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Method using ultraviolet spectroscopy to detect p-coumaric acid in high-throughput manner

A technology of p-coumaric acid and ultraviolet light, applied in the field of biochemistry, can solve the problems of low detection efficiency, difficulty in random mutation transformation of highly active TAL, low activity of tyrosine ammonia lyase and large-scale synthesis, etc., to achieve high detection efficiency Effect

Active Publication Date: 2017-06-27
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The modification of the bacterial metabolic stream requires the detection of the intermediate p-coumaric acid content at any time. Although the currently widely used high-performance liquid chromatography (HPLC) detection method is very accurate, its detection efficiency is low and cannot meet the needs of high-throughput rapid Testing needs
[0004] Existing studies have shown that in the process of synthesizing p-coumaric acid and other phenylpropanoids from L-tyrosine as a substrate, the low activity of tyrosine ammonia lyase (TAL) is the limit for the large amount of phenylpropanoids. Key Factors for Synthesis
However, due to the limitations of detection methods, it is very difficult to carry out random mutation transformation on existing highly active TALs.
Although Abell reported that the p-coumaric acid content could be detected at 315nm as early as 1987, the reaction system used was a pure buffer system, which could not be applied to the field of fermentation engineering.

Method used

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  • Method using ultraviolet spectroscopy to detect p-coumaric acid in high-throughput manner
  • Method using ultraviolet spectroscopy to detect p-coumaric acid in high-throughput manner
  • Method using ultraviolet spectroscopy to detect p-coumaric acid in high-throughput manner

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Analysis of Spectroscopy Properties

[0024] First, dimethyl sulfoxide (DMSO) was used as a solvent to prepare 200 mg / L p-coumaric acid and 50 mg / L L-tyrosine. Since L-tyrosine is the direct precursor of coumaric acid, in order to verify whether L-tyrosine and the UV absorption of coumaric acid will interfere with each other, the prepared sample was placed in the multifunctional microplate reader Cytation 3 The plate reader (BioTek) performs full-wavelength scanning from 230 to 999 nm. The results show that both substances have a maximum absorption peak at 290nm ( figure 1 ). That is, when the auxochrome group is not added, it is impossible to separate the maximum absorption peak of L-tyrosine and coumaric acid, so it is impossible to quantitatively analyze coumaric acid.

Embodiment 2

[0025] Example 2 Effect of detection wavelength on detection results

[0026] One of the important challenges in the detection of fermentation product yield is that the presence of complex components in the fermentation broth may interfere with the detection of target compounds. We use MOPS medium to cultivate wild-type E. coli BL21(DE3) for 48h. Subsequently, the fermentation broth was centrifuged at 12000 rpm for 5 minutes, and the supernatant was taken as the solvent (natural pH ≈ 4.5) to prepare 2% NaBH 4 The solution is used as an auxochrome group, and 10mg / L p-coumaric acid and 50mg / L L-tyrosine are added respectively. Subsequently, a multi-functional microplate reader was used to scan the full wavelength of 230-999nm. The scan results showed that when MOPS was used as a fermentation medium to culture E. coli BL21 (DE3) in the fermentation broth, NaBH 4 As a auxochrome group, a new absorption peak can be formed for coumaric acid at 339nm, and there is less overlap with the ...

Embodiment 3

[0028] Example 3 NBH 4 The influence of solution concentration and pH of fermentation broth on test results

[0029] (1) Use MOPS medium to cultivate wild-type E. coli BL21(DE3) for 48h, then centrifuge the fermentation broth at 12000rpm for 5min, take the supernatant as the solvent (natural pH≈4.5), and prepare the mass fractions of 1% and 2% respectively , 4% and 6% NaBH 4 The solution is used as a solvent to detect p-coumaric acid.

[0030] (2) Prepare standard solutions of p-coumaric acid with concentrations of 4, 8, 12, 16 and 20 mg / L with DMSO as the solvent. The standard solution of p-coumaric acid was compared with the NaBH of 1%, 2%, 4% and 6% obtained in step (1). 4 The solution is mixed in equal volume, and then the UV absorption intensity is detected in a multifunctional microplate reader to make a standard curve. Take the standard curve R 2 For the evaluation criteria, the results are shown in Table 1:

[0031] Table 1 NaBH 4 Concentration optimization

[0032]

[0033]...

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Abstract

The invention discloses a method using ultraviolet spectroscopy to detect p-coumaric acid in a high-throughput manner and belongs to the field of biochemistry. The method is characterized in that NaBH4 is used as the auxochrome group, the NaBH4 has reaction with p-coumaric acid to form a new ultraviolet absorption peak at 339 nanometers, and the new ultraviolet absorption peak can be separated from a direct precursor L-tyrosine absorption peak. The method has the advantages that convenience is provided for the fermentation engineering production of penylpropanoids, and the high-throughput experiment method can be possibly applied to the modification of strains synthesized by the penylpropanoids.

Description

Technical field [0001] The invention relates to a method for high-throughput detection of p-coumaric acid by ultraviolet spectroscopy, and belongs to the field of biochemistry. Background technique [0002] P-coumaric acid belongs to the phenylpropanoids, which has many physiological functions and biological activities such as antibacterial and hypolipidemic, and has been widely used as an effective ingredient in foods and medicines. Since phenylpropanoid compounds are a general term for a class of compounds formed by connecting a benzene ring with 3 carbon atoms, p-coumaric acid is a simpler compound among phenylpropanoid compounds. Therefore, p-coumaric acid, as an important precursor or intermediate product for the synthesis of more other more complex phenylpropanoids, such as flavonoids, plays an important role in metabolic engineering and synthetic biology. [0003] As an intermediate metabolite of many phenylpropanoid compounds with medicinal value, p-coumaric acid, its accu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/33
Inventor 周景文陈坚周胜虎堵国成张娟
Owner JIANGNAN UNIV
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