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A kind of quantitative detection method of L-amino acid oxidase activity

A quantitative detection and amino acid technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of complex steps, high equipment requirements, and no reports, and achieve simple and convenient operation, high sensitivity, and environmental friendliness Effect

Active Publication Date: 2015-10-07
SHANGHAI BAILANG BIOTECHOLOGY
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Xylenol orange detection H 2 o 2 The method is more sensitive, but the current method is mainly to detect H by spectrophotometry 2 o 2 Concentration, high requirements on equipment, and small detection throughput, complicated and cumbersome steps
So far, there has been no report on the use of xylenol orange to detect the H produced by the activity of LAAO enzymes. 2 o 2 Concentration, and there is no report to use xylenol orange to quantitatively detect LAAO enzyme activity

Method used

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  • A kind of quantitative detection method of L-amino acid oxidase activity
  • A kind of quantitative detection method of L-amino acid oxidase activity
  • A kind of quantitative detection method of L-amino acid oxidase activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: the preparation of xylenol orange agar plate

[0034] Weigh 0.0278g FeSO 4 Dissolve in 100mL of water to make FeSO with a concentration of 1mmol / L 4 solution; take 0.046g xylenol orange and dissolve it in 100mL of water to make a concentration of 0.6mmol / L xylenol orange solution; weigh 8g agar and dissolve it in 200mL of water to make a concentration of 40g / L agar aqueous solution; the above 100mL concentration of 1mmol / L FeSO 4 Solution, 100mL xylenol orange solution with a concentration of 0.6mmol / L and 200mL agar aqueous solution with a concentration of 40g / L were mixed, shaken evenly, boiled and dissolved, poured into a petri dish to configure 20 xylenol orange agar plates; After cooling and solidification, use a puncher to punch a small hole with a diameter of 6mm on the xylenol orange agar plate (the plate is often slightly yellow). FeSO in xylenol orange agar plates prepared above 4 The concentration is 0.25mmol / L, the concentration of xylenol ...

Embodiment 2

[0035] Embodiment 2: The effect of pH on the color development of xylenol orange agar under the action of hydrogen peroxide

[0036] (1) Mix 6N HCl aqueous solution and 6N NaOH aqueous solution to prepare mixed solutions with pH of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14, Use a pipette to pipette 50 μL of the above-mentioned mixed solution of pH 1 to 14, respectively, and transfer them into the small holes (diameter 6 mm) of the xylenol orange agar plate prepared by the method in Example 1, and observe after standing at room temperature (25°C) for 60 minutes. Xylenol orange agar color results, see figure 1 Line 1 shows.

[0037] From figure 1 It can be seen in the first line that at pH 2 and below, the xylenol orange agar plate produces an orange circle, and as the pH decreases, the orange circle becomes larger, which is mainly under strong acidic conditions, as an acid-base indicator Xylenol orange itself will be obviously orange-yellow; in the case of pH 3-11, th...

Embodiment 3

[0041] Example 3: FeSO 4 Effect of Concentration on Color Development of Xylenol Orange Agar Under the Action of Hydrogen Peroxide

[0042] Under the condition that the fixed xylenol orange concentration is 0.15mmol / L and the agar concentration is 20g / L, FeSO is prepared according to the method of Example 1. 4 Different xylenol oranges with concentrations of 0, 0.05mmol / L, 0.10mmol / L, 0.15mmol / L, 0.20mmol / L, 0.25mmol / L, 0.30mmol / L, 0.35mmol / L and 0.40mmol / L agar plate; then prepare aqueous hydrogen peroxide solution (pH 7.5) with a concentration of 50 μmol / L in water, take 50 μL respectively, and transfer them to the above-prepared 4 Concentration of xylenol orange agar plate in the small hole (diameter 6mm), room temperature for 60 minutes to observe the xylenol orange agar color development results, measure the diameter of the resulting purple circle ( figure 2 ). Such as figure 2 As shown, with FeSO in xylenol orange agar plate 4 As the concentration increases, the d...

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Abstract

The invention provides a method for quantitatively detecting the activity of L-amino acid oxidase by utilizing a xylenol orange agar flat plate. The method comprises the following steps of: punching small holes with the diameter of 4-10mm in the xylenol orange agar flat plate by using a hole puncher, then adding a solution to be detected into the small holes, standing at room temperature for 20-100min, determining the diameter of a purple-red circle, determining the concentration of hydrogen peroxide in the solution to be determined by a standard straight line made according to a log value of the concentration of the hydrogen peroxide and the diameter of the purple-red circle, and further determining the activity of the L-amino acid oxidase, wherein the solution to be detected takes an enzyme solution to be detected of the L-amino acid oxidase as an enzyme source, 0.2-20mmol / L of L-leucine is taken as a substrate, the reaction is performed for 0.5-2h under the conditions that the temperature is 25-50 DEG C and the pH value is 5-9, then the hydrogen peroxide is produced, and an obtained reaction solution is diluted to obtain the solution to be detected. The method provided by the invention has the characteristics of very high sensitivity, low cost, simplicity and convenience in operation, good stability, wide range of application, environmental friendliness and the like.

Description

(1) Technical field [0001] The invention relates to a method for quantitatively detecting 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 xylenol orange 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 flavin proteases. The enzyme can specifically oxidize the deamination of L-amino acids to generate α-keto acids, ammonia and hydrogen peroxide (H 2 o 2 ). The product α-keto acid is a key intermediate in organic synthesis, drug synthesis and biosynthesis, and has important application prospects in medicine, food, feed and other industries. 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 functions of interacting wit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/26
Inventor 余志良裘娟萍赵春田王菊周宁
Owner SHANGHAI BAILANG BIOTECHOLOGY
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