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Glucose dehydrogenase enzyme activity accurate qualitative and quantitative method

A technology of stationary phase and mobile phase, applied in the field of enzyme activity detection, to achieve mild reaction conditions, wide application range, and good stability of the method

Inactive Publication Date: 2019-12-27
HEC PHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The present invention will solve the problem of accurate qualitative and quantitative enzymatic activity of glucose dehydrogenase, firstly, it needs to solve the detection problem of NADPH, the product of enzymatic reaction of glucose dehydrogenase

Method used

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  • Glucose dehydrogenase enzyme activity accurate qualitative and quantitative method
  • Glucose dehydrogenase enzyme activity accurate qualitative and quantitative method
  • Glucose dehydrogenase enzyme activity accurate qualitative and quantitative method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1 glucose dehydrogenase enzyme activity assay step

[0054] (1) Drawing of NADPH concentration-NADPH peak area standard curve

[0055] Weigh about 25 mg of NADPH standard (Bangtai Biology) into a 25 mL volumetric flask, dissolve it with Tris-HCl buffer (100 mmol / L, pH 8.0) and set the volume to the mark, and then dilute to 0.0068 μmol / mL, 0.03402 μmol / mL , 0.06804μmol / mL, 0.3402μmol / mL, 0.6804μmol / mL standard solution series, add 320μL Tris-HCl buffer (100mmol / L, pH 8.0), 20μL glucose solution (0.754mmol / mL) and 20 μL NADP + (10mg / mL) solution mixed evenly, add 5 μL of NADPH standard solution of different concentrations above respectively, put into boiling water and boil for 30s immediately after mixing, cool immediately, equilibrate to room temperature, pass through 0.45μm filter membrane, measure NADPH at 340nm Peak area. Take the peak area of ​​NADPH as the abscissa, and the concentration of NADPH as the ordinate to draw a standard curve, see figure 1...

Embodiment 2

[0076] Durability investigation of embodiment 2HPLC detection method

[0077] HPLC normal conditions:

[0078] Chromatographic column: Welch-C30 (4.6*250mm); Wavelength: 340nm;

[0079] Column temperature: 28°C; flow rate: 1.0ml / min; running time: 15min;

[0080] Elution conditions: sodium acetate solution (pH = 5.5): pure methanol = 90:10.

[0081] Sample information: GDH-BO009.

[0082] In the case of appropriately changing column temperature, flow rate, detection wavelength, concentration of sodium acetate solution, mobile phase ratio, and pH of sodium acetate solution, evaluate the influence of single-factor chromatographic conditions on the detection results. Under each condition, NADPH standard solution, The reaction solution of the enzyme and the substrate, and the blank solution were each injected into a needle, and the chromatogram was recorded, and the area of ​​the main peak was reported, and the enzyme activity was calculated.

[0083] The enzyme activity detec...

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Abstract

The present invention relates to the technical field of enzyme activity detection, and specifically relates to a glucose dehydrogenase enzyme activity accurate qualitative and quantitative method. According to the method, an HPLC detection method of a product NADPH of a glucose dehydrogenase enzymatic reaction is first provided. In the method, a reversed phase column in which a silane bonded phaseis used as a stationary phase is used, a moving phase consists of buffer salt and an organic solvent, an ultraviolet detect wavelength is 330 to 350 nm, and a flow velocity is 0.8 to 1.2 mL / min, so as to provide the glucose dehydrogenase enzyme activity accurate qualitative and quantitative method. The detection method provided in the present invention is simple in operation, high in sensitivity,strong in accuracy, less vulnerable to insoluble particles and some pigments in a complicated sample, and is easy for standard operation.

Description

technical field [0001] The invention relates to the technical field of enzyme activity detection, in particular to a method for accurately qualitative and quantitative glucose dehydrogenase enzyme activity. Background technique [0002] Glucose Dehydrogenase (Glucose Dehydrogenase, E.C.1.1.1.47, referred to as GDH) mainly exists in the liver of animals and Acetobacter suboxidans, and can catalyze β-D-glucose to generate gluconic acid (D-glucono-δ-lactone ), accompanied by the generation of the reducing coenzyme NADPH. As early as 1975, Banauch et al. used glucose dehydrogenase as a diagnostic enzyme for clinical blood glucose determination to measure the concentration of glucose in human body fluids. Currently, glucose dehydrogenase is mainly used in clinical glucose monitoring sensors and NADPH regeneration mediated by NADP-dependent glucose dehydrogenase. [0003] At present, the main detection methods of glucose dehydrogenase activity include spectrophotometry, ion chro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02
CPCG01N30/02
Inventor 屈代鑫张嘉杰谢文平张馨月梁在华黄小龙庞嘉颖麦倩婷
Owner HEC PHARM
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