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Method for rapidly measuring activity of epimerase by using circular dichroism spectrum

A technology of epimerase and circular dichroism, applied in color/spectral characteristic measurement, measuring device, material analysis through optical means, etc., can solve the problems of no quantitative detection of CD, lack of research and reporting of CD signal, etc. Achieve the effect of good linear relationship, small error range and good reproducibility

Active Publication Date: 2020-01-31
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Small peptide is an asymmetric molecule and also has optical activity, but the CD signal generated by small peptides is still lacking in research and reports, and there is no technology to apply CD to the quantitative detection of small peptide enantiomers

Method used

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  • Method for rapidly measuring activity of epimerase by using circular dichroism spectrum
  • Method for rapidly measuring activity of epimerase by using circular dichroism spectrum
  • Method for rapidly measuring activity of epimerase by using circular dichroism spectrum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1, the preparation of dipeptide standard substance

[0055] With buffer 20mM Tris-HCl (pH8.0), 10mM MgCl 2 As a solvent, prepare 10mM standard solutions of AE, Ae, AD, Ad, AM, Am, AR, Ar, AK, Ak, AP, and Ap respectively.

[0056] With buffer 20mM Tris-HCl (pH8.0), 10mM MgCl 2 As a solvent, prepare 0.6, 1.0, 1.5, 2.0, 20, 40, 60, 80, 100mM AE standard solutions.

[0057] With buffer 20mM Tris-HCl (pH8.0), 10mM MgCl 2 As a solvent, prepare 0.6, 1.0, 1.5, 2.0, 20, 40, 60, 80, 100mM Ae standard solution respectively.

[0058] With buffer 20mM Tris-HCl (pH8.0), 10mM MgCl 2 As a solvent, prepare 0.8, 1.0, 2.0, 5.0, 10, 20, 40, 60mM AD standard solution respectively.

[0059] With buffer 20mM Tris-HCl (pH8.0), 10mM MgCl 2 As a solvent, prepare 0.8, 1.0, 2.0, 5.0, 10, 20, 40, 60mM Ad standard solution respectively.

Embodiment 2

[0060] Embodiment 2, the detection of circular dichroism spectrum

[0061] Get the 10mM AE, Ae, AD, Ad, AM, Am, AR, Ar, AK, Ak, AP, Ap standard substance solution prepared in Example 1, and carry out circular dichroism detection, and the detection method is as follows:

[0062] Take 400 μL dipeptide standard solution, add it to a micro cuvette, and measure the CD spectrum of each dipeptide standard solution with a circular dichroism spectrometer. The detection wavelength is 190nm-250nm, the detection step is 2nm, and the response time is 1s. The speed is 500nm / min, each sample is scanned 3 times, the average value is calculated, and the response value of the same solution other than the dipeptide is deducted, and the CD signal is expressed in millidegrees (mdeg).

[0063] Test results such as figure 1 As shown, the L-type dipeptides of AE / Ae, AD / Ad, AM / Am, AR / Ar, and AK / Ak are mirror images of the CD spectra of the corresponding D-type dipeptides. Specifically, AE has a posit...

Embodiment 3

[0065] Embodiment 3, the drafting of CD signal intensity-AE / Ae concentration standard curve

[0066] Get the AE and Ae standard solution of 0.6, 1.0, 1.5, 2.0, 20, 40, 60, 80, 100mM prepared in Example 1, detect according to the detection method of circular dichroism spectrum in Example 2, the result is as follows figure 2 As shown, the CD spectra on the concentration gradients of AE and Ae are mirror images, AE has a positive peak at 226nm, and Ae has a negative peak at 226nm, and the peak shape is intact. When the concentration of dipeptide enantiomers changed, different CD spectral response values ​​were shown, and the peaks all appeared at 226nm.

[0067] The concentration of the D-type dipeptide is calculated as the negative number of the concentration of the L-type dipeptide, for example, "10mM Ae" is calculated as "-10mM AE". With dipeptide concentration as independent variable and CD signal intensity as dependent variable, make a standard curve y=ax+b,

[0068] That...

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Abstract

The invention relates to a method for rapidly measuring the activity of epimerase by using a circular dichroism spectrum. According to the method, epimerase is added into a dipeptide enantiomer initial solution for catalytic reaction; a CD signal intensity change of the dipeptide enantiomer solution before and after the catalytic reaction is detected by using a circular dichroism spectrum; if anychange happens, the epimerase is indicated to have activity; and otherwise, e epimerase is indicated not to have activity. According to the method provided by the invention, no derivative reaction isneeded; the operation is simple, the reproducibility is high, the error range is small, the sensitivity is high, the linear range is wide, and measurement can be carried out continuously, and in-situdetection can be realized. Therefore, a new field is provide for research on chiral recognition.

Description

technical field [0001] The invention relates to a method for rapidly measuring epimerase activity by using circular dichroism spectrum, belonging to the field of biotechnology. Background technique [0002] Most amino acids have chiral centers and exist as D / L enantiomers. The two enantiomers have different biochemical properties and physiological activities, and play different roles in biological processes. Small peptides refer to oligopeptides composed of 2 to 3 amino acids. Due to the different chirality of amino acids, small peptides also exist in the form of D / L enantiomers, such as L-Ala-D-Glu and L-Ala -L-Glu are enantiomers of each other. Epimerase is a class of enzymes that catalyze the transposition reaction of the three-dimensional configuration of groups bound to the same carbon atom. Among them, L-Ala-D / L-Glu epimerase catalyzes L-Ala-D Interconversion of -Glu with L-Ala-L-Glu. The gene YcjG of Escherichia coli K12 encodes a typical L-Ala-D / L-Glu epimerase Y...

Claims

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Application Information

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IPC IPC(8): G01N21/19G01N21/25G01N21/75
CPCG01N21/19G01N21/25G01N21/75G01N2021/755
Inventor 陈秀兰杨洁钟帅于洋宋晓妍张玉忠苏海楠秦启龙李平一
Owner SHANDONG UNIV
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