Application of NADP type glucose dehydrogenase in detection of NAD kinase activity and detection method

Abstract

The invention provides application of NADP type glucose dehydrogenase in detection of NAD kinase activity and a detection method, and belongs to the technical field of enzyme activity detection. The invention provides application of NADP (Nicotinamide Adenine Dinucleotide Phosphate) type glucose dehydrogenase in detection of NAD (Nicotinamide Adenine Dinucleotide) kinase activity. The amino acid sequence of the NADP type glucose dehydrogenase is shown as SEQ ID NO: 1. The reaction rate of the NADP type glucose dehydrogenase obtained in a recombinant expression mode and NADP < + > is high, the NADP type glucose dehydrogenase hardly reacts with NAD < + >, and the activity of the NAD kinase is calculated according to the generation rate of NADPH by performing enzyme coupling reaction with the NAD kinase. The detection result shows that the glucose dehydrogenase can be used for detecting the activity of NAD kinase.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme activity detection, and particularly relates to the application of NADP-type glucose dehydrogenase in detecting NAD kinase activity and a detection method therefor. Background Art

[0002] NAD kinase (NADK) is widely present in animals, plants, microorganisms and cultured cells, and is the only enzyme found in organisms that can catalyze the phosphorylation of NAD + to generate NADP + It can catalyze the phosphorylation reaction of NAD(H) with ATP or inorganic polyphosphate [poly(P)] as the phosphoryl donor to generate NADP(H). Therefore, NAD kinase plays an important role in the synthesis of NADP(H) and the regulation of the balance between NAD(H) and NADP(H).

[0003] Currently, the detection principle of the general NADK activity detection kit is that NADP + can be reduced by glucose-6-phosphate dehydrogenase to NADPH, and NADPH has a characteristic absorption peak at 340 nm. By measuring the increase rate of NADPH (the change in absorbance value) at 340 nm, the activity of NADK can be reflected. Since the coenzyme specificity of most glucose dehydrogenases is relatively low, they not only react with NADP + but also react with NAD + Therefore, when detecting NAD kinase activity, most glucose dehydrogenases cannot reflect the activity of NAD kinase. Summary of the Invention

[0004] In view of this, the present invention provides the application of NADP-type glucose dehydrogenase in detecting NAD kinase activity. The coenzyme specificity of the glucose dehydrogenase GDH is NADP-type, and it can accurately reflect the activity of NAD kinase.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides the application of NADP-type glucose dehydrogenase in detecting NAD kinase activity. The amino acid sequence of the NADP-type glucose dehydrogenase is as shown in SEQ ID NO: 1.

[0007] Preferably, the glucose dehydrogenase is prepared by expression of a recombinant strain;

[0008] The host bacterium of the recombinant strain includes Escherichia coli Rosetta(DE3).

[0009] The present invention provides a kit for detecting the activity of NAD kinase, comprising NADP-dependent glucose dehydrogenase and a substrate;

[0010] The amino acid sequence of the NADP-dependent glucose dehydrogenase is as shown in SEQ ID NO: 1.

[0011] Preferably, the substrate comprises adenosine triphosphate, NAD + and glucose.

[0012] The present invention provides a method for detecting the enzyme activity of NAD kinase. The NADP-dependent glucose dehydrogenase, the substrate and the sample of the NAD kinase to be detected in the kit are mixed for reaction, and the enzyme activity of the NAD kinase is obtained according to the generation rate of NADPH during the mixing reaction.

[0013] Preferably, the system of the mixing reaction comprises components with the following concentrations: Tris-HCl 43.75 mM, magnesium ion 2 mM, adenosine triphosphate 2 mM, NAD + 0.2 mM, glucose 5 mM, NADP-dependent glucose dehydrogenase 0.0021875 mg / mL, and the sample of the NAD kinase to be detected ≤ 5 mg / mL.

[0014] Preferably, the time of the mixing reaction is greater than 5 s.

[0015] Preferably, the temperature of the mixing reaction is 30 °C.

[0016] Preferably, the generation rate of NADPH is obtained by continuously monitoring the rising rate of the absorbance of NADPH at 340 nm during the linear period of the mixing reaction;

[0017] The calculation formula of the generation rate of the NADPH is as shown in Formula I:

[0018] Generation rate of NADPH (v) = [(A1 - A0) × V ÷ (ε × d)] ÷ (T1 - T0) Formula I;

[0019] wherein, T0 is the starting time of the linear period of the mixing reaction, T1 is the ending time of the linear period of the mixing reaction, A0 is the absorbance value at 340 nm at T0, A1 is the absorbance value at 340 nm at T1, V is the total volume of the system of the mixing reaction; ε is the molar extinction coefficient of NADPH ε340, ε340 = 6220 L / (mol·cm); d is the optical path length of the cuvette.

[0020] Preferably, 1 enzyme activity unit of the NAD kinase is the amount of enzyme required to produce 1 μmol of NADPH within 1 min under the condition of 30 °C.

[0021] The present invention has the following advantages compared with the prior art:

[0022] The present invention provides an application of NADP-dependent glucose dehydrogenase in detecting the activity of NAD kinase. The amino acid sequence of the NADP-dependent glucose dehydrogenase is shown as SEQ ID NO: 1. The coenzyme specificity of the NADP-dependent glucose dehydrogenase GDH is of the NADP type, and it can carry out an enzyme-coupled reaction with NAD kinase for detecting the activity of NAD kinase. When detecting the activity of NAD kinase, NADK present in the sample catalyzes the phosphorylation of NAD + to generate NADP + ; NADP + can be reduced to NADPH by the NADP-dependent glucose dehydrogenase of the present invention. NADPH has a characteristic absorption peak at 340 nm. By measuring the increasing rate of NADPH (the change in absorbance value) at 340 nm, the activity of NADK can be reflected. In the examples of the present invention, the coenzyme characteristics of the NADP-dependent glucose dehydrogenase were detected. The results showed that the reaction rate of the NADP-dependent glucose dehydrogenase with NADP is very high, while the reaction rate with NAD + is almost 0. Therefore, when the NADP-dependent glucose dehydrogenase is used to detect the activity of NAD kinase, it can accurately reflect the activity of NAD kinase.

[0023] Based on the fact that when the NADP-dependent glucose dehydrogenase is used to detect the activity of NAD kinase, it can accurately reflect the activity of NAD kinase, the present invention provides a kit for detecting the activity of NAD kinase, which includes the NADP-dependent glucose dehydrogenase and a substrate. The kit of the present invention can accurately detect the activity of NAD kinase.

[0024] The present invention provides a method for detecting the enzyme activity of NAD kinase. The NADP-dependent glucose dehydrogenase, the substrate and the sample of the NAD kinase to be detected in the kit are mixed for reaction, and the enzyme activity of the NAD kinase is obtained according to the generation rate of NADPH during the reaction. The results of the examples of the present invention show that the method can accurately detect the enzyme activity and specific activity of NAD kinase. In addition, the detection equipment required for the method of the present invention is only an ultraviolet spectrophotometer, and the operation is simple, and it can be applied to the detection of a large number of enzyme activities. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the schematic diagram of detecting the activity of NAD kinase by the enzyme-coupled method;

[0026] Figure 2 is the protein detection electrophoresis diagram of the recombinantly expressed NADP-dependent glucose dehydrogenase;

[0027] Figure 3 is the activity detection result diagram of the NADP-dependent glucose dehydrogenase recombinantly expressed in BL21(DE3);

[0028] Figure 4 The figure shows the detection results of the activity of NADP-dependent glucose dehydrogenase recombinantly expressed in Rosetta(DE3);

[0029] Figure 5 The figure shows the detection results with different amounts of NADK added. Specific implementation manners

[0030] The present invention provides an application of NADP-dependent glucose dehydrogenase in detecting the activity of NAD kinase, and the amino acid sequence of the NADP-dependent glucose dehydrogenase is as shown in SEQ ID NO: 1.

[0031] In the present invention, the NADP-dependent glucose dehydrogenase is glucose 1-dehydrogenase, which specifically catalyzes only NADP + , and cannot catalyze NAD + reaction. The NADP-dependent glucose dehydrogenase is preferably prepared by expression with a recombinant strain; the host bacterium of the recombinant strain preferably includes Escherichia coli Rosetta(DE3). The recombinant strain preferably contains a recombinant vector. The backbone vector of the recombinant vector preferably includes pBAD-HISA(Amp). The preparation method of the recombinant vector preferably includes cloning the coding gene of the NADP-dependent glucose dehydrogenase into the NcoI and XhoI multiple cloning sites of the backbone vector. The nucleotide sequence of the coding gene of the NADP-dependent glucose dehydrogenase is as shown in SEQ ID NO: 2. Examples of the present invention compared the effects of different host bacteria on the enzyme activity of the recombinantly expressed NADP-dependent glucose dehydrogenase. The results showed that when the host bacterium was Escherichia coli Rosetta(DE3), the activity of the recombinantly expressed NADP-dependent glucose dehydrogenase was higher than that when the host bacterium was Escherichia coli BL21(DE3).

[0032] In the present invention, the NADP-dependent glucose dehydrogenase (GDH) can perform an enzyme-coupled reaction with NAD kinase to detect the activity of NAD kinase. When detecting the activity of NAD kinase, NADK present in the sample catalyzes the phosphorylation of NAD + to generate NADP + ; NADP + can be reduced to NADPH by the NADP-dependent glucose dehydrogenase of the present invention. NADPH has a characteristic absorption peak at 340 nm. By measuring the increase rate of NADPH (the change in absorbance value) at 340 nm, the magnitude of NADK activity can be reflected ( Figure 1 ). Examples of the present invention detected the coenzyme characteristics of the NADP-dependent glucose dehydrogenase. The results showed that the reaction rate of the NADP-dependent glucose dehydrogenase with NADP was very high (reaching the end point within 1 minute), while with NAD +The reaction rate is almost zero. Therefore, when the NADP-type glucose dehydrogenase is used to detect the NAD kinase activity, it can accurately reflect the activity of the NAD kinase. In the embodiments of the present invention, the enzyme-coupled analysis method is used to detect the NAD kinase activity with the NADP-type glucose dehydrogenase, and the detection results are consistent with those of the commercial kit, and can accurately reflect the NAD kinase activity.

[0033] Based on the fact that when the NADP-type glucose dehydrogenase is used to detect the NAD kinase activity, it can accurately reflect the activity of the NAD kinase, the present invention provides a kit for detecting the NAD kinase activity, which includes the NADP-type glucose dehydrogenase and a substrate.

[0034] In the present invention, the amino acid sequence and the preparation method of the NADP-type glucose dehydrogenase are preferably the same as those in the above application, and will not be elaborated here. The substrate preferably includes adenosine triphosphate, NAD + and glucose. The kit preferably further includes a buffer solution. The buffer solution preferably includes Tris-HCl and magnesium ions. The buffer solution can maintain a stable pH environment, which is beneficial to accurately reflecting the NAD kinase activity during the detection.

[0035] The present invention provides a method for detecting the enzyme activity of the NAD kinase. The NADP-type glucose dehydrogenase, the substrate and the sample of the NAD kinase to be detected in the kit are mixed and reacted, and the enzyme activity of the NAD kinase is obtained according to the generation rate of NADPH during the reaction.

[0036] In the present invention, when preparing the mixed reaction system, it is preferably to mix the substrate and the buffer solution first, then add the NADP-type glucose dehydrogenase, and finally add the sample of the NAD kinase to be detected. The total volume of the mixed reaction system is preferably 1000 μL, the total volume of the substrate and the buffer solution is preferably 875 μL, the volume of the NADP-type glucose dehydrogenase is preferably 25 μL, and the volume of the NAD kinase sample is preferably 100 μL. The mixed reaction system preferably includes the following components at the following concentrations: Tris-HCl 43.75 mM, magnesium ions 2 mM, adenosine triphosphate 2 mM, NAD + 0.2 mM, glucose 5 mM, NADP-type glucose dehydrogenase 0.0021875 mg / mL, and the sample of the NAD kinase to be detected ≤ 5 mg / mL. In the mixed reaction system, the NADP-type glucose dehydrogenase can specifically react with NADP + and does not react with NAD + , and the generation rate of NADPH can accurately reflect the activity of the NAD kinase.

[0037] In the present invention, the temperature of the mixed reaction is preferably 30 °C. If the absorbance value at the initial 5 s of the reaction is greater than 1, it is preferred to dilute the NAD kinase sample with buffer Tris-HCl before measurement. The time of the mixed reaction is preferably greater than 5 s, more preferably 15 - 600 s, and further preferably 20 - 300 s.

[0038] In the present invention, the generation rate of NADPH is obtained by continuously monitoring the rising rate of the absorbance of NADPH at 340 nm during the linear period of the mixed reaction;

[0039] The calculation formula for the generation rate of NADPH is as shown in Formula I:

[0040] Generation rate of NADPH (v) = [(A1 - A0) × V ÷ (ε × d)] ÷ (T1 - T0) Formula I;

[0041] Wherein, T0 is the starting time of the linear period of the mixed reaction, T1 is the ending time of the linear period of the mixed reaction, A0 is the absorbance value at 340 nm at T0, A1 is the absorbance value at 340 nm at T1, V is the total volume of the system of the mixed reaction; ε is the molar extinction coefficient of NADPH ε340, ε340 = 6220 L / (mol·cm);

[0042] d is the optical path length of the cuvette.

[0043] In the present invention, during the mixed reaction, it is preferred to continuously monitor the absorbance value at 340 nm by spectrophotometry to obtain a time-absorbance curve, continuously select the absorbance values in the linear period of the mixed reaction (the difference in absorbance between two points is equal) in the time-absorbance curve, and obtain the generation rate of NADPH from the rising rate of the absorbance in this linear period of the mixed reaction. The linear period of the mixed reaction is preferably the 5th - 600th s of the mixed reaction, more preferably 15 - 400 s, further preferably 20 - 300 s, and most preferably 40 - 240 s.

[0044] In the present invention, 1 enzyme activity unit of the NAD kinase is preferably the amount of enzyme required to produce 1 μmol of NADPH within 1 min under the condition of 30 °C. Therefore, the enzyme activity of the NAD kinase can be obtained according to the generation rate of NADPH.

[0045] In the present invention, the method can preferably also be used to calculate the specific activity of the NAD kinase. The calculation formula for the specific activity of the NAD kinase is preferably as shown in Formula II:

[0046] Specific activity of NAD kinase = v / m Formula II;

[0047] Wherein, v is the generation rate of NADPH, and m is the mass of the NAD kinase in the system of the mixed reaction.

[0048] Embodiments of the present invention show that the method can accurately detect the enzyme activity and specific activity of NAD kinase.

[0049] To further illustrate the present invention, the following describes in detail the application of a NADP-dependent glucose dehydrogenase in the detection of NAD kinase activity and the detection method provided by the present invention in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0050] Example 1

[0051] Preparation of NADP-dependent glucose dehydrogenase GDH

[0052] 1. Nucleic acid sequence and amino acid sequence of NADP-dependent glucose dehydrogenase GDH

[0053] The nucleotide sequence of the encoding gene of NADP-dependent glucose dehydrogenase GDH is shown in SEQ ID NO:1: ATGCCGGCGCCGTATAAAGATCGTTTTGCGGGCAAAAAAGTGCTGGTGACCGGCGCGAGCCAGGGCATTGGCGAAGCGACCGCGCTGCGTTTTGCGGAAGAAGGCGCGCAGGTGGCGCTGAACGGCCGTAAAGAAGATAAACTGATTGCGGTGCGTGAAAAACTGCCGAAAGTGAGCGGCGGCGAACATCCGATTGCGACCGGCGATATTAGCAAAGAAGATGATGTGAAACGTCTGGTGGCGGAAAGCATTAAAGCGATGGGCGGCCTGGATGTGCTGGTGTGCAACGCGGGCTATCAGATTCCGAGCCCGAGCGAAGATATTAAACTGGAAGATTTTGAAGGCGTGATGGCGGTGAACGTGACCGGCGTGATGCTGCCGTGCCGTGAAGTGATTCGTTATTGGCTGGAAAACGGCATTAAAGGCACCATTATTGTGAACAGCAGCGTGCATCAGATTATTCCGAAACCGCATTATCTGGGCTATAGCGCGAGCAAAGGCGCGGTGGGCAACATTGTGCGTACCCTGGCGCTGGAATATGCGACCCGTGGCATTCGTGTGAACGCGGTGGCGCCGGGCGCGATTGTGACCCCGATTAACATGAGCTGGATTGATGATCCGGAACAGTATAAAGCGGTGAGCAGCCATATTCCGATGAAACGTCCGGGCGAAAGCCGTGAAATTGCGGATGCGATTACCTTTCTGGCGGCGGAAGATAGCACCTATATTACCGGCCAGACCCTGTATGTGGATGGCGGCCTGACCCTGTATGGCGATTTTGAAAACAACTGGAGCAGCTGA。

[0054] The amino acid sequence of NADP-dependent glucose dehydrogenase GDH is shown in SEQ ID NO:2:

[0055] MPAPYKDRFAGKKVLVTGASQGIGEATALRFAEEGAQVALNGRKEDKLIAVREKLPKVSGGEHPIATGDISKEDDVKRLVAESIKAMGGLDVLVCNAGYQIPSPSEDIKLEDFEGVMAVNVTGVMLPCREVIRYWLENGIKGTIIVNSSVHQIIPKPHYLGYSASKGAVGNIVRTLALEYATRGIRVNAVAPGAIVTPINMSWIDDPEQYKAVSSHIPMKRPGESREIADAITFLAAEDSTYITGQTLYVDGGLTLYGDFENNWSS。

[0056] 2. Method for constructing a genetically engineered strain recombinantly expressing NADP-dependent glucose dehydrogenase. The gene encoding NADP-dependent glucose dehydrogenase was cloned into the NcoI and XhoI multiple cloning sites of the backbone vector pBAD-HisA (Amp) (induced by 0.2% arabinose), and the recombinant vector expressing NADP-dependent glucose dehydrogenase was separately transformed into BL21(DE3) and Rosetta(DE3) for expression.

[0057] pBAD-HISA was purchased from Tsingke Biotechnology Co., Ltd., BL21(DE3) was purchased from Sangon Biotech (Shanghai) Co., Ltd., and Rosetta(DE3) was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0058] 3. Expression and purification of NADP-dependent glucose dehydrogenase GDH

[0059] PBS buffer: 2 mM disodium hydrogen phosphate, 0.4 mM potassium dihydrogen phosphate, 0.54 mM potassium chloride, 27.4 mM sodium chloride, pH 7.2 - 7.4.

[0060] The expression steps of NADP-dependent glucose dehydrogenase are as follows:

[0061] a) Inoculate the seed solution of the genetically engineered strain into 500 mL of LB medium (containing the corresponding resistance) at an inoculation amount of 1%.

[0062] b) Incubate in a shaker (37 °C, 220 rpm) for 3 - 5 h until the OD 600 is approximately 0.6 - 0.8;

[0063] c) Add L-arabinose with a final concentration of 0.2% and induce overnight at 25 °C to obtain the fermentation broth.

[0064] The purification steps of NADP-dependent glucose dehydrogenase GDH are as follows.

[0065] a) Centrifugation: Centrifuge the above fermentation broth at 6000 rpm for 30 min, discard the supernatant and retain the cells.

[0066] b) Disruption: Add PBS buffer (pH 7.2 - 7.4) to the cells and disrupt the cells using a high-pressure homogenizer.

[0067] c) Centrifugation: Centrifuge the disrupted solution at 15000 rpm for 30 min, remove the cell debris, and collect the supernatant.

[0068] d) Filtration: Further remove large particle impurities from the supernatant after centrifugation through a 0.22 μm filter membrane.

[0069] e) Ni column purification: Equilibrate the column: Wash with PBS (pH 7.2 - 7.4) buffer; Load the sample: Load the filtered crude enzyme solution; Equilibrate the column: Wash with PBS buffer containing 10 mM imidazole; Obtain the target protein: Gradient elute with PBS buffer (pH 8.0) containing 250 mM imidazole; Preserve the column: After obtaining the target protein NADP-dependent glucose dehydrogenase, wash with ultrapure water and store in 20% ethanol solution.

[0070] f) Protein concentration: The size of NADP-dependent glucose dehydrogenase is approximately 28 KDa, see the protein gel diagram in Figure 2 , select a 10 KDa ultrafiltration tube to remove imidazole by centrifugation and concentrate the protein.

[0071] g) Protein concentration detection: Detect the protein concentration using a Bradford kit.

[0072] 4. The enzyme activity detection method of NADP-dependent glucose dehydrogenase (GDH) is as follows:

[0073] Prepare the detection system:

[0074] NAD + The total volume of the detection system is 1000 μL, including 975 μL of the detection reagent and 25 μL of NADP-dependent glucose dehydrogenase. The detection reagent is a 50 mM Tris-HCl solution containing NAD + and glucose (Glu), with a pH value of 7.4. The final concentrations of each component in the detection system are NAD + 200 μM, Glu 5 mM, and NADP-dependent glucose dehydrogenase 0.034 mg / ml or 0.0021875 mg / ml;

[0075] NADP + The total volume of the reaction system is 1000 μL, including 975 μL of the detection reagent and 25 μL of NADP-dependent glucose dehydrogenase. The detection reagent is a 50 mM Tris-HCl solution containing NADP +A 50 mM Tris-HCl solution of glucose (Glu) with a pH value of 7.4. The final concentrations of each component in the detection system are NADP + 200 μM, Glu 5 mM, and NADP-dependent glucose dehydrogenase 0.034 mg / ml or 0.0021875 mg / ml;

[0076] Detect the enzyme kinetic curve: Use an ultraviolet spectrophotometer to detect, set the time to continuously monitor for 10 min, and the wavelength to 340 nm to obtain the enzyme kinetic curve.

[0077] The results show that the NADP-dependent glucose dehydrogenase recombinantly expressed by BL21(DE3) has weak activity ( Figure 3 ), and the NADP-dependent glucose dehydrogenase recombinantly expressed by Rosetta(DE3) has high activity ( Figure 4 ). The NADP-dependent glucose dehydrogenase produced by Rosetta(DE3) is denoted as GDH023, and GDH023 is used for subsequent experiments.

[0078] Example 2

[0079] Investigate the coenzyme characteristics of GDH023

[0080] According to the reaction detection results of GDH023 with NAD + and NADP + in the method of Example 1 ( Figure 4 ), it can be seen that GDH023 with a final concentration of 0.0021875 mg / ml (added concentration of 0.0875 mg / ml, added volume of 25 μL) has a very high reaction rate with NADP + (reaching the end point within 1 min), and the reaction rate with NAD + is almost 0. It has strong coenzyme characteristics and is an NADP-dependent glucose dehydrogenase, which can be used for subsequent determination of the activity of NAD kinase.

[0081] Example 3

[0082] NADK addition amount and detection time

[0083] a) Prepare the detection solution: Add 38.08 mg of MgCl2, 202.87 mg of ATP, NAD + 26.5 mg, and 180.16 mg of Glu to 175 mL of 50 mM Tris-HCl buffer, and adjust the pH value to 7.4;

[0084] b) Use an ultraviolet spectrophotometer to detect the enzyme kinetic curve. Set the time to 10 min and the wavelength to 340 nm; add 875 μL of the detection solution to a 1 mL cuvette, click "blank"; then add 25 μL of 0.0875 mg / ml GDH023 and click "start detection", and immediately add 100 μL, 75 μL or 25 μL of 11.11 mg / mL NADK enzyme sample solution, shake well, continuously monitor for 10 min, and save the data.

[0085] The enzyme kinetic curves after continuously monitoring for 10 min after adding different volumes of NADK-1 enzyme are as Figure 5 shown. According to Figure 5 it can be seen that when the amount of NAD kinase in the system is 100 μL and the detection time is 10 min, the end absorbance value is 0.963.

[0086] Example 4

[0087] Enzyme activity detection of NADK sample 1 (NADK-1)

[0088] a) Prepare the detection solution: Add 38.08 mg of MgCl2, 202.87 mg of ATP, 26.5 mg of NAD + and 180.16 mg of Glu to 175 mL of 50 mM Tris-HCl buffer, and adjust the pH value to 7.4;

[0089] b) Use an ultraviolet spectrophotometer to detect the enzyme kinetic curve. Set the time to 10 min and the wavelength to 340 nm; add 875 μL of the detection solution to a 1 mL cuvette, click "blank"; then add 25 μL of 0.0875 mg / ml GDH023 and click "start detection", and immediately add 100 μL of 11.11 mg / mL NADK-1 enzyme solution, shake well, continuously monitor for 10 min, and save the data.

[0090] Monitor the enzyme kinetic curve for 10 min, continuously select the absorbance values in the linear phase of the enzyme kinetic curve, and calculate the generation rate of NADPH based on the change value of the unit absorbance in this linear phase, and then obtain the enzyme activity of NAD kinase. The calculation formula for the generation rate of NADPH is shown in Equation I:

[0091] Generation rate of NADPH (v) = [(A1 - A0) × V ÷ (ε × d)] ÷ (T1 - T0) Equation I;

[0092] Among them, T0 is the starting time of the linear period of the mixing reaction, T1 is the ending time of the linear period of the mixing reaction, A0 is the absorbance value at 340 nm at T0, A1 is the absorbance value at 340 nm at T1, V is the total volume of the system of the mixing reaction; ε is the molar extinction coefficient of NADPH ε340, ε340 = 6220 L / (mol·cm);

[0093] d is the optical path length of the cuvette.

[0094] The calculation formula for the specific activity of NAD kinase is shown in Formula II:

[0095] Specific activity of NAD kinase = v / m Formula II;

[0096] Among them, v is the generation rate of NADPH, and m is the mass of NAD kinase in the system of the mixing reaction.

[0097] Based on the data of the linear period and the molar extinction coefficient of NADPH ε340 = 6220 L / (mol·cm), (A1 - A0) is 0.288, V is 1 mL, d is 1 cm; (T1 - T0) is 3.334 min, the enzyme activity of NADK sample 1 enzyme (NADK-1) can be obtained as 0.013887898, and the specific enzyme activity is 12.5 U / g.

[0098] Example 5

[0099] Enzyme activity detection of NADK sample 2

[0100] The detection method is the same as that in Example 4, except that the batches of NAD kinase are different.

[0101] Continuously monitor the enzyme kinetic curve for 10 min, and calculate the specific activity according to the method in Example 4. The specific activity of NADK sample 2 enzyme (NADK-2) can be obtained as 202.16 U / mg.

[0102] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. Other embodiments can also be obtained according to these embodiments without creative work, and these embodiments all belong to the protection scope of the present invention.

Claims

1. Use of an NADP-type glucose dehydrogenase in detecting NAD kinase activity, wherein the amino acid sequence of the NADP-type glucose dehydrogenase is as shown in SEQ ID NO:

1.

2. The application according to claim 1, wherein The glucose dehydrogenase is prepared by expression of a recombinant strain; The host bacterium of the recombinant strain includes Escherichia coli Rosetta(DE3).

3. A kit for detecting NAD kinase activity, comprising an NADP-type glucose dehydrogenase and a substrate; The amino acid sequence of the NADP-type glucose dehydrogenase is as shown in SEQ ID NO:

1.

4. The kit according to claim 3, wherein The substrate includes adenosine triphosphate, NAD + and glucose.

5. A method for detecting the enzyme activity of NAD kinase, characterized in that, Mix the NADP-type glucose dehydrogenase, the substrate and the sample of the NAD kinase to be detected in the kit according to claim 4, and obtain the enzyme activity of the NAD kinase according to the generation rate of NADPH during the mixing reaction.

6. The method according to claim 5, characterized in that The system of the mixed reaction comprises components with the following concentrations: Tris-HCl 43.75 mM, magnesium ions 2 mM, adenosine triphosphate 2 mM, NAD + 0.2 mM, glucose 5 mM, NADP-dependent glucose dehydrogenase 0.0021875 mg / mL, and the NAD kinase sample to be tested ≤ 5 mg / mL.

7. The method according to claim 5, characterized in that The time of the mixing reaction is greater than 5 s.

8. The method according to claim 5, wherein The temperature of the mixing reaction is 30 °C.

9. The method according to claim 5, characterized in that The generation rate of NADPH is obtained by continuously monitoring the rising rate of the absorbance of NADPH at 340 nm during the linear period of the mixing reaction; The calculation formula of the generation rate of NADPH is as shown in Formula I: Generation rate of NADPH (v) = [(A1 - A0) × V ÷ (ε × d)] ÷ (T1 - T0) Formula I; Wherein, T0 is the starting time of the linear period of the mixing reaction, T1 is the ending time of the linear period of the mixing reaction, A0 is the absorbance value at 340 nm at T0, A1 is the absorbance value at 340 nm at T1, V is the total volume of the mixing reaction system; ε is the molar extinction coefficient of NADPH ε340, ε340 = 6220 L / (mol·cm); d is the optical path length of the cuvette.

10. According to the method according to any one of claims 5 to 9, 1 enzyme activity unit of the NAD kinase is the amount of enzyme required to produce 1 μmol of NADPH within 1 min under the condition of 30 °C.

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