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Method for detecting flavoprotein based on three-dimensional fluorescence spectrum

A three-dimensional fluorescence, flavoprotein technology, applied in the field of enzyme protein detection, can solve the problems affecting the accurate, rapid and high-sensitivity of flavoprotein to characterize the target protein, and achieve the effects of high accuracy, high sensitivity and simple operation.

Inactive Publication Date: 2021-08-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to detect the flavoprotein quickly and accurately, and solve the technical problem that the impurity protein in the flavoprotein crude enzyme liquid fermented by genetically engineered bacteria affects the flavoprotein accurate, fast and highly sensitive qualitative target protein

Method used

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  • Method for detecting flavoprotein based on three-dimensional fluorescence spectrum
  • Method for detecting flavoprotein based on three-dimensional fluorescence spectrum
  • Method for detecting flavoprotein based on three-dimensional fluorescence spectrum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1. Utilize the method of three-dimensional fluorescence spectrum to detect flavoprotein

[0040] Detection of sarcosine oxidase (SOX enzyme)

[0041] 1. Prepare 5 μmol FAD solution, store it away from light, take 3mL FAD solution, and use the F-7000 fluorescence spectrometer to collect the FAD spectrum under the condition of PMT voltage of 700V. image 3 As shown, the results show that the FAD fluorescence characteristic peaks are in the range of λex=330-500nm and λem=500-600nm, and the maximum fluorescence intensity of the FAD fluorescence peak appears at λex / λem=396 / 456nm.

[0042] 2. Preparation of recombinant sarcosine oxidase (SOX) pure enzyme

[0043] The sarcosine oxidase gene (GenBank: AY626822.2) derived from Bacillus sp.BSD-8 was cloned into the vector pET28a, and the recombinant expression plasmid pET28a-sox was transformed into E.coli BL21(DE3) for inducible expression. Pick a single colony from the solid plate, insert it into LB medium, and cul...

Embodiment 2

[0054] Example 2. Using three-dimensional fluorescence spectroscopy to quantify sarcosine oxidase

[0055] 1. Determination of the position of the endogenous fluorescence characteristic peak of pure sarcosine oxidase

[0056] Sarcosine oxidase pure enzyme liquid samples with different protein concentrations were selected according to the gradient to measure the FAD fluorescence intensity at 700PMT voltage, and the sarcosine oxidase protein concentration (c, mg / mL) was compared with the FAD fluorescence intensity (ΔF, a.u.) Linear fitting, the concentration of SOX active protein has a good linear relationship in the range of 0.05-1.0 mg / mL, according to the linear relationship between the fluorescence intensity of FAD in the pure enzyme solution of sarcosine oxidase and the concentration of sarcosine oxidase protein, such as Figure 5 As shown, the regression equation I F =44.7+1674c, the R of the regression equation 2 = 0.9929.

[0057] 2. In order to verify the accuracy of...

Embodiment 3

[0062] Example 3. Using three-dimensional fluorescence spectroscopy to detect the presence of cholesterol oxidase in the crude enzyme solution of cholesterol oxidase

[0063] 1. Determination of the position of the endogenous fluorescence characteristic peak of the pure enzyme of cholesterol oxidase

[0064] The cholesterol oxidase gene (GenBank: MK757498.1) derived from Burkholderia cepacia ZWS15 was cloned into the vector pET28a, and the recombinant expression plasmid was transformed into E.coli BL21(DE3) for inducible expression. The constructed engineered bacteria E.coli BL21(DE3)-pET20b(+)-COD was single-colonized into the medium, cultured overnight at 37°C, 200rpm, inserted into LB medium, and cultured overnight at 37°C. Insert TB culture medium according to 5% inoculum amount, cultivate at 37°C, 200r / min until the cell concentration OD 600 After about 0.6-0.8, add 20% lactose induction solution, 28°C, 200rpm, induce culture for 20h. 8000r / min, centrifuge at 4°C for 5m...

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Abstract

The invention discloses a method for detecting flavoprotein based on three-dimensional fluorescence, which belongs to the field of zymoprotein detection and aims to solve the technical problem that impure protein in a flavin protein crude enzyme liquid fermented by genetically engineered bacteria affects accurate, rapid and high-sensitivity qualitative target protein of flavin protein. The invention provides a method for detecting flavoprotein based on three-dimensional fluorescence spectrum. FAD / FMN is specific coenzyme in flavoprotein, existence of flavoprotein in crude enzyme liquid can be quickly and intuitively judged according to FAD / FMN three-dimensional fluorescence characteristic peaks, and according to a linear relation between fluorescence intensity of the coenzyme FAD / FMN fluorescence characteristic peaks in flavoprotein and protein concentration, the flavin protein concentration in the crude enzyme liquid can be rapidly measured, and reference is provided for other rapid protein detection and analysis.

Description

technical field [0001] The invention relates to a method for detecting flavoprotein by using three-dimensional fluorescence spectrum, belonging to the field of enzyme protein detection. Background technique [0002] Flavin proteins are a class of enzymes that bind to flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) coenzymes, and perform redox, bioenergy synthesis, biological Important biological functions such as degradation, apoptosis, neurodevelopment, and biosynthesis. For example, sarcosine oxidase is widely used in the determination of creatinine level in clinical trials, it can accelerate the biodegradation of N-methyl organic nitrogen pesticides in the agricultural field, and it is used in the food field to detect organic acids in wine samples; cholesterol oxidation The enzyme can be used for the determination of cholesterol content in clinical samples, and can also be used for the preparation of biocatalyzed steroid hormone drug prodrugs. [0003] ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6402G01N21/6486G01N2021/6417
Inventor 张玲孟繁松林文萱杨海麟
Owner JIANGNAN UNIV
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