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Method for quantitatively determining activity of L-amino acid oxidase by using Prussian blue plate

A technology of Prussian blue and amino acids is applied in the field of quantitative detection of L-amino acid oxidase activity by using Prussian blue agar plates, which can solve the problems of high equipment requirements, complicated operation, high price and the like, and achieves simple and convenient operation, wide applicability and low cost. low effect

Active Publication Date: 2013-02-06
SHANGHAI BAILANG BIOTECHOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although this kit is relatively sensitive, stable and widely used, it requires high equipment, complicated operation, and extremely expensive (each reaction costs about tens of RMB)

Method used

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  • Method for quantitatively determining activity of L-amino acid oxidase by using Prussian blue plate
  • Method for quantitatively determining activity of L-amino acid oxidase by using Prussian blue plate
  • Method for quantitatively determining activity of L-amino acid oxidase by using Prussian blue plate

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Embodiment 1

[0029] The preparation of embodiment 1 Prussian blue agar plate

[0030] Weigh 1g of FeCl 3 ·6H 2 O was dissolved in 50mL of water to configure solution A; weigh 1g of potassium ferricyanide (red blood salt, K 3 Fe(CN) 6 ) was dissolved in 50mL of water to prepare solution B; 50mL of solution A and 50mL of solution B were mixed and shaken to form solution C; 900mL of MM agar (yeast extract 3g / L, peptone 5g / L, agar 20g / L, pH 7.2, solvent is water), then mix 100mL of solution C with 900mL of MM agar and shake well, sterilize at 115°C for 30min, pour it into a Petri dish to prepare 50 Prussian blue agar plates; After the agar is cooled and solidified, use a puncher to punch a small hole with a diameter of 6 mm on the Prussian blue agar plate (the plate is often slightly light green). The composition of the final concentration of the MM agar solution is more preferably yeast extract 3g / L, peptone 5g / L, agar 20g / L, pH 7.5, and the solvent is water.

Embodiment 2

[0031] Embodiment 2 The influence of hydrogen peroxide on the color performance of Prussian blue agar under different pH values

[0032] (1) Use 6N HCl aqueous solution and 6N NaOH aqueous solution to prepare aqueous solutions with pHs of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14, and pipette Pipette 50 μL of the above-mentioned pH 1-14 aqueous solution, respectively, and transfer them into the small holes (diameter 6 mm) of the Prussian blue agar plate prepared by the method in Example 1, place it at room temperature (25°C) for 30 minutes, and observe the color development results of the Prussian blue agar ,See figure 1 Line 1 shows.

[0033] From figure 1 It can be seen in the first line that at pH4, the Prussian blue agar plate produces a small blue circle, and as the pH decreases, the blue circle becomes larger, indicating that Prussian blue agar is more sensitive to pH4 and below; in the case of pH5~8 Under pH conditions, there is no color change on the Prussian...

Embodiment 3

[0036] The blue circle diameter size on the Prussian blue agar plate under the different hydrogen peroxide concentrations of embodiment 3

[0037]Prepare hydrogen peroxide standard solutions (pH about 6) with concentrations of 0.5mmol / L, 1mmol / L, 2mmol / L, 5mmol / L, 10mmol / L, 20mmol / L, 25mmol / L and 30mmol / L respectively with water, Take 50 μL each, and transfer it into the 6mm diameter hole of the Prussian blue agar plate prepared by the method in Example 1 (do three parallels for each hydrogen peroxide concentration), and observe the color development result of the Prussian blue agar after standing at room temperature for 30 minutes ( figure 2 shown). From figure 2 It can be seen that under the action of different concentrations of hydrogen peroxide, blue circles were formed on the Prussian blue agar plates, and the parallelism of the experiment was ideal; moreover, as the concentration of hydrogen peroxide increased, the diameter of the blue circles increased; When the hyd...

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Abstract

The invention discloses a method for quantitatively detecting the activity of L-amino acid oxidase by using a Prussian blue agar plate, which comprises the following steps: with a punch, punching a small hole having a diameter of 4-10 mm on the Prussian blue agar plate; then, adding a liquid to be determined into the small hole, and standing at room temperature for 10-30 minutes, wherein hydrogen peroxide generated by the liquid to be determined forms a blue ring around the small hole; and determining the diameter of the blue ring, determining the release amount of hydrogen peroxide according to the hydrogen peroxide and a standard straight line made of the diameter of the blue ring, and then deducing to obtain the activity of L-amino acid oxidase, wherein the liquid to be determined is a reaction liquid obtained by reacting an enzyme source and a substrate under the conditions of a temperature of 25-50 DEG C and a pH value of 5-9 for 0.5-2 hours, the enzyme source is an enzyme obtained through the fermentation culture of Pseudoalteromonas sp. B3, and the substrate is L-amino acid. The method has the characteristics of low cost, simple and convenient operation process, wide applicability, high stability and the like.

Description

(1) Technical field [0001] The invention relates to a method for testing the activity of L-amino acid oxidase, in particular to a method for quantitatively detecting the activity of L-amino acid oxidase by using a Prussian blue agar plate. (2) Background technology [0002] L-amino acid oxidase (LAAO for short, enzymatic code: EC1.4.3.2) is a kind of flavin adenine dinucleotide (FAD) or mononucleotide (FMN) as the prosthetic flavoprotease. This enzyme can specifically catalyze the oxidative deamination of L-amino acids to generate α-keto acids, ammonia and hydrogen peroxide. Therefore, it can be applied to the quantitative analysis of L-amino acids, the resolution of LD-amino acids and the preparation of α-keto acids. In recent years, it has been reported in the literature that LAAO itself has biological activities such as antibacterial, insecticidal, and antiviral, and also has the effects of interacting with platelets, cytotoxicity, and inducing apoptosis. It has extreme...

Claims

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Application Information

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IPC IPC(8): C12Q1/26C12R1/38
Inventor 余志良周宁裘娟萍
Owner SHANGHAI BAILANG BIOTECHOLOGY
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