid sample;
Vtotal volume: total volume of reaction; vSample volume: sample reaction volume.
9. The method of claim 6, wherein the room temperature reaction time T is 4 min.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101186640A (en) * | 2007-11-30 | 2008-05-28 | 宁夏回族自治区水产研究所 | Transcription factor for regulating and controlling catfish degradation pyrethrin pesticide and its coding gene and application |
| CN102618590A (en) * | 2012-05-07 | 2012-08-01 | 江南大学 | Method for preparing (R)-2-hydroxy-4-phenyl ethyl butyrate by catalyzing with recombinant carbonyl reductase |
| CN102634566A (en) * | 2012-04-20 | 2012-08-15 | 宁波大学 | Method for measuring activity of cellobiase in copepods |
| CN106754777A (en) * | 2016-12-28 | 2017-05-31 | 江苏阿尔法药业有限公司 | A kind of GDH mutant and its preparation method and application |
| CN108132318A (en) * | 2016-12-01 | 2018-06-08 | 重庆本贝得生物工程技术研究院有限公司 | A kind of isolation and purification method of NADPH |
| CN108303475A (en) * | 2017-12-25 | 2018-07-20 | 新绎健康科技有限公司 | A kind of detection method of energy matter |
-
2019
- 2019-02-16 CN CN201910118351.5A patent/CN110618203A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101186640A (en) * | 2007-11-30 | 2008-05-28 | 宁夏回族自治区水产研究所 | Transcription factor for regulating and controlling catfish degradation pyrethrin pesticide and its coding gene and application |
| CN102634566A (en) * | 2012-04-20 | 2012-08-15 | 宁波大学 | Method for measuring activity of cellobiase in copepods |
| CN102618590A (en) * | 2012-05-07 | 2012-08-01 | 江南大学 | Method for preparing (R)-2-hydroxy-4-phenyl ethyl butyrate by catalyzing with recombinant carbonyl reductase |
| CN108132318A (en) * | 2016-12-01 | 2018-06-08 | 重庆本贝得生物工程技术研究院有限公司 | A kind of isolation and purification method of NADPH |
| CN106754777A (en) * | 2016-12-28 | 2017-05-31 | 江苏阿尔法药业有限公司 | A kind of GDH mutant and its preparation method and application |
| CN108303475A (en) * | 2017-12-25 | 2018-07-20 | 新绎健康科技有限公司 | A kind of detection method of energy matter |
Non-Patent Citations (4)
| Title |
|---|
| MASAYUKI AKIMOTO 等: "The in vitro metabolism of desglymidodrine,an active metabolite of prodrug midodrine by human liver microsomes", 《EUROPEAN JOURNAL OF DRUG METABOLISM AND PHARMACOKINETICS》 * |
| 于刚 等: "高效液相色谱法测定3-羟基-3-甲基戊二酸单酰辅酶Ａ还原酶抑制剂的活性", 《分析化学》 * |
| 吕圭源 等: "肿节风提取物抗肿瘤的实验研究", 《中草药》 * |
| 许理 等: "高效液相色谱法测定鱼腥草素对鸭肝脂肪酸合成酶活性的影响", 《分析化学》 * |
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