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Fluorescent detecting method for nitrile hydrolitic enzyme activity

A nitrilase and fluorescence detection technology, which is applied in the field of fluorescence detection of nitrilase activity, can solve the problems of lack of fluorescent probe detection theory and methods, limit large-scale application, low sensitivity, etc., achieve stable properties, simple equipment, The effect of high purity

Inactive Publication Date: 2007-04-11
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But these two kinds of methods all have bigger defects in themselves: the former is the NH released after the nitrile is hydrolyzed 3 Act with phenol / hypochlorous acid reagent and measure its spectrophotometry at 640nm. The biggest disadvantage of this method is its low sensitivity and the need for corrosive reagents, which limits its large-scale application
However, no matter at home or abroad, the theory and method of detecting and analyzing nitrilase by fluorescent probes are extremely lacking. So far, there is no fluorescent probe that can be directly used to detect nitrilase activity.

Method used

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  • Fluorescent detecting method for nitrile hydrolitic enzyme activity
  • Fluorescent detecting method for nitrile hydrolitic enzyme activity
  • Fluorescent detecting method for nitrile hydrolitic enzyme activity

Examples

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Embodiment 1

[0023] Get 3.90g of salicylaldehyde and 1.86g of hydroxylamine hydrochloride and add them into 0.066mol of sodium hydroxide solution. Then the reaction mixture was stirred and reacted in a water bath at 50°C for 2 hours to obtain a homogeneous solution of a color (yellow), and the pH was adjusted with 36% acetic acid until neutral, and it was placed in an ice bath, there will be Crystals were precipitated, then filtered, washed with water, and air-dried to obtain 2.50 g of the product (salicylaldoxime), with a yield of 67.8%. Then take 1.00 g of salicylaldoxime and 0.50 g of acetic anhydride into the reactor, heat, stir and reflux with an oil bath (160° C.), and react for 10 hours. After heating to reflux, the reaction solution is rotary distilled, unreacted acetic anhydride is distilled off, and 10% NaOH solution is added to the remaining liquid after distillation until the liquid is a clear liquid, and then an appropriate amount of absolute ethanol is added, Make the liquid...

Embodiment 2

[0030] In the NaOH solution (2mol / ml) of 30ml, successively add the 5-chloro salicylaldehyde of 2.00g and the hydroxylamine hydrochloride of 1.00g, (at this moment, the gained solution is not homogeneous, contains the solid of a small amount of particle wherein, so, in reaction A small amount of absolute ethanol was added dropwise in the reaction solution to dissolve a small amount of solid particles in the reaction solution) and the reaction mixture was stirred and reacted in a water bath at 50°C for 2 hours, and then 36% acetic acid was added to adjust the pH to neutral, with white The powdery solid is precipitated, and then the reactor is placed in an ice-water bath to stand still for a while, and then filtered, and the obtained white powdery solid is 5-chlorosalicylaldoxime, and the yield of this product is more than 90%. Then take 1.00g of 5-chloro salicylaldoxime and 0.50g of acetic anhydride into the reactor, heat, stir and reflux with an oil bath (160° C.), and react fo...

Embodiment 3

[0036] Take 1.00g of 5-fluorosalicylaldehyde and 0.50g of hydroxylamine hydrochloride, add successively to 30ml of NaOH (2mol / ml) solution, and stir the reaction mixture in a water bath at 50°C for 2 hours, then add 36% acetic acid to adjust When the pH is neutral, a white powdery solid is precipitated, and then the reactor is placed in an ice-water bath to stand still for a while, and then filtered, and the white powdery solid obtained is 5-fluorosalicylaldoxime, and the yield of this product is 95% . Then take 1.00g of 5-fluorosalicylaldoxime and 0.50g of acetic anhydride into the reactor, heat, stir and reflux with an oil bath (160° C.), and react for 8 hours. After heating to reflux, the reaction solution is rotary distilled, unreacted acetic anhydride is distilled off, and 10% NaOH solution is added to the remaining liquid after distillation until the liquid is a clear liquid, and then an appropriate amount of absolute ethanol is added, Make the liquid uniform (yellow in...

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Abstract

The fluorescent detecting method for nitrile hydrolase activity includes hydrolyzing fluorescent probe with nitrile hydrolase and measuring the fluorescence strength to obtain nitrile hydrolase activity data. The fluorescent probe is made of salicyl nitrile compound in the structure as shown. The present invention has the advantages of simple preparation of fluorescent probe, stable property and high purity of the fluorescent probe, and high detection sensitivity, precision and speed.

Description

(1) Technical field [0001] The invention relates to a fluorescent detection method for nitrilase activity, in particular to a fluorescent detection method for nitrilase activity using salicylnitrile compounds as fluorescent probes. (2) Background technology [0002] Nitrilases are a very important class of hydrolases, which can directly hydrolyze nitrile groups into carboxylic acids and ammonium. Studies have shown that it participates in the biosynthesis of many important substances in organisms, such as plant hormones and 3-carboxyindole. In addition, nitrilase, as a biocatalyst, plays an extremely important role in the asymmetric synthesis of carboxylic acids and their derivatives. [0003] At present, high performance liquid chromatography is basically used for the detection of nitrilase activity. However, this method is time-consuming and laborious, and is not suitable for high-throughput detection. Recently, some new detection methods, such as Berthelot method and F...

Claims

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Application Information

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IPC IPC(8): G01N21/64G01N1/28C12Q1/34C07C255/53
Inventor 朱勍
Owner ZHEJIANG UNIV OF TECH
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