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Method for rapidly detecting activity of nitrilase

A nitrilase and activity technology, applied in the field of biochemical analysis, can solve problems such as low sensitivity and low throughput, and achieve the effects of simple operation process, simple equipment and low price

Inactive Publication Date: 2016-05-04
SHANGHAI INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] For the above-mentioned technical problems in the prior art, the invention provides a kind of method for fast detection nitrilase activity, described this method for fast detection nitrilase activity solves the problem of detecting nitrilase activity in the prior art. The technical problems of low throughput and low sensitivity of the method

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  • Method for rapidly detecting activity of nitrilase
  • Method for rapidly detecting activity of nitrilase
  • Method for rapidly detecting activity of nitrilase

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Embodiment 1 nitrilase reaction process and sample processing

[0024] Resuspend 50 mg of Escherichia coli resting cells containing the nitrilase gene in 5 mL of 0.1M phosphate buffer solution, add an appropriate amount of 1-cyano-cyclohexyl acetonitrile (final concentration 50 mM), and place in a 30°C, 180r / min Shaker reaction 8h. Terminate the reaction with methanol, centrifuge at 4°C to obtain a supernatant, and then dilute it 5 times with double distilled water, which is the sample of nitrile compounds hydrolyzed by nitrilase.

Embodiment 2

[0025] Embodiment 2 hydroxylamine hydrochloride concentration optimization

[0026] Take 1ml of the 5-fold diluted 1-cyano-cyclohexylacetonitrile sample hydrolyzed by nitrilase into a 5ml EP tube, first add 3mL of ethanol solution of hydroxylamine hydrochloride, the concentrations are 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM and 9mM, followed by adding 0.2mL of DCC ethanol solution, the concentration is 0.2M. The mixture was shaken and mixed, heated in a water bath, and heated at 30°C for 20min. After the water bath is completed, add 0.05ml of ferric chloride solution in ethanol, the concentration of which is 0.8M. After developing the color for 10 minutes, measure the absorbance value at 520nm with an ultraviolet spectrophotometer. The results are shown in Table 1. When the added amount increased from 1mM to 4mM, the absorbance increased continuously, and continued to increase the amount to 9mM, and the absorbance decreased continuously. Therefore, its optimum dosage is 4mM. ...

Embodiment 3 3

[0029] Embodiment 3 ferric chloride concentration optimization

[0030] Take 1ml of the 1-cyano-cyclohexylacetonitrile sample diluted 5 times into a 5ml EP tube, first add 3ml of ethanol solution of hydroxylamine hydrochloride, the concentration is 4mM, and then add 0.2ml of ethanol solution of DCC , and its concentration is 0.20M. The mixture was shaken and mixed, heated in a water bath, and heated at 30°C for 20min. After the water bath is completed, add 0.05ml of ferric chloride solution in ethanol, the concentration of which is 0.10M, 0.20M, 0.30M, 0.40M, 0.50M, 0.60M, 0.70M, 0.80M, 0.90M and 1.0MmM, After 10 min of color development, the absorbance value at 520 nm was measured with a UV spectrophotometer. The results show that when the content of ferric chloride increases from 0.1M to 0.5M, the corresponding absorption value increases continuously, but when the concentration of ferric chloride continues to increase to 1.0M, the corresponding absorption value basically t...

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Abstract

The invention discloses a method for rapidly detecting the activity of nitrilase. The method comprises the following steps: adding hydroxylamine hydrochloride and an ethanol solution of dicyclohexylcarbodiimide into a nitrilase reaction sample, vibrating the mixture to evenly mixing the solution, heating the solution by a water bath, then adding an ethanol solution of iron trichloride, fully carrying out color development, measuring the absorbance value at 520 nm by a ultraviolet spectrophotometer, and calculating the activity of nitrilase according to the absorbance value; the measured relative standard deviation (RSD) and recovery rate are 1-2% and 99-102%, and the results are identical with those of HPLC method. The provided method has the advantages of simpleness, rapidness, safety, cheapness, and precision, and can be applied to rapid screening and high flux screening of nitrilase.

Description

technical field [0001] The invention belongs to the field of biochemical analysis and relates to a nitrilase, in particular to a method for rapidly detecting the activity of the nitrilase. Background technique [0002] Nitrilases are a very important class of hydrolytic enzymes, which can directly hydrolyze nitriles into carboxylic acids or amines, and participate in the biosynthesis of important substances in various organisms. In addition, nitrilase can be used as a biocatalyst and plays an important role in the asymmetric synthesis of carboxylic acids and their derivatives. [0003] Over the past few decades, many methods for the determination of nitrilase activity have been developed, including high performance liquid chromatography, gas chromatography, and capillary electrophoresis. However, these methods are complex and time-consuming (ApplEnvironMicrobiol, 2007, 73(19):6053-6057). Among the traditional methods for screening nitrilases, thin-plate chromatography is s...

Claims

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Application Information

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IPC IPC(8): G01N21/33
CPCG01N21/33
Inventor 徐毅杨美燕马宝娣陈建波潘宝悦束亮亮许来罗小龙
Owner SHANGHAI INST OF TECH
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